Ebook The cutaneous lymphoid proliferations - A comprehensive textbook of lymphocytic infiltrates of the skin (2nd edition): Part 1

(BQ) Part 1 book The cutaneous lymphoid proliferations - A comprehensive textbook of lymphocytic infiltrates of the skin presents the following contents: Introduction to the classification of lymphoma; the therapy of cutaneous T cell lymphoma; molecular analysis in cutaneous lymphoid proliferation; benign lymphocytic infiltrates; reactive lymphomatoid tissue reactions mimicking cutaneous T and B cell lymphoma,...

Professor of Pathology and Laboratory Medicine

Department of Pathology, Cornell University

Weill Cornell Medicine

New York, NY, USA

Clinical Professor of Dermatology, Pathology, and Surgery

Director of Dermatopathology at the University of Oklahoma and Regional Medical Laboratory

President of Pathology Laboratory Associates

Tulsa, OK, USA

Clinical Professor of Pathology and Dermatology, Harvard Medical School

Director of Melanoma Program, Dermatology, Brigham and Women’s Hospital

Co-Director of Melanoma Program, Dana-Farber and Brigham and Women’s Cancer Center

Director, Mihm Cutaneous Pathology Consultative Service

Brigham and Women’s Hospital

Boston, MA, USA

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10 9 8 7 6 5 4 3 2 1

Acknowledgments, viii

1 Introduction to the Classification of Lymphoma, 1

Kiel Lukes–Collins, and Working Formulation

Appendix: Definitions of key terms and techniques, 9

2 The Therapy of Cutaneous T Cell Lymphoma, 14

Benjamin H Kaffenberger, Mark A Bechtel, and Pierluigi Porcu

Introduction, 14

Diagnostic work-up and staging procedures, 14

CTCL therapies, 15

Goals of therapy in advanced-stage CTCL, 16

Extracorporeal photopheresis (ECP), 17

Interferons, 17

Retinoids, 17

Immunotoxins, 18

Monoclonal antibodies, 18

Histone deacetylase inhibitors (HDACi), 19

Antibody drug conjugates (ADC), 19

Cytotoxic chemotherapy, 19

Investigational therapies, 20

TLR agonists and cytokines, 20

Allogeneic hematopoietic stem cell transplantation (allo-HSCT), 20

Immunoglobulin and T cell receptor structure, 23

PCR design for determination of clonality, 24

Detection of PCR products for clonality, 24

Spongiotic and eczematous dermatitis, 37

Other spongiotic/eczematous tissue reactions, 40Other causes of subacute eczematous dermatitis, 40Interface dermatitis: cell-poor vacuolar interface dermatitis, 42Interface dermatitis: lichenoid pattern, 46

Diffuse and nodular lymphocytic dermal infiltrates without atypia, 51

Diffuse and nodular lymphocytic infiltrates associated with autoimmune disease, 53

Angiomatous Variants of Pseudolymphoma, 67Case vignettes, 69

References, 86

6 Precursor Lesions of Cutaneous T Cell Lymphoma, 89

Cutaneous T cell lymphoid dyscrasia, 89Large plaque parapsoriasis, 90

Hypopigmented interface T cell dyscrasia: a unique indolent T cell dyscrasia, 91

Pigmented purpuric dermatosis (PPD), 92Pityriasis lichenoides, 94

Idiopathic erythroderma (pre-Sézary), 96Syringolymphoid hyperplasia with alopecia, 96Folliculotropic T cell lymphocytosis/pilotropic T cell dyscrasia, 97

Idiopathic follicular mucinosis/alopecia mucinosa, 98Keratoderma-like T cell dyscrasia, 99

Atypical lymphocytic lobular panniculitis, 100Case vignettes, 102

References, 132

7 Marginal Zone Lymphoma and Other Related Post Germinal Center B Cell Lymphoproliferative Disorders of The Skin, 134

Marginal zone lymphoma, 134Blastic marginal zone lymphoma, 140Epidermotropic marginal zone lymphoma, 140Castleman disease, 141

Primary cutaneous plasmacytoma, 142Case vignettes, 145

References, 166

8 Primary Cutaneous Follicle Center Cell Lymphoma, 169

9 Primary Cutaneous Diffuse Large B-Cell Lymphoma

Including the Leg Type and Precursor B Cell Lymphoblastic

Lymphoma, 187

Primary cutaneous diffuse large B cell lymphoma, 187

Systemic diffuse large B cell lymphomas with a propensity to

involve the skin, 197

Intravascular anaplastic large cell lymphoma, 219

Benign intravascular proliferations of histiocytes and reactive

Sézary syndrome and erythrodermic mycosis fungoides, 243

Large cell transformation of mycosis fungoides, 251

Extracutaneous involvement in mycosis fungoides, 254

Case vignettes, 259

References, 271

13 CD30-Positive Lymphoproliferative Disorders Including

Lymphomatoid Papulosis, Borderline CD30-Positive

Lymphoproliferative Disease, Anaplastic Large Cell

Lymphoma, and T-Cell-Rich CD30-Positive Large B Cell Lymphoma, 274

Introduction, 274 Lymphomatoid papulosis, 274

CD8+ lymphomatoid papulosis, including the type

D variant, 278

Type E lymphomatoid papulosis

(Case vignette 15), 278

Borderline CD30-positive lymphoproliferative disorders

(type C LYP) (Case vignette 9), 279

Lymphomatoid papulosis with a rearrangement of

chromosome 6p25.3, 279 Cutaneous anaplastic large cell lymphoma, 280 Small cell ALCL, 282

Additional unusual histologic variants of anaplastic large cell

lymphoma, 282

Breast-implant-associated anaplastic large cell

lymphoma, 282 Intravascular anaplastic large cell lymphoma, 282

Sarcomatoid anaplastic large cell lymphoma

(Case vignette 14), 283 CD30-positive large B cell lymphoma, 285 Case vignettes, 286

References, 309

14 CD4+ Peripheral T Cell Lymphoma, Not Otherwise Specified, Including Primary Cutaneous Cd4+ Small/ Medium-Sized Pleomorphic T Cell Lymphoma, 312

Introduction, 312Primary cutaneous CD4+ small/medium-sized pleomorphic

T cell lymphoma, 312CD30-negative large cell T cell lymphoma, 313Cutaneous follicular helper T cell lymphoma, 314Overview of overall prognosis of primary cutaneous peripheral

T cell lymphoma, unspecified, 315Evolution of the nomenclature of primary cutaneous CD4+ small/medium-sized pleomorphic T cell lymphoma, 319Case vignettes, 320

References, 333

15 Subcutaneous Panniculitis-Like T Cell Lymphoma, 334

Clinical features, 334Morphology, 336Phenotype, 337Molecular studies, 337Differential diagnosis, 337Case vignettes, 340References, 349

16 CD8 T Cell Lymphoproliferative Disease of the Skin, 351

Overview, 351Introduction, 351Classification of primary CD8+ cutaneous

T cell lymphomas, 352Histomorphology of primary cutaneous CD8+ T cell lymphoma: primary cutaneous aggressive epidermotropic CD8+ T cell lymphoma, and CD8+ variants of

peripheral T cell lymphoma, NOS, including primary

cutaneous CD8+ granulomatous T cell lymphoma, 353

CD8 variant of lymphomatoid papulosis and other related

CD30-positive T cell lymphoproliferative disorders of CD8

subtype, 354

Light microscopic findings, 354

Indolent CD8 positive lymphoid proliferation of the face and

other body sites including acral surfaces, 355

17 Nasal and Related Extranodal Natural Killer Cell/T Cell

Lymphomas and Blastic Plasmacytoid Dendritic Cell

Neoplasm, 377

Introduction, 377

Biology of NK and NK-like T cells, 377

NK/T-cell lymphoma, 379

Nasal NK/T cell lymphoma, 379

Nasal type NK/T cell lymphoma, 380

Aggressive NK cell lymphoma, 380

Role of Epstein–Barr virus in the evolution of NK/T cell

lymphomas, 382

Blastic plasmacytoid dendritic cell neoplasm, 382

CD56-positive γ δ lymphoma involving the subcutaneous fat,

383

Chronic granular lymphocytosis/large granular cell leukemia, 384

Natural killer-like CD4+ T cell lymphoma, 384

EBV-associated NK/T cell lymphomas of the elderly, 385

Hydroa vaccineforme (HV)-like lymphoma, 385

Cutaneous intravascular NK T cell lymphoma, 386

Hydroa vacciniforme-like EBV-associated T cell

lymphoproliferative disease/mosquito bite hypersensitivity, 416

EBV+ cutaneous B cell lymphoproliferative disorder of the

elderly, 420

EBV-associated mucocutaneous ulcer, 421

EBV + T cell lymphoproliferative disease of the elderly, 421

General principles regarding EBV-associated

lymphomagenesis, 421

Pathogenetic link between EBV-associated B cell lymphoma

and iatrogenic immune dysregulation related to either

methotrexate or cyclosporine, 421Case vignettes, 423

References, 432

20 Hodgkin Lymphoma of the Skin, 435

Clinical features, 435Subtypes of Hodgkin lymphoma, 436References, 447

21 Chronic Lymphocytic Leukemia of B Cell and T Cell Prolymphocytic Leukemia, 449

B cell chronic lymphocytic leukemia, 449

T cell prolymphocytic leukemia, 452Case vignettes, 455

References, 471

22 Adult T Cell Leukemia/Lymphoma, 473

Clinical features, 473Pathology, 474Phenotypic studies, 475Pathogenesis, 475Infective dermatitis of childhood, 476Case vignettes, 477

References, 484

23 Angioimmunoblastic Lymphadenopathy/

Angioimmunoblastic T Cell Lymphoma, 486

Clinical features, 486Light microscopic findings, 487Phenotypic studies, 488Molecular studies, 488Pathogenesis, 489Case vignettes, 491References, 497

24 Lymphomatoid Granulomatosis, 499

Introduction, 499Clinical features, 499Histopathology, 500Histogenesis, 501Clonality studies, 501Differential diagnosis, 501Treatment, 502

Case vignette, 503References, 506

25 Cutaneous Infiltrates of Myeloid Derivation 507

Introduction, 507Leukemia cutis, 507Clonal histiocytopathy syndromes, 509Histiocytopathy of factor XIIIA perivascular dermal dendritic cell origin, 514

Case vignettes, 517References, 537Index, 541

The Cutaneous Lymphoid Proliferations: A Comprehensive Textbook of Lymphocytic Infiltrates of the Skin, Second Edition Cynthia M Magro, A Neil Crowson and Martin C Mihm.

© 2016 John Wiley & Sons, Inc Published 2016 by John Wiley & Sons, Inc.

1

Introduction to the Classification of Lymphoma

Kiel Lukes–Collins, and Working Formulation

classifications

Since the first edition of this book, further clinical, morphologic

and genetic research has continued to shed light on the different

aspects of lymphoma A significant revision of the classification

was published in 2008 (Swerdlow et al., 2008), which highlights

the extensive advances that have been made over the decades

in understanding these hematologic disorders The prominent

aspects of this approach will be considered, as well as the basis for

the new recommendations A review of some suggestions for

fur-ther classifications of T cell lymphomas will also be detailed The

earliest classification schemes were based on architectural criteria;

specifically, lymphomas were categorized in terms of those that

assumed a diffuse versus a nodular growth pattern (Rappaport et

al., 1956; Lennert et al., 1975; Lennert, 1978; Lennert and Feller,

1992) In the 1960s, the Rappaport classification scheme, prior to

the advent of immunophenotyping, added a consideration of the

cell type In that classification scheme, the large lymphocytes were,

not surprisingly, mistaken for histiocytes Thus, for example, that

scheme recognized a diffuse histiocytic lymphoma, which we now

know to derive from lymphocytes and to be, most often, a diffuse

large B cell lymphoma With the use of immunophenotyping, and

the recognition of the distinction between T and B lymphocytes

and histiocytes, new approaches to lymphoma classification

emerged One such scheme, designated the Kiel classification

(see Table 1.1), graded lymphoid neoplasms into low-grade

ver-sus high-grade lymphomas and attempted to relate the cell types

identified in any particular lymphoma to their non-neoplastic

counterparts in the benign lymph node (Gerard-Marchant et

al., 1974; Lennert et al., 1975; Lennert, 1978, 1981; Stansfield

et al., 1988; Lennert and Feller, 1992) Popular in the Western

hemisphere from the mid-1970s to the mid-1980s, the Lukes–Collins classification emphasized immunophenotypic profiling (Lukes and Collins, 1974)

In the early 1980s, the International Working Formulation categorized lymphoid neoplasms into low, intermediate, and high grade malignancies based on clinical aggressiveness in con-cert with light microscopic findings The goal was to produce

a categorization of hematologic malignancies regardless of site

of origin that was clinically useful, yet had scientific merit and diagnostic reproducibility (the non-Hodgkin pathological clas-sification project 1982) Although the Kiel classification pres-aged the Working Formulation, this newer classification scheme

did not emphasize B and/or T cell ontogeny per se; this was in

contradistinction to the updated Kiel classification (Table 1.2) Among the low-grade malignancies were small lymphocytic lymphoma, chronic lymphocytic leukemia, small cleaved fol-licular lymphoma, and follicular lymphoma of mixed cell type The intermediate-grade tumors included malignant lymphoma

of follicle center cell origin with a predominance of large cells, diffuse lymphoma of small cleaved cells, and diffuse lymphoma

of mixed and/or cleaved or noncleaved large cell type The high-grade tumors were the diffuse immunoblastic, lymphoblastic, and Burkitt’s lymphoma The cytomorphology

Source: Lennert, 1981 Reproduced with permission of Springer.

Table 1.1 Kiel classification of lymphomas

and architecture were clearly of cardinal importance and, in

es-sence, took precedence over the cell of origin in this

classifica-tion scheme

By the mid-1990s there was sufficient data gleaned from

im-munohistochemistry, cytogenetics, and molecular techniques

to better categorize these tumors as distinct clinical and

patho-logical entities manifesting reproducible phenotypic, cytogenetic,

and molecular features, all defining critical determinants in the

clinical course and prognosis To attempt to evaluate whether a

new classification scheme could be devised, a panel of 19

hemat-opathologists from Europe and the United States met to

evalu-ate the current classification systems to consider whether a

synthesis of the prior efforts could be made into a more usable

and practical device to aid pathologists and clinicians The

clas-sifications under consideration were the Kiel classification

(Gerard-Marchant et al., 1974; Lennert et al., 1975; Lennert, 1978,

1981; Stansfield et al., 1988; Lennert and Feller, 1992), the Lukes–

Collins classification (Lukes and Collins, 1974), and the Working

Formulation (non-Hodgkin lymphoma pathologic classification

project, 1982) What ultimately eventuated from this meeting

was the Revised European–American Classification of Lymphoid

Neoplasms (REAL classification) (see Table 1.3) It represented a

synopsis of the existing hematologic literature, allowing

categori-zation based on distinctive forms of hematopoietic and lymphoid

malignancy separated on the basis of their peculiar clinical, light

microscopic, phenotypic, molecular, and cytogenetic profiles

(Har-ris et al., 1994; Cogliatti and Schmid, 2002).

Table 1.2 Working Formulation

Small lymphocytic Consistent with chronic lymphocytic leukemia;

plasmacytoid

Malignant lymphoma, follicular

Predominantly small cleaved diffuse areas; sclerosis

Malignant lymphoma, follicular

Mixed, small cleaved and large cell diffuse areas;

sclerosis

Predominantly large cell Diffuse areas; sclerosis

Malignant lymphoma, diffuse

Small cleaved Sclerosis

Malignant lymphoma, diffuse

Mixed, small and large cell Sclerosis; epithelioid cell component

Malignant lymphoma, diffuse

Large cell Cleaved; noncleaved; sclerosis

Large cell, immunoblastic Plasmacytoid; clear cell; polymorphous; epithelioid cell component

Malignant lymphoma

Lymphoblastic convoluted; nonconvoluted

Malignant lymphoma

Small noncleaved Burkitt’s; follicular areas

Mycosis fungoides Histiocytic lymphoma Extramedullary plasmacytoma Unclassifiable

Other

Source: Non-Hodgkin lymphoma pathologic classification project, 1982 Reproduced

with permission of John Wiley & Sons.

Table 1.3 Revised European–American Lymphoma classification (REAL)

Precursor B cell neoplasm

Precursor B-lymphoblastic leukemia/lymphoma

Mature (peripheral) B cell neoplasms

B cell chronic lymphocytic leukemia/small lymphocytic lymphoma

B cell prolymphocytic leukemia Lymphoplasmacytic lymphoma Splenic marginal zone B cell lymphoma (+/−villous lymphocytes) Hairy cell leukemia

Plasma cell myeloma/plasmacytoma Extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type

Nodal marginal zone lymphoma (+/−monocytoid B-cells) Follicle center lymphoma, follicular,

Mantle cell lymphoma Diffuse large cell B cell lymphoma Mediastinal large B cell lymphoma Primary effusion lymphoma Burkitt’s lymphoma/Burkitt’s cell leukemia

T cell and natural killer cell neoplasms Precursor T cell neoplasm

Precursor T lymphoblastic lymphoma/leukemia

Mature (peripheral) T cell and NK cell neoplasms

T cell prolymphocytic leukemia

T cell granular lymphocytic leukemia Aggressive NK cell leukemia Adult T cell lymphoma/leukemia (HTLV-1+) Extranodal NK/T cell lymphoma, nasal type Enteropathy-type T cell lymphoma Hepatosplenic γ /δ T cell lymphoma

Mycosis fungoides/Sézary syndrome Anaplastic large cell lymphoma, T/null cell, primary cutaneous type Peripheral T cell lymphoma, not otherwise characterized Angioimmunoblastic T cell lymphoma

Anaplastic large cell lymphoma, T/null cell, primary systemic type

Source: Harris et al., 2000 Reproduced with permission of Oxford University Press.

WhO, reaL, eOrtC, and the Combined WhO/ eOrtC classifications

The new WHO classification was a modest revision of the REAL classification, once again amalgamating reproducible clinical, light microscopic, phenotypic, molecular, and cytogenetic features into

a coherent scheme (Jaffe et al., 2001; Cogliatti and Schmid, 2002)

The concept of a classification scheme based purely on morphology was now considered archaic However, the WHO/REAL classifica-tion was deficient from the perspective of cutaneous hematologic dyscrasias, as will be alluded to presently (Cogliatti and Schmid, 2002) (Table 1.3) Hence, in 1997 the European Organization for the Research and Treatment of Cancer (EORTC) established a scheme for the classification of cutaneous lymphomas (see Table 1.4) This classification scheme was met with criticism for reasons that will be discussed Among the distinct clinical and pathological entities that were recognized by the EORTC classification were my-cosis fungoides, including specific variants, lymphomatoid papulo-sis, large cell CD30-positive lymphoma, large cell CD30-negative lymphoma, panniculitis-like T cell lymphoma, marginal zone B cell lymphoma, primary cutaneous follicle center cell lymphoma, pri-mary cutaneous large B cell lymphoma of the leg, and primary cu-

taneous plasmacytoma (Willemze et al., 1997) (Table 1.4) The main

problem with this classification scheme was not the specific

enti-ties per se or even their purported clinical behavior The difficulty

was that there were a number of cutaneous hematologic dyscrasias

that either were not included in this classification scheme or were

phenotypically and biologically disparate, yet had to be forced into

the same category For example, both diffuse large B cell

lympho-mas of the trunk without features of follicle center cell origin and

CD30-negative large cell T cell lymphoma would be categorized as

CD30-negative large cell lymphomas However, they are different

from a prognostic perspective, the former being indolent and the

latter being an aggressive form of lymphoma Adult T cell

leuke-mia lymphoma, nasal and extranodal NK/T cell lymphoma, nasal

type, angioimmunoblastic T cell lymphoma, and T prolymphocytic

leukemia commonly involve the skin as part of a disseminated

lym-phomatous process, yet they were not recognized in this

classifica-tion scheme (Cogliatti and Schmid, 2002; Willemze et al., 2005).

Those who were proponents of the updated WHO classification

(i.e., the REAL classification) contended that the WHO scheme

was superior to the EORTC classification of cutaneous lymphomas

However, in the REAL/WHO classification scheme, there was only

recognition of a few distinctive forms of cutaneous lymphoma,

namely, mycosis fungoides, Sézary syndrome, and panniculitis-like

T cell lymphoma All of the other lymphomas were in the context

of disease not specifically involving the skin, albeit recognizing that

the diagnostic terms rendered could certainly be applied to various

cutaneous lymphomas, including anaplastic large cell lymphoma,

peripheral T cell lymphoma, not otherwise specified, NK/T cell

lym-phoma, extranodal marginal zone lymlym-phoma, follicular lymlym-phoma,

diffuse large B cell lymphoma, and extramedullary plasmacytoma

Furthermore, all of the systemic and/or extracutaneous lymphomas

that commonly involved the skin, such as adult T cell leukemia

lymphoma were recognized by the WHO (Harris et al., 1994; Jaffe

et al., 2001) Thus, the advantage of this classification scheme was

that it encompassed a much broader spectrum of hematologic

dis-eases having the potential to involve the skin The problem was the

radical difference in prognosis between the various lymphomas at

extracutaneous sites relative to their behavior when presenting as

primary cutaneous neoplasms Perhaps the best example of this is primary cutaneous follicle center lymphoma and primary cutane-ous diffuse large cell B cell lymphoma, which can represent indo-lent forms of malignancy in the skin The same potentially benign clinical course may apply to primary cutaneous anaplastic large cell lymphoma and localized peripheral T cell lymphoma in the skin, when dominated by small- and medium-sized lymphocytes

To address the deficiencies in both the WHO and EORTC schemes as they apply to cutaneous hematologic disorders, a group

of dermatologists and pathologists met in Lyon, France and Zurich, Switzerland in 2003 and 2004 The result was a publication that rep-resents an amicable marriage, falling under the designation of the joint WHO–EORTC classification for cutaneous lymphomas (Jaffe

et al., 2001; Cogliatti and Schmid, 2002; Burg et al., 2005; Slater,

2005; Willemze et al., 2005) (see Table 1.5) The WHO–EORTC

classification recognizes 10 types of cutaneous T cell lymphoma and

4 forms of cutaneous B cell lymphoma, with clinical outcomes for those neoplasms designated as primary cutaneous lymphomas be-ing recognized as distinct and separate from their extracutaneous counterparts For example, diffuse large B cell lymphoma of follicle center cell origin is an indolent lymphoma while the “leg” type is

an intermediate-prognosis lymphoma The WHO–EORTC sification scheme also recognizes hematodermic neoplasm, which

clas-is a nonlymphoid tumor; hematodermic neoplasm now falls under the designation of blastic plasmacytoid dendritic cell neoplasm Furthermore, it does include systemic lymphomas that commonly involve the skin, such as adult T cell leukemia lymphoma and in-travascular large B cell lymphoma The main deficiencies are the failure to include certain lymphoid neoplasms that characteristically involve the skin, namely, primary cutaneous B cell lymphoblastic lymphoma, angioimmunoblastic lymphadenopathy, lymphomatoid granulomatosis, and T cell prolymphocytic leukemia In addition, while it does consider folliculotropic mycosis fungoides, there is

no mention of syringotropic mycosis fungoides The scheme does not address primary cutaneous post-transplant lymphoproliferative

Table 1.4 EORTC Classification for Primary Cutaneous Lymphomas

MF + follicular mucinosis

Pagetoid reticulosis Immunocytoma (marginal

zone B-cell lymphoma) Large cell CTCL, CD30 +

CTCL, pleomorphic small/

medium-sized

Subcutaneous panniculitis-like

T-cell lymphoma

CTCL, cutaneous T-cell lymphoma; CBCL, cutaneous B-cell lymphoma; MF, mycosis

fungoides; SS, Sezary syndrome.

Source: Willemze et al., 1997 Reproduced with permission of American Society of

Hematology.

Table 1.5 WHO–EORTC Classification of Cutaneous Lymphomas

Cutaneous T cell and NK cell lymphomas

Mycosis fungoides Mycosis fungoides variants and subtypes Folliculotropic mycosis fungoides Pagetoid reticulosis

Granulomatous slack skin Sézary syndrome Adult T cell leukemia/lymphoma Primary cutaneous CD30+lymphoproliferative disorders Primary cutaneous anaplastic large cell lymphoma Lymphomatoid papulosis

Subcutaneous panniculitis-like T cell lymphoma Extranodal NK/T cell lymphoma, nasal type Primary cutaneous peripheral T cell lymphoma, unspecified Primary cutaneous aggressive epidermotropic CD8+T cell lymphoma (provisional) Cutaneous γ/δT cell lymphoma (provisional)

Primary cutaneous CD4+ small/medium sized pleomorphic T cell lymphoma (provisional)

Cutaneous B cell lymphomas

Primary cutaneous marginal zone B cell lymphoma Primary cutaneous follicle center lymphoma Primary cutaneous diffuse large B cell lymphoma, leg type Primary cutaneous diffuse large B cell lymphoma, other Intravascular large B cell lymphoma

Precursor hematologic neoplasm

CD4+/CD56+ hematodermic neoplasm (blastic NK cell lymphoma)

Source: Willemze et al., 1997 Reproduced with permission of American Society of

Hematology.

disease (PTLD) and methotrexate associated lymphoproliferative

disease, although most of these in fact would fall in the category of

diffuse large B cell lymphoma or anaplastic large cell lymphoma As

regards to PTLD, polymorphic variants and plasmacytic hyperplasia,

however, would not be recognized In contrast, the WHO

consid-ers these categories of iatrogenic dyscrasia (Jaffe et al., 2001) Other

Epstein–Barr virus (EBV)-related disorders, such as plasmablastic

lymphoma and hydroa vacciniforme-like lesions are not considered

It does not recognize those primary cutaneous small/medium sized

pleomorphic T cell lymphomas that are rarely of the CD8 subset and

which are to be distinguished prognostically from primary

cutane-ous aggressive epidermotropic CD8-positive T cell lymphoma The

designation of peripheral T cell lymphoma, type unspecified, can

denote an aggressive form of cutaneous T cell lymphoma, however

The more accurate designation is that of CD30 negative large T cell

lymphoma and one could argue that the latter designation would be

more apposite While the new scheme does consider hematodermic

neoplasm a tumor of monocytic derivation, there is no consideration

of granulocytic sarcoma, the histiocytopathies, or mast cell disease

The endogenous T cell dyscrasias that may presage lymphoma such

as syringolymphoid hyperplasia with alopecia, atypical lymphocytic

lobular panniculitis, pigmented purpuric dermatosis, and pityriasis

lichenoides are not part of the classification scheme Despite these

deficiencies, it is to date the most accurate classification scheme for

the categorization of hematologic diseases expressed in the skin

(Burg et al., 2005; Willemze et al., 2005).

Since the 2006 WHO/EORTC classification of cutaneous

lym-phoma, further modifications have not been made of this

clas-sification scheme, although there are a number of emerging

lymphoproliferative disorders, all of which we will consider in this

latest edition of the book, including the new variants of

lymphoma-toid papulosis, indolent CD8 lymphoid proliferation, EBV+

lym-phoproliferative disease of the elderly, indolent variants of gamma

delta T cell lymphoma, and double-hit lymphoma However, an

important modification made by the International Society for

Cu-taneous Lymphoma/EORTC for the TNM classification of

myco-sis fungoides (MF) and Sézary syndrome was published in 2007

(Kim et al., 2007) It was the advancement in the understanding

of the pathophysiology, including the cytogenetic and molecular

basis of MF/SS that emerged as the impetus for the revised TNM

classification of MF/SS presented in Table 1.6 The basic principles

are identical to those outlined in the 1979 classification scheme In

the revised classification scheme, T0, as defined by lesions that are

clinically and or histopathologically suspicious for MF/SS no longer

exists Another modification reflects the designated T1 and T2

sub-script as “a” for cases that are exclusively in the context of patch

stage MF and “b” for cases that manifest a patch/plaque stage

over-lap For skin, patch indicates any size skin lesion without significant

elevation or induration Presence/absence of hypo- or

hyperpig-mentation, scale, crusting, and/or poikiloderma is noted A plaque

indicates any size skin lesion that is elevated or indurated

Pres-ence or absPres-ence of scale, crusting, and/or poikiloderma is noted

The percentage of the skin involved is another important staging

determinant In the 1979 classification, it was assumed that the

palm represented 1% of the body surface area; however, the revised

updated classification scheme indicates that the palm represents

approximately 0.5% of the body surface area Another

methodol-ogy for calculating percentage of body surface involved addresses

the percentage of the skin involved in 12 specific regions and then

tabulates the cumulative percentages In the revised

classifica-tion scheme, ulceraclassifica-tion does not define a criterion for warranting

Table 1.6 ISCL/EORTC revision to the classification of mycosis fungoides and Sézary syndrome

TNMB stages Skin

T1 Limited patches, papules, and/or plaques covering < 10%

of the skin surface May further stratify into T1a (patch only) versus T1b (plaque ± patch).

T2 Patches, papules or plaques covering ≥ 10% of the skin

surface May further stratify into T2a (patch only) versus T2b(plaque ± patch).

T3 One or more tumors (≥ 1-cm diameter)

T4 Confluence of erythema covering ≥ 80% body surface area

Node

N0 No clinically abnormal peripheral lymph nodes; biopsy not

required

N1 Clinically abnormal peripheral lymph nodes; histopathology

Dutch grade 1 or NCI LN0-2

N1a Clone negative

N1b Clone positive

N2 Clinically abnormal peripheral lymph nodes; histopathology

Dutch grade 2 or NCI LN3

N2a Clone negative

N2b Clone positive

N3 Clinically abnormal peripheral lymph nodes; histopathology

Dutch grades 3-4 or NCI LN4; clone positive or negative

Nx Clinically abnormal peripheral lymph nodes; no histologic

confirmation

Visceral

M0 No visceral organ involvement

M1 Visceral involvement (must have pathology confirmation and

organ involved should be specified)

Blood

B0 Absence of significant blood involvement: ≤ 5% of peripheral

blood lymphocytes are atypical (Sézary) cells

B0a Clone negative

B0b Clone positive B1 Low blood tumor burden: > 5% of peripheral blood

lymphocytes are atypical (Sézary) cells but does not meet the criteria of B2

B1a Clone negative

B1b Clone positive B2 High blood tumor burden: ≥ 1000/μL Sézary cells with positive

clone

N, node; B, blood; T, tumor; M, metastatic; ISCL, International Society of Cutaneous

Lymphoma; EORTC, European Organization for the Research and Treatment of Cancer.

the designation of tumor stage MF To qualify as tumor stage MF requires at least one tumor 1.5 cm in diameter The total number

of lesions, total volume of lesions, largest size lesion, and region

of body involved is documented Erythroderma qualifies as T4, independent of whether or not the biopsy shows neoplastic T cell infiltration They isolate only two histologic features of prognos-tic significance, namely variants of MF showing folliculotropism, which are classified as representing either a T1 or T2 form of the disease The second histologic feature is one of large cell transfor-mation, defined as a biopsy specimen showing large cells (≥ 4 times the size of a small lymphocyte) in 25% or more of the dermal in-filtrate The large cells are then evaluated for expression of CD30, given the prognostic significance of cases showing CD30-positive large cell transformation versus cases of large cell transformation that are CD30 negative The lymph node alterations range from dermatopathic lymphadenitis (N1) and collections of atypical lym-phocytes (N2), to one of frank effacement of the lymph node (N3) Atypical lymphocytes may be small (6–10 μm) or large (> 11.5 μm) cells; the cells exhibit irregularly folded, hyperconvoluted nuclei In the revised ISCL/EORTC classification, clonality in the lymph node

in the absence of any histologic abnormalities does not alter the staging Abnormal peripheral lymph node(s) indicates any palpable

peripheral node that on physical examination is firm, irregular,

clustered, fixed or 1.5 cm or larger in diameter Node groups

ex-amined on physical examination include cervical, supraclavicular,

epitrochlear, axillary, and inguinal Central nodes, which are not

generally amenable to pathologic assessment, are not currently

considered in the nodal classification unless used to establish N3

histopathologically Peripheral blood involvement has been

recate-gorized whereby B0 represents 5% or less circulating Sézary cells, B2

is now defined as a clonal rearrangement of the TCR in the blood

and either 1.0 K/μL or more Sézary cells or one of two phenotypic

criteria being T cells with CD4/CD8 of 10 or more, or an increase

in circulating CD4+ T cells that show a loss of CD7 or CD26

rep-resenting 40% or 30%, respectively, of the peripheral blood CD4 T

cells B1 is defined as more than 5% Sézary cells, but either less than

1.0 K/μL absolute Sézary cells or absence of a clonal rearrangement

of the TCR, or both (Kim et al., 2007).

In addition, the International Society of Cutaneous Lymphoma

and the EORTC created a risk stratification for cutaneous

lym-phoma other than MF and Sézary syndrome In this risk

stratifi-cation scheme, they proposed a TNM classifistratifi-cation for non-MF/

SS cutaneous lymphomas, as summarized in Table 1.7 The authors

emphasized the importance of a complete history/review of

sys-tems (e.g., +/− B-symptoms, organ-specific signs) and a thorough

physical examination Among the important laboratory values are a

complete blood count with differential, and a comprehensive blood

chemistry measurement, including lactate dehydrogenase (LDH)

They recommend appropriate imaging studies, including the neck

for evaluation of the cervical lymph nodes in cases showing

sig-nificant head and neck involvement Biopsies of suspicious

extra-cutaneous sites are encouraged They also suggest a bone marrow

biopsy and aspirate should be performed in patients at risk of

mar-row involvement, especially in more aggressive forms of lymphoma,

such as natural killer (NK)/T cell, aggressive CD8 + T cell and γ/δ T

cell lymphoma and diffuse large B cell lymphoma, leg type) A bone

marrow is not required in cases of indolent lymphoproliferative

dis-ease A negative marrow involvement would further confirm that

the skin involvement is primary and not secondary to a primary

extracutaneous presentation A lumbar puncture and spinal fluid

assessment is recommended for patients with NK/T cell lymphoma

(Kim et al., 2007) A bone biopsy is recommended for all cases of

diffuse large B cell lymphoma of leg type Some physicians suggest

a bone marrow assessment in cases of primary cutaneous follicle center lymphoma because of the reported incidence of bone mar-row involvement in 10% of cases, which in turn is associated with

an inferior survival The international extranodal lymphoma study group emphasize three clinical parameters that are of prognostic value, namely elevated LDH, the presence of two or more lesions, and a cutaneous tumor that manifests a nodular morphology in the setting of primary cutaneous marginal zone lymphoma and

primary cutaneous follicle center lymphoma.(Senff et al., 2008)

The frequency and the clinical pathological spectrum of mas of the skin diagnosed between the years of 2006 and 2013 at a major referral center in Austria, as categorized according to the two main recent classification schemes, namely the WHO/EORTC and the TNM ISCL/EORTC classifications, was recently published in

lympho-2015 Eighty-three percent of their cases fell into the cutaneous T cell lymphoma category with 60% of these cases being represented

by mycosis fungoides, followed in decreasing order by itive lymphoproliferative disease, primary cutaneous CD4+ small/medium-sized pleomorphic T cell lymphoma, Sézary syndrome and subcutaneous panniculitis-like T cell lymphoma Not surpris-ingly, the most common B cell lymphomas were marginal zone lymphoma, primary cutaneous follicle center lymphoma and dif-fuse large B cell lymphoma of leg type Their experience in terms

CD-30pos-of disease frequency, clinical features, and prognosis mirrors most major academic centers In their study they also found a male pre-dominance, an increasing incidence of cutaneous lymphoma inci-dence with age, and a greater age of onset of B cell lymphoma in

women compared to men (Eder et al., 2015).

While there have not been any further updates of the 2006 WHO classification of cutaneous lymphoma, the 4th edition of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues was published in 2008 by the International Agency for Re-

EORTC-search on Cancer (Swerdlow et al., 2008; Jaffe et al., 2009; Campo

et al., 2011) It was a modification of the earlier WHO classification

of hematologic disorders based on the exact same philosophy as that which formulated the earlier WHO classification In particular, he-matologic disorders were considered as distinct clinicopathological entities where the combination of the clinical features, morphology, phenotypic profile, molecular features, and cytogenetics defined the entity with a precision that reflects the striking advances in our under-standing of the genetic and epigenetic basis of disease Compared to the earlier WHO classification of lymphoma, a far greater number of primary cutaneous lymphomas were recognized

In the category of mature B cell neoplasms, the two primary neous forms of B cell lymphoma that are recognized in the new 2008 classification of hematologic dyscrasias are diffuse large b cell lym-phoma of leg type and primary cutaneous follicle center lymphoma

cuta-In the category of mature T and NK cell neoplasms, mycosis goides, Sézary syndrome, primary cutaneous gamma delta T cell lym-phoma, primary cutaneous CD30-positive T cell lymphoproliferative disease, primary cutaneous CD4+ small/medium sized pleomorphic

fun-T cell lymphoma and subcutaneous panniculitis-like fun-T cell phoma are described The variants of mycosis fungoides recognized include follicular MF, pagetoid reticulosis, and granulomatous slack skin Each lymphoma is presented as a distinct clinical pathological entity with unique clinical and histologic features, a distinctive phe-notypic, molecular and cytogenetic, and oncogenic gene profile The evolution of the current classification to one of precision at the exact

lym-Table 1.7 TNM Classification for lymphomas other than MF and SS

T

T1: Solitary skin involvement

T1a: a solitary lesion ≤5 cm diameter

T1b: a solitary >5 cm diameter

T2: Regional skin involvement: multiple lesions limited to one body region or

two contiguous body regions

T2a: all disease encompassing in a ≤15-cm-diameter circular area

T2b: all disease encompassing in a >15 ≤30-cm-diameter circular area

T2c: all disease encompassing in a >30-cm-diameter circular area

T3: Generalized skin involvement

T3a: multiple lesions involving two noncontiguous body regions

T3b: multiple lesions involving at least three body regions

N

N0: No clinical or pathologic lymph node involvement

N1: Involvement of one peripheral lymph node region that drains an area of

current or prior skin involvement

N2: Involvement of two or more peripheral lymph node regions or involvement

of any lymph node region that does not drain an area of current or prior

skin involvement

N3: Involvement of central lymph nodes

M

M0: No evidence of extracutaneous non-lymph node disease

M1: Extracutaneous non-lymph node disease present

MF, mycosis fungoides; SS, Sezary syndrome; T, tumor; N, node; M, metastatic.

Table 1.8 The 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid

Tissues

MYELOPROLIFERATIVE NEOPLASMS

Chronic myelogenous leukaemia, BCR-ABL 1 positive

Chronic neutrophilic leukaemia

Mast cell leukaemia

Mast cell sarcoma

Extracutaneous mastocytoma

Myeloproliferative neoplasm, unclassifiable

MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA AND

ABNORMALITIES OF PDGFRA, PDGFRB OR FGFR1

Myeloid and lymphoid neoplasms with PDGFRA rearrangement

Myeloid neoplasms with PDGFRB rearrangement

Myeloid and lymphoid neoplasms with FGFR1 abnormalities

MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASMS

Chronic myelomonocytic leukaemia

Atypical chronic myeloid leukaemia, BCR-ABL1 negative

Juvenile myelomonocytic leukaemia

Myelodysplastic/myeloproliferative neoplasm, unclassifiable

Refractory anaemia with ring sideroblasts associated with marked thrombocytosis

Refractory anaemia with ring sideroblasts

Refractory cytopenia with multilineage dysplasia

Refractory anaemia with excess blasts

Myelodysplastic syndrome, unclassifiable

Childhood myelodysplastic syndrome

Refractory cytopenia of childhood

ACUTE MYELOID LEUKAEMIA (AML) AND RELATED PRECURSOR NEOPLASMS

AML with recurrent genetic abnormalities

AML with t(8;21)(q22;q22); RUNX1-RUNX1T1

AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) CBFB-MYH11

Acute promyelocytic leukaemia with t(15;17)(q22;q12); PML-RARA

AML with t9(;11)(q22;q23); MLLT3-MLL

AML with t(6;9)(p22;q34); DEK-NUP214

AML with inv (3)(q31q26.2) or t (3;3)(q31;q26.2); RPN1-ENV1

AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MLK1

AML with mutated NPM1

AML with mutated CEBPA

AML with meylodysplasia-related changes

Therapy-related myeloid neoplasms

Acute myeloid leukaemia, NOS

AML with minimal differentiation

AML without maturation

AML with maturation

Acute myelomonocytic leukaemia

Acute monoblastic and monocytic leukaemia

Acute erythroid leukaemia

Acute megakaryoblastic leukaemia

Acute basophilic leukaemia

Acute panmyelosis with myelofibrosis

Myeloid sarcoma

Myeloid proliferations related to Down syndrome

Transient abnormal myelopoiesis

Myeloid leukaemia with associated Down syndrome

Blastic plasmacytoid dendritic cell neoplasm

ACUTE LEUKAEMIAS OF AMBIGUOUS LINEAGE

Acute undifferentiated leukaemia

Mixed phenotype acute leukaemia with t(9;22)(q34;q11.2); BCR-ABL1

Mixed phenotype acute leukaemia with t(v;11q23); MLL rearranged

Mixed phenotype acute leukaemia, B/myeloid, NOS

Mixed phenotype acute leukaemia, T/myeloid, NOS

Natural killer (NK) cell lymphoblastic leukaemia/lymphoma

PRECURSOR LYMPHOID NEOPLASMS

B lymphoblastic leukaemia/lymphoma

B lymphoblastic leukaemia/lymphoma, NOS

B lymphoblastic leukaemia/lymphoma with recurrent genetic abnormalities

B lymphoblastic leukaemia/lymphoma with t(9;22)(q34;q11.2)BCR-ABL1

B lymphoblastic leukaemia/lymphoma with t(v;11q23); MLL rearranged

B lymphoblastic leukaemia/lymphoma with t(12;21)(p13;q22); TEL-AML1 (ETV6-RUNX1)

B lymphoblastic leukaemia/lymphoma with hyperdiploidy

B lymphoblastic leukaemia/lymphoma with hyperdiploidy (hypodiploid ALL)

B lymphoblastic leukaemia/lymphoma with t(5;14)(q31;q32); IL3-IGH

B lymphoblastic leukaemia/lymphoma with t(1;19)(q23;p13.3); E2A-PBX1 (TCF3-PBX1)

T lymphoblastic leukaemia/lymphoma MATURE B-CELL NEOPLASMS

Chronic lymphocytic leukaemia/ small lymphocytic lymphoma B-cell prolymphocytic leukaemia

Splenic marginal zone lymphoma Hairy cell leukaemia

Splenic B-cell lymphoma/leukaemia, unclassifiable Splenic diffuse red pulp small B-cell lymphoma Hairy cell leukaemia-variant

Lymphoplasmacytic lymphoma Waldenström macroglobulinemia Heavy chain diseases

Alpha heavy chain disease Gamma heavy chain disease

Mu heavy chain disease Plasma cell myeloma Solitary plasmacytoma of bone Extraosseous plasmacytoma Extranodal marginal zone lymphoma of mucosa-associated lympoid tissue (MALT lymphoma)

Nodal marginal zone lymphoma Paediatric nodal marginal zone lymphoma Follicular lymphoma

Paediatric follicular lymphoma Primary cutaneous follicle center lymphoma Mantle cell lymphoma

Diffuse large C-cell lymphoma (DLBCL), NOS T-cell/histiocyte rich large B-cell lymphoma Primary DLBCL of the CNS

Primary cutaneous DLBCL, leg type EBV positive DLBCL of the elderly DLBCL associated with chronic inflammation Lymphomatoid granulomatosis

Primary mediastinal (thymic) large B-cell lymphoma Intravascular large B-cell lymphoma

ALK positive large B-cell lymphoma Plasmablastic lymphoma Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease Primary effusion lymphoma

Burkitt lymphoma B-cell lymphomas, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma

B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma

MATURE T-CELL AND NK-CELL NEOPLASMS

T-cell prolymphocytic leukaemia T-cell large granular lymphocytic leukaemia Chronic lymphoproliferative disorder of NK-cells Aggressive NK cell leukaemia

Systemic EBV positive T-cell lymphoproliferative disease of childhood Hydroa vacciniforme-like lymphoma

Adult T-cell leukaemia/lymphoma Extranodal NK/T cell lymphoma, nasal type Enteropathy-associated Tcell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides

Sézary syndrome Primary cutaneous CD30 positive T-cell lymphoproliferative disorders Lymphomatoid papulosis

Primary cutaneous anaplastic large cell lymphoma Primary cutaneous gamma-delta T-cell lymphoma Primary cutaneous CD8 positive aggressive epidermotropic cytotoxic T-cell lymphoma

Primary cutaneous CD4 positive small/medium T-cell lymphoma Peripheral T-cell lymphoma, NOS

Angioimmunoblastic T-cell lymphoma Anaplastic large cell lymphoma, ALK positive Anaplastic large cell lymphoma, ALK negative

HODGKIN LYMPHOMA

Nodular lymphocyte predominant Hodgkin lymphoma

Classical Hodgkin lymphoma

Nodular sclerosis classical Hodgkin lymphoma

Lymphocyte-rich classical Hodgkin lymphoma

Mixed cellularity classical Hodgkin lymphoma

Lymphocyte-depleted classical Hodgkin lymphoma

HISTIOCYTIC AND DENDRITIC CELL NEOPLASMS

Histiocytic sarcoma

Langerhans cell histiocytosis

Langerhans cell sarcoma

Interdigitating dendritic cell sarcoma

Follicular dendritic cell sarcoma

Fibroblastic reticular cell tumour

Indeterminate dendritic cell tumour

Disseminated juvenile xanthogranuloma

POST-TRANSPLANT LYMPHOPROLIFERATIVE DISORDERS (PTLD)

Early Lesions

Plasmacytic hyperplasia

Infectious mononucleosis-like PTLD

Polymorphic PTLD

Monomorphic PTLD (B- and T/NK-cell types)*

Classical Hodgkin lymphomas type PTLD*

NOS, not otherwise specified.

The italicized histologic types are provisional entities, for which the WHO Working

Group felt there was insufficient evidence to recognize as distinct diseases at this time.

*These lesions are classified according to the leukaemia or lymphomas to which they

correspond, and are assigned the respective ICD-O code.

genomic level is a dichotomous contrast to the original nascent sification scheme, which recognized only cell size and architecture A summary of the classification scheme is presented in Table 1.8

clas-Summary

Tables 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 1.7 summarize the classification schemes as they have evolved over time It should be apparent to the reader that the most recent classification scheme is certainly apropos, but still not globally inclusive Each of the conditions listed in the clas-sification scheme are discussed in the ensuing chapters, emphasiz-ing the approach that should be given to each hematologic dyscrasia Specifically, the entities are presented in the context of an integration

of clinical, light microscopic, phenotypic, molecular, and cytogenetic data, and, where appropriate, additional considerations are given re-garding pathobiology Each cutaneous disorder truly has its own fin-gerprint; in this regard we have considered many of the individual hematologic disorders in their own respective chapters and/or consid-ered no more than a few entities in a given chapter to emphasize the truly distinctive nature of so many of these disorders In addition, we consider other forms of lymphoid dyscrasia that commonly involve the skin, recognizing that they are rare conditions and are still not part of the WHO–EORTC classification scheme

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appendix: Definitions of key terms and

techniques

t cell antibodies

CD1a (T6, Leu6, OKT6, O10): An immature T cell antigen, found

on cortical thymocytes and Langerhans cells, but not mature T

cells

CD2 (T11, Leu5, OKT11, MT910): A pan T cell antigen that

cor-responds to the sheep erythrocyte rosette receptor It is present on

all normal mature T cells

CD3 (Leu4, T3, OKT3,SP7, PS1, Polyclonal): A pan-T cell antigen

that is composed of five polypeptide chains covalently linked to the

T cell receptor All elements of the CD3/T cell receptor must be

present for cell surface expression Most anti-CD3 antibodies are

directed toward the epsilon chain of the CD3/T cell receptor

com-plex The majority of mature T cells are CD3 positive The CD3

an-tigen is first expressed in the cell cytoplasm and then on the surface

NK cells will manifest only cytoplasmic expression

TCR-1, BF-1: They are antibodies that recognize the α/β

het-erodimer of the human T cell antigen receptor It is expressed on

normal mature peripheral blood T lymphocytes and on 50–70% of

cortical thymocytes The vast majority of T cell malignancies are

derived from T cells of the αβ subtype.

TCR-gamma 1: An antibody that recognizes the γ/δ heterodimer

portion of human T cell antigen receptor It is present on a minor

subset of CD3-positive T cells in peripheral blood, thymus, spleen,

and lymph node

CD5 (T1, Leu1, OKT1, CD5/54/F6, 4C7): A pan T cell antigen

present on the majority of thymocytes and mature peripheral blood

T cells; a loss of CD5 expression in T cells is indicative of ensuing

neoplasia The CD5 antigen is present on a small subset of normal

B cells representing nạve B cells with endogenous autoreactive

features and which have been implicated in innate immunity It

is also expressed on neoplastic B cell lymphoma cells of chronic

lymphocytic leukemia, small lymphocytic lymphoma, rare cases of

marginal zone lymphoma, and mantle zone lymphoma

CD43 (DF-T1): This T-cell-associated antigen is expressed by

normal T cells, granulocytes, and a subset of plasma cells, but not

normal B cells CD43 expression by a B cell is a feature of B cell

neoplasia Primary cutaneous diffuse large B cell lymphomas,

mar-ginal zone lymphomas, and follicle center cell lymphomas can be

CD43-positive

CD7 (Leu9, DK24): A pan T cell marker that is expressed by the

majority of peripheral T cells The expression of CD7 is an event

that occurs relatively early in T cell ontogeny prior to

rearrange-ment of the TCR-β chain The CD7 antigen is expressed by both

mature and immature T cell neoplasms The CD7 antigen may not

be expressed by memory T cells manifesting selective homing to the

skin Although a substantial reduction of this marker is

character-istic for mycosis fungoides can be seen in other forms of peripheral

T cell lymphoma, it is also diminished in the prelymphomatous T

cell dyscrasias and many reactive dermatoses, albeit to a lesser

de-gree than in mycosis fungoides There is variation in the intensity of

staining based on the detection system

CD62L (LECAM-1, LAM-1, MEL-14): CD62L is part of the family of

selectins that comprises three subcategories: L-selectin, E-selectin, and

P-selectin designated as CD62L, CD62E, and CD62P, respectively All

of the selectins exhibit a similar glycan contributing to their adhesion

function and participating in the interactions between inflammatory

cells and endothelium CD62L is expressed on blood monocytes, blood

neutrophils, subsets of natural killer cells, and T and B lymphocytes,

including those of näıve phenotype Virgin T cells in human eral blood uniformly express CD62L, whereas among the memory/ef-fector population, the three predominant subsets are CD62L+/CLA+, CD62L+/CLA−, and CD62L−/CLA−

periph-CD4 (Leu3a, OKT4, MT310): A helper/inducer cell antigen It is

expressed by the majority of peripheral blood T cells and 80–90% of cortical thymocytes Cortical thymocytes that are CD4-positive usu-ally coexpress CD8 The majority of T cell neoplasms are of the CD4

subset γδ T cells and NK cells are CD4negative CD4 is also expressed

by monocytes including, in the context of histiocytic proliferative orders, myelomonocytic dyscrasias and hematodermic neoplasm

dis-CD8 (Leu 2a, C8/144B): A suppressor/cytotoxic cell antigen

The CD8 antigen is a 32 kilodalton heterodimeric protein that is expressed by approximately 30% of peripheral blood mononuclear cells and 60–85% of cortical thymocytes (P/F) Cortical thymocytes

coexpress CD4 γδ Cells are frequently CD8-negative A small

per-centage of peripheral T cell lymphomas are of the CD8 subset, such

as primary cutaneous CD8-positive epidermotropic cytotoxic T cell

lymphoma, some γδ T cell lymphomas, and panniculitis-like T cell

lymphoma Rarely, classic lesions of cutaneous T cell lymphoma (i.e., mycosis fungoides) will be CD8-positive CD8 cells may be suppressive or cytotoxic in nature The latter express cytotoxic pro-teins such as TIA and granzyme

CD26: The protein encoded by the DPP4 gene is an antigenic

enzyme expressed on the surface of most cell types and is ated with immune regulation, signal transduction and apoptosis

associ-It is an intrinsic membrane glycoprotein and a serine exopeptidase that cleaves X-proline dipeptides from the N-terminus of polypep-tides The neoplastic cells of Sézary syndrome do not express CD26 and hence this particular marker is of value in the assessment of the peripheral blood in patients who are suspected as having Sézary syndrome

CD52 (VTH34.5, Campath-1G): Expressed in lymphocytes,

monocytes, eosinophils, thymocytes, and macrophages It is pressed on most B and T cell lymphoid-derived malignancies; ex-pression on myeloma cells is variable

ex-Cutaneous Lymphocyte Antigen (HECA-452): Expressed in

mem-ory T lymphocytes with preferential homing proportion to the skin endothelial cells and epithelial cells

Fox P3 (236A/F7): Constitutive high expression of FOXP3 mRNA

has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of FOXP3 in CD4+CD25− cells imparts a Treg phenotype in these cells

TCL1 oncogene: The TCL1 locus on the chromosome 14q32.1 is

associated with the development of leukemia when there is a location and or an inversion resulting in juxtaposition to various regulating elements of the T cell receptor Tcl1 positivity is observed amidst the neoplastic cells in blastic plasmacytoid dendritic cell ne-oplasm, adult T cell leukemia, and T cell prolymphocytic leukemia

trans-NFATc : Calcineurin/Nuclear factor of activated T cells (NFAT)

signaling plays a critical role in peripheral T-cell activation following TCR engagement In resting cells, inactive NFAT transcription fac-tors are located in the cytoplasm Pathway activation leads to NFAT dephosphorylation, nuclear translocation, and activation of its transcriptional targets In reactive lymphocytic infiltrates and early lesions of mycosis fungoides, the expression of NFAT is primarily confined to the cytoplasm With advanced mycosis fungoides and

or other forms of cutaneous T cell lymphoma, such as peripheral

T cell lymphoma, type unspecified, there is acquisition of nuclear expression of NFAT within the nucleus Of particular relevance

is the finding that the catalytic domain of PLCG1 is frequently

mutated in tumoral samples of cutaneous T cell lymphoma and is

associated with the nuclear expression of NFAT

PD-1: Programmed death-1 (PD-1/CD279) cell surface

pro-tein, an inhibitory member of the CD28 costimulatory

recep-tor superfamily, is expressed mainly in the subset of B cells,

NK T cells, activated monocytes dendritic cells, activated T

lymphocytes, and follicular helper T cells The PD-1 pathway

exerts its function through inhibiting TCR-mediated T cell

proliferation and cytokine secretion, via its two ligands PD-L1

(B7-HICD274), and PD-L2 (B7-DC/CD273) PD1 is expressed

in certain T cell malignancies of putative follicular helper T

cell origin, including angioimmunoblastic lymphoma, primary

cutaneous CD4+ small/medium-sized pleomorphic T cell

lym-phoma, and peripheral T cell lymphoma with a follicular pattern

In addition, in Sézary syndrome, the neoplastic cell populace is

characteristically PD1 positive

TOX: Thymocyte selection-associated high-mobility group box

fac-tor (TOX) is another critical regulafac-tor of early T-cell development,

spe-cifically during the transition from CD4+ CD8+ precursors to CD4+ T

cells However, upon completion of this process, it is tightly suppressed

and mature CD4+ cells do not have significant TOX expression, except

follicular helper T cells There is significant upregulation of nuclear

TOX expression in the neoplastic epidermotropic T cells of mycosis

fungoides Nuclear expression of TOX is not an absolute criterion of

malignancy as it can be seen in reactive lymphocytes, although the

ex-tent and intensity of intraepidermal and dermal nuclear TOX

expres-sion amidst T cells is less in reactive inflammatory dermatoses Since

TOX is upregulated in follicular helper T cells, it is common to see very

strong expression of TOX in cases of primary cutaneous CD4+ small/

medium-sized pleomorphic T-cell lymphoma

plasma cell markers

CD138 (MI15): CD138/syndecan-1 protein backbone is a single

chain molecule of 30.5 kDa Five putative GAG attachment sites

ex-ist in the extracellular domain GAG fine structure appears to reflect

the cellular source of the syndecan Expression of CD138 in

hu-man hematopoietic cells is restricted to plasma cells in normal bone

marrow Early B cell precursors in human bone marrow are CD138

negative CD138 is also expressed in endothelial cells, fibroblasts,

keratinocytes, and normal hepatocytes

Natural killer cell-associated markers

CD16 (DJ130c): A natural killer cell and myelomonocytic antigen

It is expressed by all resting natural killer cells, neutrophils, and

ac-tivated macrophages It is also the antibody receptor for antibody

dependent cellular cytotoxicity

CD56 (MOC1, T199, C5.9): A natural killer cell antigen This antigen

is expressed by all resting and activated natural killer cells, a subset of

cytotoxic T cells that mediates non-major histocompatibility complex

(non-MHC) restricted cytotoxicity, and dendritic monocytes

How-ever, it is expressed by other cell types including CD T cells and

plas-macytoid dendritic cells, and myeloid leukemic cells can express CD56

Cytotoxic protein markers

CD10 (CALLA): This B cell antigen was originally thought to be

a tumor-specific marker expressed by neoplastic cells of acute phoblastic leukemia The CD10 antigen can be expressed by fol-licular lymphomas, B cell lymphoblastic lymphomas, normal T cells undergoing apoptosis and certain T cell malignancies namely in the context of angioimmunoblastic lymphadenopathy

lym-CD19 (HD37): The lym-CD19 antigen is expressed initially at the time

of immunoglobulin heavy chain gene rearrangement Anti-CD19 antibodies stain almost all cases of non-T cell acute lymphoblastic leukemia, as well as mature B cell leukemias and lymphomas Re-stricted to use in flow cytometry or frozen tissues

CD20 (B1, L26, Leu16): A pan B cell antigen that is expressed at

the time of light chain gene rearrangement Anti-CD20 ies react with 50% of immature B cell lymphoblastic leukemia cells CD20 is not expressed by plasma cells It can occasionally be ex-pressed by neoplastic T cells and there is also a population of nor-mal T cells that weakly expresses CD20

antibod-CD22 (4 KB128, To15): A pan B cell antigen that is very similar

to the CD20 antigen

Bcl-1: Bcl-1/cyclin D1 belongs to the G1 cyclins and plays a key

role in cell cycle regulation during the G1/S transition by cooperating with cyclin-dependent kinases (CDKs) Its overexpression may lead to growth advantage for tumor cells by way of enhanced cell cycle progres-sion, and it has been reported in various human cancers, for example, esophageal, breast, and bladder carcinomas Among hematolymphoid malignancies, cyclin D1 overexpression resulting from translocational activation has also been recognized in a subset of B chronic lym-phocytic leukemia (BCLL), multiple myeloma, splenic marginal zone lymphoma, hairy cell leukemia, and mantle cell lymphoma

Bcl-2: The bcl-2 family of proteins (bcl-2, bcl-w, bcl-xL, bcl-2 related protein A1, etc.) regulates outer mitochondrial membrane permeabil-ity Bcl-2, bcl-w, bcl-xL, and bcl-2 related protein A1 are antiapoptotic

members that prevent release of cytochrome c from the mitochondrial

intermembrane space into the cytosol Bcl-2 and bcl-xL are present on the outer mitochondrial membrane and are also found on other mem-branes in some cell types Bcl-w is required for normal sperm matura-tion In the context of its value in lymphoid infiltrates, it is ubiquitously expressed by small mature lymphocytes Normal germinal center cells are bcl-2 negative In contrast, neoplastic germinal center cells can be bcl-2 positive and are typically positive in nodal follicular lymphoma

In primary cutaneous diffuse large cell lymphomas, bcl-2 expression is

an adverse prognostic variable

Bcl-6: Bcl-6 protein is expressed in B cell lymphomas of follicle

center B cell origin

Bcl-10: Apoptosis regulator B cell lymphoma 10 (bcl-10) may

show aberrant nuclear expression in primary cutaneous marginal zone lymphomas associated with extracutaneous dissemination

CD79a: CD79a is expressed during all phases of B cell ontogeny

and in this regard, CD79a is positive in B cells in both early- and late-stage B cell ontogeny It is expressed prior to the expression

of CD20 and is retained in the postgerminal B cell after CD20 is

no longer expressed CD79a is involved in B cell receptor ment whereby a genetic deletion of CD79a can prevent and halt

develop-B cell development Since CD79a is expressed at all stages of develop-B cell ontogeny, it is a valuable marker in concert with CD20; a decre-ment in the expression of CD79a would potentially signify B cell neoplasia

PAX5: The PAX5 gene is a transcription factor that exhibits a highly

conserved DNA binding motif that defines an important factor in the

early development of B cells It has been postulated that dysregulation

of the PAX5 gene contributes to lymphomagenesis It is expressed in

mature B cells including Hodgkin lymphoma There are rare cases of

its expression in anaplastic large cell lymphoma

Myelomonocytic markers including dendritic

cell markers

CD15 (C3D-1): It is normally expressed on neutrophils and most

forms of nonlymphoid acute leukemia It is aberrantly expressed

by Reed–Sternberg cells of Hodgkin lymphoma along with chronic

lymphocytic leukemia and lymphoblastic lymphoma

CD68 (PGM1, KP1): This antigen is found on monocytes,

granu-locytes, mast cells, and macrophages

CD34 (QBEnd10): The CD34 antigen is a single-chain

transmem-brane glycoprotein that is associated with human hematopoietic

progenitor cells It is present on immature hematopoietic

precur-sor cells and TdT-positive B cells and T lymphoid precurprecur-sors CD34

expression decreases as these hematopoietic precursors undergo

progressive maturation CD34 myeloid progenitors can

differenti-ate into two major myeloid subsets in the skin: Langerhans cells and

dermal interstitial dendrocytes While these mature

antigen-pre-senting cells are CD34 negative, the dermal dendritic and

Langer-hans cell precursors manifest a CD34+ CD14+ CD116+ phenotype

The quantity of CD34+ progenitor cells in the marrow is closely

associated with advancement of disease in patients with chronic

idiopathic myelofibrosis Expectedly, patients with myelofibrosis

can develop paraneoplastic Sweet’s-like reactions whereby the

pres-ence of CD34+ cells in the infiltrate could be a harbinger of a more

accelerated clinical course (personal observations) CD34+

hemat-opoietic stem cells are the source of dermal fibrocytes involved in

wound healing and representing the implicated fibrogenic cell of

nephrogenic systemic fibrosis

CD43: CD43 antigen is expressed by T cell lymphomas and about

30% of B cell lymphomas CD43 is expressed on the membrane and

in the cytoplasm of T cells and cells of myeloid lineage, including

monocytes CD43 expression by a B cell is a phenotypic aberration

indicative of B cell neoplasia

CD123: The protein encoded by this gene is an interleukin-3

(IL-3)-specific subunit of a heterodimeric cytokine receptor The

receptor is composed of a ligand-specific α subunit and a signal

transducing β subunit shared by the receptors for IL-3, colony

stimulating factor 2 (CSF2/GM-CSF), and interleukin-5 (IL-5)

The binding of this protein to IL3 depends on the β subunit The

β subunit is activated by the ligand binding and is required for the

biological activities of IL-3 This gene and the gene encoding the

colony-stimulating factor 2 receptor α chain (CSF2RA) form a

cy-tokine receptor gene cluster in an X–Y pseudoautosomal region on

chromosomes X or Y It is positive in acute myelogenous leukemia

and blastic plasmacytoid dendritic cell tumor

CD83: This protein is a member of the Ig superfamily

manifest-ing expression on mature dendritic cells of all types, includmanifest-ing

plas-macytoid dendritic cells and Langerhans cells

CD11c: CD11c transmembrane protein expressed at high levels

on dendritic cells and monocytes that are likely destined to become

dendritic cells It is also positive on hairy cell leukemia cells and

chronic lymphocytic leukemia cells

MXA: MXA is a surrogate marker for the type-I-rich

micro-environment It is expressed in plasmacytoid dendritic cells and

hence can be expressed in neoplastic cells of the blastic toid dendritic cell tumor In addition, myeloid dendritic cells can express MXA including in the context of a neoplastic counterpart characteristic of clonal myeloid dendritic cell dyscrasia, a marker of chronic myeloproliferative disease (i.e myelofibrosis, chronic my-elodysplastic syndrome, myelomonocytic leukemia)

plasmacy-Lysozyme: Lysozyme is also referred to as muramidase It is a

hydrolytic glycosidase with potential antibacterial properties It is found in high concentrations in various bodily secretions and is present at high levels in egg whites Lysozyme is expressed in mac-rophages and neutrophils It is also expressed by earlier precursor cells of myelomonocytic derivation and hence is positive in mye-loid, monocytic and myelomonocytic acute leukemias

CD163: CD163 is a scavenger receptor for the hemoglobin

hap-toglobin complex and is expressed in macrophages Certain nally differentiated monocytes with dendritic cell properties may not be positive for CD163; for example, Langerhans cells do not express CD163 Acute myeloid leukemia with monocytic differen-tiation can, however, exhibit positivity for CD163

termi-Langerin: Langerin is a transmembrane receptor specific for

Langerhans cells, manifesting localization to the Birbeck granule, where it plays a role in the internalization of antigen prior to anti-gen presentation to T cells It is not expressed on indeterminate cells

en route to the lymph node, but rather is expressed on immature Langerhans cells, which reside in the epidermis

CD14 This molecule functions as a toll receptor and is a

marker of terminally differentiated monocytes that are likely destined to become dendritic cells It performs a critical func-tion in the detection of bacterial lipopolysaccharide While the dominant expression is by macrophages and other related ma-ture monocytes, there is weak expression amidst neutrophils The differentiation of the CD14 positive monocyte into a my-eloid dendritic cell and other dendritic cell types occurs in the setting of a cytokine milieu rich in interleukin 4 and granlocyte macrophage colony-stimulating factor

CD117: Mast/stem cell growth factor receptor(SCFR), also known

asproto-oncogene c-CD117 falls under the alternative designations of tyrosine protein kinase and is a receptor tyrosine kinase protein that is encoded by the KIT gene It is expressed in mast cells and in melano-cytes, but it is also expressed by hematopoietic stem cell precursors This latter cell type is normally present at very low levels in the pe-ripheral blood; however, certain agents, such as granulocyte colony-stimulating factor can lead to mobilization to the peripheral blood and extramedullary organ sites CD117 is a proto-oncogene that is overex-pressed in myeloid leukemias and of course is extensively positive in benign and neoplastic mast cell infiltrates

Myeloperoxidase: Myeloperoxidase is a peroxidase enzyme that is

abundantly expressed in neutrophils at high levels Over and above its expression in mature granulocytes, is its positivity in neutrophil precursors In this regard it is expressed in the setting of myeloid leukemia Myeloperoxidase is also expressed in activated macro-phages and therefore can be found in certain histiocyte-rich inflam-matory conditions, such as Kikuchi’s disease, and in the setting of histiocytoid Sweet’s syndrome

Follicular dendritic cell markers

CD21: CD21 also falls under the designation of the C3d receptor and

Epstein Barr virus receptor It is expressed on all mature B cells and follicular dendritic cells It forms a complex with CD19 and CD81 defining the coreceptor B complex It interacts with antigen and

optimizes the B cell response to antigen CD21 is of value in the

as-sessment of the follicular dendritic network in B cell proliferations, as

significant disruption of the orderly follicular dendritic network in a

germinal center is a feature of follicle center lymphoma and marginal

zone lymphoma

CD23: While there is no literature precedent on either the

ex-pression of CD23 in lesions of primary cutaneous B cell lymphoma,

CD23 expression in non-neoplastic lymphoid cells is well described,

occurring in nạve B cells, monocytes and follicular dendritic cells

In human tonsillar tissue, CD23 is a precentroblast marker; it is

ex-pressed on nạve B cells both in the mantle zone and early germinal

center phase It is upregulated in the early stages of B cell activation

by interleukin 4 and functions as an IgE receptor and lymphocyte

growth factor CD23 also plays a role in the augmentation of B cell

proliferation and of antigen presentation Human B lymphocytes

induced from a resting state to one of blastic transformation

dem-onstrate CD23 expression

CD35: CD35 also falls under the designation of Complement

receptor type 1 (CR1) representing a glycoprotein found on

eryth-rocytes, leukocytes, glomerular podocytes, hyalocytes, and splenic

follicular dendritic cells The protein is important in the mediation

of interactions between effector cells and immune complexes

con-taining activated complement It plays a critical role in the removal

of complement opsonized immune complexes It is a negative

regu-lator of the complement cascade, resulting in inhibition of both the

classic and alternative pathways

activation/proliferation markers

CD25 (Tac, ACT-1): An activation marker that detects the α chain of

the interleukin-2 receptor The CD25 antigen is a 55 kilodalton

glyco-protein that is expressed by activated B and T lymphocytes and weakly

by histiocytes The CD25 antigen is strongly expressed by cutaneous T

cell neoplasms undergoing transformation The CD25 antigen is also

expressed by the Reed–Sternberg cells of Hodgkin lymphoma

CD30 (Ber-H2, Ki-1): An antigen (glycoprotein) associated with

activation of hematopoietic cells of B, T, and monocyte origin

CD71 (Ber-T9): An activation antigen that defines the transferrin

receptor It is expressed on activated T cells, bone marrow blasts,

normal histiocytes, and intermediate- and higher-grade

lympho-mas, the Reed–Sternberg and Hodgkin cells of Hodgkin lymphoma,

and other nonhematopoietic rapidly growing neoplasms

DR: Expressed normally on B lymphocytes; however,

HLA-DR is negative on quiescent T lymphocytes It is expressed on

acti-vated T lymphocytes

Ki-67 (MIB-1): The Ki-67 antibody detects a nuclear-associated

antigen that is expressed by proliferating, but not resting cells

Ki-67 staining correlates with morphologic grade, whereby a higher

number of staining cells are associated with a poor survival

panels on paraffin-embedded tissue

CD52: clone, YTH34.5 or Campath-1G; concentration, 1:500 Fox P3: clone, 236A/E7; concentration, 1:100 CLA clone, HECA-

452; concentration, 1:25

B cell:

CD20CD79CD21CD23CD10CD5CD43Cyclin D1Bcl-1Bcl-2Bcl-6Oct-2Mum-1CD30

mRNA κ/λ to ascertain light chain restriction

TdTPAX5

Cytotoxic markers:

TIAPerforinGranzyme

Plasma cell markers:

mRNA κ/λ

CD138

Natural killer cell:

CD56CD16

Myeloid:

CD34CD43CD68Leder (Chloroacetate esterase) histochemical stainTdT

CD99CD15

Hodgkin specific:

CD15CD40 clone, 11E9; concentration, 1:10Fascin clone, 55K-2; concentration, 1:500CD30

CD45 RoPAX5

CD30+ lymphoproliferative disease:

CD2CD3CD4CD5CD8CD30TIAgranzymeepithelial membrane antigenanaplastic lymphoma kinaseclusterin

cases The 5′ IRF4 CTD-2308G5 probe is labelled with Cyanine3 (R for red) and the 3′ IRF4 RP11-164H16 probe with SpectrumGreen (G for green) After hybridization of 5′ and 3′ IRF4 probes, the normal diploid

pattern is one of two fusion signals (2F); a chromosomal break point

at the vicinity of IRF4 is associated with 1F-1R-1G pattern (1F-1 split),

defining a translocation in this area of the genome

MYC breakapart probe: The LSI MYC dual color, breakapart

rear-rangement probe is a mixture of two probes that hybridize to

op-posite sides of the region located 3′ of MYC This region is involved

in the vast majority of breakpoints for t(8;22)(q24;q11) and t(2;8)

(p11;q24) Translocation involving the CMYC gene can be expected

to occur in the vast majority (>90%) of Burkitt’s lymphoma and atypical Burkitt’s lymphoma

MYC IgH fusion probe: The LSI IGH/MYC, CEP 8 tricolor,

dual-fusion translocation probe is designed to detect the

juxtaposi-tion of immunoglobulin heavy chain (IGH) locus and MYC gene

region sequences The IGH probe contains sequences homologous

to essentially the entire IGH locus, as well as sequences extending about 300 kb beyond the 3′ end of the IGH locus The large MYC probe extends approximately 400 kb upstream of MYC and about

350 kb 3′ beyond MYC A cell harboring the reciprocal t(8;14)

with the 8q24 breakpoint well within the MYC probe target is pected to produce a pattern of one orange, one green, two orange/green fusions, and two aqua signals Translocation involving the

ex-C-MYC gene can be expected to occur in the vast majority (>90%)

of Burkitt’s lymphoma and atypical Burkitt’s lymphoma

Bcl-2 IgH fusion probe: The LSI IGH/bcl-2 dual-color,

dual-fu-sion translocation probe (Vysis) is designed to detect the

juxtapo-sition of immunoglobulin heavy chain (IGH) locus and bcl gene

sequences It is detected in most lymphomas harboring a t(14;18)

Cyclin D1 IgH fusion probe: The LSI IGH/CCND1 dual-color,

dual-fusion XT translocation probe (Vysis) is designed to detect

the juxtaposition of immunoglobulin heavy chain (IGH) locus and

CCND1 gene sequences It will detect most t(11;14)-bearing cells

and is therefore seen in the majority of mantle cell lymphomas

MALT1 breakapart probe: The LSI MALT1 dual-color, breakapart

rearrangement probe consists of a mixture two FISH DNA probes The first probe, a 460 kb probe labeled in SpectrumOrange™, flanks

the 5′ side of the MALT1 gene The second probe, a 660 kb probe labeled in SpectrumGreen™, flanks the 3′ side of the MALT1 gene

It will detect cells with t(18q21) and/or aneuploidy of chromosome

18 Translocation involving the MALT1 gene can be expected to

occur in approximately 25–50% of extranodal marginal zone phomas, but is quite uncommon in nodal-based marginal zone lymphoma and primary cutaneous marginal zone lymphoma

lym-MALT1 IgH fusion probe: The LSI IGH/lym-MALT1 color,

dual-fusion translocation probe is composed of a mixture of a 1.5 Mb SpectrumGreen™ labeled IGH probe and a 670 kb SpectrumOrange™ labeled MALT1 probe The IGH probe contains sequences homolo-

gous to essentially the entire IGH locus, as well as sequences ing about 300 kb beyond the 3′ end of the IGH locus The LSI MALT1

extend-probe contains sequences that extend from a point telomeric to the

D18S531 locus, through the MALT1 and HAK genes, and end

proxi-mally at a point centromeric to the HAK locus This probe is useful in identifying the IGH/MALT1 t(14;18)(q32;q21) translocation.

API2 MALT1 fusion probe: The LSI API2/MALT1 dual-color,

dual-fusion translocation probe is composed of a mixture of a trumGreen™ labeled IGH probe and a SpectrumOrange™ labeled

Spec-MALT1 probe This probe is useful in identifying the API2/Spec-MALT1

t(11;18)(q21;q21) translocation It will detect cells with a t(11;18)(q21;q21) translocation

Special techniques

reverse transcriptase in situ hybridization assays

Epstein–Barr virus-associated latent small nuclear RNA (EBER): EBER-1

and EBER-2, present in both the productive and various forms of latent

EBV infection We employ EBER rather than LMP-1 since EBER is

present in both the latent and lytic phases of infection while LMP-1 is

typically not present in the lytic stage EBER-1 and EBER-2 are present

in much higher copy numbers than LMP-1, potentially providing us

with higher sensitivity than testing LMP-1 protein

Viral thymidine kinase (vTK assay): EBV thymidine kinase detected

with the probes 5′-GAACCCGCATGCTCTCCTT-3′ and

5′-TCT-GGATGATGCCCAAGACA-3′, respectively, detects lytic infection

HHV8: Detection of HHV8 RNA is accomplished using primers

specific for the T0.7 viral message, which is expressed in latent and

active infection

Fluorescent in-situ hybridization (FISh)

MYC amplification and translocation, and trisomy 8: For MYC

amplifi-cation, a ratio of the total number of MYC signals to the total number

of CEP8 signals, in at least 60 interphase nuclei with nonoverlapping

nuclei in the tumor cells, is determined Cells with no signals or with

signals of only one color are disregarded Tumor cells displaying at least

two centromeric chromosome 8 signals and multiple MYC signals,

with a MYC/CEP8 ratio ≥2, are considered consistent with

amplifi-cation of the MYC gene Overamplifiamplifi-cation of C-MYC is not

associ-ated with any particular hematologic malignancy, but would only be

expected in those with a more aggressive course and would not be a

feature of a benign lymphoid cell population Tumor cells displaying

multiple centromeric chromosome 8 signals and an approximately

equal number of MYC signals with a somewhat random distribution

of both probe signals are considered polysomy 8

Summary of antibodies, clones, and dilutions

Antibody Clone Ig class Dilutions Pretreatment incubation Primary AB Manufacturer

Novacastra CD7 C BC.37 IgG2b 1:80 Citra Plus 30

minutes DakoCytomation, Carpinteria, CA

minutes

Vision Biosystems;

Novacastra

ALK-1 breakapart probe: The LSI ALK (anaplastic lymphoma

kinase) dual color, breakapart rearrangement probe contains two

differently labeled probes on opposite sides of the breakpoint of the

ALK gene This region is involved in the vast majority of breakpoints

for known 2p23 rearrangements that occur in t(2;5) and its variants

The translocation (2;5)(p23;q35) is identified in approximately 50%

of cases of anaplastic large cell lymphoma (noncutaneous) The

ab-sence of the translocation (2;5)(p23;q35) does not exclude the

diag-nosis of anaplastic large cell lymphoma and in primary cutaneous

anaplastic large cell lymphoma it is primarily not seen

Interferon regulatory factor 4-breakapart dual color probes:

Trans-locations involving the multiple myeloma oncogene-1/interferon

regulatory factor-4 (IRF4) locus on 6p25 in primary cutaneous

ana-plastic large cell lymphoma and a subset of lymphomatoid papulosis

The Cutaneous Lymphoid Proliferations: A Comprehensive Textbook of Lymphocytic Infiltrates of the Skin, Second Edition Cynthia M Magro, A Neil Crowson and Martin C Mihm.

© 2016 John Wiley & Sons, Inc Published 2016 by John Wiley & Sons, Inc.

14

Introduction

Mycosis fungoides (MF), the most common type of cutaneous T

cell lymphoma (CTCL), generally is characterized by an indolent

presentation and by a low probability of progression Patients with

limited patches or plaques (Stage IA) have a <10% risk of

develop-ing progressive disease and have median survival similar to that of

age-matched controls without the disease (Kim et al., 1996) In a

subset of patients with Stage IB-IIA, however, CTCL may progress

to more extensive disease and in a small minority of cases it may

present de novo with tumors, erythroderma, and peripheral blood

or visceral involvement (Kim et al., 2003) In these circumstances

CTCL is associated with a significant risk of disease-related

mortal-ity and shorter survival (Lu et al., 2001; Kim et al., 2003; Vonderheid

and Bernengo, 2003; Tancrede-Bohin et al., 2004) Sézary syndrome

(SS), a rare and unique primary leukemic form of CTCL, presents

with erythroderma and peripheral blood lymphocytosis

(Vonder-heid and Bernengo, 2003) Prognosis for these patients is poor, with

5-year survival at best 30% (Tables 2.1 and 2.2)

In the absence of molecular biomarkers, there are currently no

broadly applicable molecular tools for risk stratification in CTCL

Twist 1, a transcription factor that inhibits p53 and

C-MyC-induced apoptosis, correlates with later stages of CTCL and Sézary

syndrome, however it has not been validated as a risk factor of

pro-gression (Goswami et al., 2012) Advanced clinical stage, older age,

elevated lactate dehydrogenase (LDH) levels, and peripheral blood

eosinophilia are all associated with poor prognosis (Tancrede-

Bohin et al., 2004) Histopathologically, the presence of over 25% of

large, atypical, CD30-positive cells, indicates large cell tion, which has a poor prognosis, and is further worsened by older

transforma-age or tumor sttransforma-age disease (Diamandidou et al., 1998) Conversely,

the expression of the chemokine receptors CCR4, CXCR3, and CXCR4 on malignant T cells is generally restricted to earlier stage lesions and loss of these markers with increased levels of CCR7, a lymph node homing receptor, is observed in patients with tumor

stage disease and lymphadenopathy (Lu et al., 2001; Kallinich et al.,

2003) Finally, loss of epidermotropism in advanced forms of MF, such as SS, is a common finding, and skin biopsies in these patients may not show any tumor cells within the epidermis

A number of defects of adaptive and innate immunity can be

ob-served during the clinical progression of CTCL (Kim et al., 2005)

Chronic and excessive production of Th2 cytokines, such as IL-4, IL-5, and IL-10, is believed to be an important mechanism by which malignant T cells circumvent antitumor responses Gradual loss of

Th1 cytokines (IL-12 and IFN-γ), CD8-positive cytotoxic T cells

(CTL), and natural killer (NK) cells are observed in advanced-stage

CTCL (yoo et al., 2001; French et al., 2005) Low absolute numbers

of peripheral blood or skin CD8-positive T cells, measured by flow cytometry or immunohistochemistry, have been reported to be an

accurate predictor of survival (Abeni et al., 2005), and therapy with

retinoids has led to increased CD8-positive T cells in responders

Diagnostic work-up and staging procedures

Staging evaluation of CTCL patients should include a sive physical examination, a complete blood count, a comprehen-sive metabolic panel, and the quantification of circulating malignant

comprehen-T cells by flow cytometry In patients presenting with typical

MF, bone marrow aspirate and biopsy, contrast-enhanced puted tomography (CT) scan, and whole body positron emission

com-the com-therapy of Cutaneous t Cell Lymphoma

Benjamin h Kaffenberger, Mark a Bechtel, and pierluigi porcu

Table 2.1 CTCL therapies

Skin-directed

 Topical corticosteroids

 Topical nitrogen mustards: mechlorethamine and carmustine (BCNU)

 Topical retinoids: bexarotene

 Topical imiquimod

 PUVA (psoralen plus ultraviolet A) phototherapy

 Narrowband UVB phototherapy

 Electron beam radiotherapy

 Denileukin diftitox (not currently available)

 Chemotherapy: Gemcitabine and doxorubicin

 Hematopoietic stem cell transplantation

Table 2.2 Therapy by stage of mycosis fungoides

Stage IA-IB Topical steroids, topical mechlorethamine,

topical BCNU, narrowband UVB, PUVA, total skin electron beam radiotherapy, topical or systemic bexarotene

Stage IIB and above Interferon-α, bexarotene, PUVA, combinations

of topical and systemic treatment, HDACi – romidepsin or vorinostat, brentuximab vedotin, pralatrexate, chemotherapy, alemtuzumab, extracorporeal photophoresis (ECP), hematopoietic stem cell transplantation

tomography (PET) scanning should only be performed if diffuse

lymphadenopathy, unexplained peripheral blood findings, or

clinical signs or symptoms of visceral involvement are present In

patients with non-MF-type CTCL, a comprehensive staging and

diagnostic work-up to verify that the disease is limited to the skin

should always be performed The role of T cell receptor (TCR)

re-arrangement analysis in the routine staging evaluation of CTCL

remains to be established Detection of clonal TCR-β

rearrange-ments in typical skin lesions of patients with suspected CTCL for

diagnosis and discovery of identical clones in the peripheral blood,

lymph nodes, or bone marrow is unequivocal evidence of

extracu-taneous extension However, in the absence of histopathological or

immunophenotypical evidence of disease, the clinical significance,

and therefore the treatment implications, of finding TCR-β

rear-rangements in the bone marrow, lymph nodes, or peripheral blood

of CTCL patients is the subject of continuous debate (Delfau-Larue

et al., 1998; Assaf et al., 2005).

CtCL therapies

The therapeutic choices for the treatment of CTCL depend on the

stage of the disease and the general health and age of the patient

Although the therapies may be effective in controlling the disease,

they have not been shown to prolong life

Because CTCL can involve skin, blood, bone marrow, and

lymph nodes, proper staging is critical in the management of this

disease The treatment of CTCL can be divided into skin-directed

and systemic therapies (see Table 2.1) Therapeutic choices may be

challenging due to limited randomized clinical trials for CTCL

Therapeutic approaches using combination therapies may have

synergistic benefits and may reduce toxicity of the single agents

Although combined modalities may increase disease-free survival,

they do not change overall survival (Duvic et al., 2003) The primary

goals of therapy are to improve the quality of life, induce

disease-free remission, and prolong life Localized skin-directed therapies

can be very successful in managing localized disease Patients with

more widespread disease need total skin-directed therapy or

sys-temic therapy

Skin-directed therapies

Topical corticosteroids

The use of topical corticosteroids is often effective in controlling

early stage mycosis fungoides (see Table 2.2) Limited patch and thin

plaque disease responds most consistently A complete response in

60% of patients was reported in early-stage disease with topical

cor-ticosteroids (Zacheim et al., 1988) Generally, cost-effective topical

steroids include hydrocortisone 2.5% cream/ointment for facial and

intertriginous sites, triamcinolone 0.1% cream/ointment for the

majority of the body, and fluocinonide 0.05% cream/ointment for

recalcitrant areas and small body surface areas The benefit of

utiliz-ing topical steroids over topical immune modulators, retinoids, and

nitrogen mustards lies in their inexpensive nature The side effects

of topical steroids include cutaneous atrophy, telangiectasias, striae,

and, rarely, suppression of the pituitary adrenal axis with systemic

absorption in high concentrations and high body surface area

applications

Topical chemotherapy

Topical nitrogen mustards, such as mechlorethamine and

carmus-tine (BCNU) intercalate between DNA strands and inhibit DNA

replication They have been demonstrated to be effective in the management of early stages of mycosis fungoides

For over 30 years mechlorethamine had to be compounded for use

in the treatment of mycosis fungoides Studies of the compounded forms demonstrated complete remissions in approximately 60–80%

of patients with early patch and plaque-stage disease Most lasting remissions occurred in patients with patch or plaque-stage mycosis fungoides without palpable lymphadenopathy (Vonder-

long-heid et al., 1989).

Compounding requires preparing 10 mg of mechlorethamine in 50–60 mL of water and applying with a brush to the entire skin surface, except eyelids, lips, and rectal and vaginal orifices This is repeated daily for 6–12 months If patients develop a hypersensi-tivity reaction manifested as cutaneous erythema and pruritus, the treatment can be briefly interrupted After the hypersensitivity re-action subsides, a more dilute solution (10 mg in 500 mL of wa-ter) can be initiated as tolerated The concentration can be slowly increased over time After a complete remission is achieved, the treatments can be gradually tapered, but no tapering schedule has demonstrated superior clinical efficacy Mechlorethamine can also

be applied in a mineral oil (Aquaphor®) base and may be less tating However, as the gel form is now FDA approved, compound pharmacists will no longer be able to dispense the old form without

irri-a good reirri-ason (e.g the pirri-atient found the gel birri-ase to be too drying

or irritating)

Topical carmustine (BCNU) has proved effective in early stage mycosis fungoides It can be applied in a stock solution of BCNU in alcohol or prepared in an ointment base with white petrolatum A complete response was documented in 86% of patients with Stage

IA and 47% with Stage IB disease The median time for a complete response was 11.5 weeks Approximately 18% of patients were re-

lapse-free at five years (Zackheim et al., 1990) The cutaneous side

effects of topical carmustine include skin tenderness, erythema, and hyperpigmentation Many patients develop increased telangiecta-sias and thus this should be avoided on the face Allergic contact dermatitis and primary contact irritation develop in a similar way

to topical mechlorethamine Myelosuppression can develop with topical use and should be carefully monitored with complete blood counts

Mechlorethamine is now FDA approved for MF in a 0.02% gel, preventing the need to compound these agents This was after a large multicenter trial showed noninferiority to the compounded

ointment (Lessin et al., 2013) However, this study did demonstrate

the gel form possessed a more rapid onset of action Unfortunately about 20% of patients in both arms withdrew due to skin irrita-tion Mechlorethamine can also be used as adjunctive therapy with other modalities, but should be avoided when using ultraviolet light therapy due to an increased risk of skin cancer

Topical retinoids

Bexarotene gel, a synthetic retinoid X agonist, has been approved for the treatment of mycosis fungoides The retinoid receptors (RAR and RXR) are members of a family of transcription factors belonging to the nuclear hormone receptor family The nuclear hormone receptor family also includes thyroxine receptor, vitamin

D receptor, and peroxisome proliferator-activated receptor arotene induces an RAR–RXR heterodimer complex that activates gene promoter regions encoding transcription factors, structural proteins, and cell receptors This results in transcriptional modu-lation of cell function and differentiation, growth inhibition, and apoptosis

Bex-The safety and efficacy of topical bexarotene gel was examined

in an open-label Phase III study of 50 patients with refractory or

persistent early-stage CTCL A partial response was noted in 54%

of patients and a complete response in 10% (Heald et al., 2003)

In a Phase I/II study of bexarotene gel, 61 patients with early stage

CTCL were treated Partial responses were noted in 42% of patients

and complete responses in 21% (Brenenman et al., 2002).

Bexarotene gel is initiated at a dose of 1% topical gel every other

day and gradually increased in frequency to four times a day at 1–2

week intervals The drug can cause a retinoid erythema and irritant

dermatitis It may take several weeks to note a clinical response

The immunologic effects of bexarotene are related to a decreased

expression of TH2 cytokines and decreased monocyte macrophage

inflammatory mediators There is a decrease in abnormal

keratino-cyte proliferation and induction of apoptosis of abnormal T cells

Topical imiquimod

Imiquimod 5% cream has been demonstrated to be helpful in limited

patch and plaque-stage mycosis fungoides (Suchin et al., 2002; Deeths

et al., 2005) Imiquimod is a topical immunomodulator via toll-like

re-ceptor 7 activation, which induces localized interferon production In

one study, three of six patients with Stage IA to IIB mycosis fungoides

were reported to have histologic clearing and significant clinical

im-provement with imiquimod 5% cream applied three times per week

for 12 weeks Patients responded best to a concomitant therapy with

PUVA, systemic retinoids, or systemic interferon (Deeths et al., 2005).

Phototherapy

Psoralen plus UVA exposure (PUVA) has become a standard

ther-apy for patch and plaque-stage CTCL (Herrmann et al., 1995) A

long-term study of CTCL and PUVA at Northwestern showed 66

of 104 patients with clinical Stages IA to IIA achieving complete

remission, which in many cases was long term Disease-free

sur-vival rates at 5 years and 10 years for Stage IA disease were 56% and

30%, respectively For Stage IB/IIA, the 5 year disease-free survival

was 74% and 10 year disease-free survival was 50% However, the

15-year survival rate for Stage IA was 82% and for Stage IB/IIA was

58% (Querfeld et al., 2005) Once the patient achieves a maximal

response to treatment, maintenance therapy is usually required to

prevent relapse Persistent or recurrent lesions particularly favor

UV-shielded sites PUVA therapy can be combined with oral

bex-arotene, interferon-α, and radiotherapy.

The side effects of PUVA include erythema, burn injury, increased

frequency of melanoma and nonmelanoma skin cancers, and

cata-racts Protection of the eyes with ultraviolet light-absorbing goggles

is necessary for 24 hours following ingestion of oral psoralens

Narrowband UVB is an effective treatment for patch and thin

plaque mycosis fungoides It is less effective than PUVA for thick

plaque disease In one study, patients with IA and IB mycosis

fun-goides achieved complete remission in 81% of cases (Dierderen

et al., 2003).

Narrowband UVB does not require photoprotection of eyes,

un-like PUVA, and may have a lower risk of secondary skin cancers It

may be less effective than PUVA for thicker plaques Narrowband

UVB also requires multiple treatments per week initially, which

make compliance an important issue

Radiation therapy

Total skin electron beam (TSEB) therapy is often very effective

for patients with early stages of mycosis fungoides An energy of

4–6 MeV is delivered to the skin with penetration limited to the upper dermis The limited penetration of electrons spares deeper parenchymal tissues, bone marrow, and the gastrointestinal tract Approximately 3600 cGy are delivered in fractionated doses over 8–12 weeks Separate exposures may be necessary for difficult to reach areas, including the palms, soles, axillae, perineum, and scalp Complete remissions were reported in 98% of patients with Stage T1 disease, 71% with Stage T2 disease, and 36% with Stage T3 dis-ease Approximately 50% of patients with Stage T1 and 20% of with

Stage T2 were disease-free at 10 years (Hoppe et al., 1979) Relapse

is highest in patients with tumors, lymphadenopathy, and visceral involvement With the highly fractionated approach, patients can receive a second course of electron beam therapy to reinduce re-mission However, unlike phototherapy, it cannot be used continu-ously and relapses will occur Hence, this method is often used for clearance before attempting a hematopoietic stem cell transplant Otherwise, after stopping the TSEB, other skin-directed therapies should be added for maintenance

The toxic side effects of TSEB therapy may be acute or chronic Acute side effects include erythema, edema, bullae, and desqua-mation Adnexal structures that may be affected include the hair follicles and sweat glands Patients consequently may experience hypohidrosis, alopecia, and shedding of nails Chronic side effects include hyperpigmentation, telangiectasias, cutaneous atrophy, xerosis, and anhidrosis As the total dose of radiation increases, so does the risk for squamous cell carcinoma

Cutaneous lymphomas are often very radiosensitive and local radiotherapy can be utilized for patients with individual tumors or thick plaques It may be used in combination with other modalities, such as PUVA Approximately 800–3000 cGy can be delivered in fractionated doses

Photodynamic therapy

Photodynamic therapy (PDT) has been reported to be beneficial in the treatment of mycosis fungoides PDT involves photosensitiza-tion with 5-aminolevulinic acid and irradiation with a noncoherent light source Activation of the photosensitizer leads to the formation

of oxygen radicals, which are highly reactive and cytotoxic PDT has the potential to inhibit the proliferation of malignant T lymphocytes (Ammann and Hunziker, 1995) This may be a useful treatment mo-dality in patients who have reached a partial remission with more conventional therapies The therapy can be directed to therapy- resistant lesions, but is not an option for large surface areas

Goals of therapy in advanced-stage CtCL

It is now established that by the time CTCL has advanced to mor lesions, the malignant T cells have acquired multiple molecu-lar abnormalities in the pathways that regulate normal cell growth and survival These defects are responsible for the high resistance

tu-to drug-induced apoptu-tosis displayed by malignant T cells and the brief and partial responses typically seen with conventional therapy

in CTCL Intensive cytotoxic chemotherapy, as used in other forms

of lymphoma and leukemia, may actually shorten survival in some patients by virtue of its myelosuppressive effect and its depression

of T-cell-mediated immunity and provides few durable responses Thus, the focus of systemic therapy in CTCL has gradually evolved from conventional cytotoxic chemotherapy to agents that induce T cell apoptosis by inhibiting survival pathways or targeting surface markers, and enhance the patient’s ability to mount an immune re-sponse, while avoiding neutropenia

extracorporeal photopheresis (eCp)

ECP is a form of systemic therapy, originally developed by Edelson

et al (1987), that is FDA approved for the treatment of SS In ECP,

patients ingest oral 8-methoxypsoralen (8-MOP); then peripheral

blood lymphocytes are collected by leukopheresis, exposed ex vivo

to ultraviolet A (UVA) light, and reinfused back into the patient

ECP is initially performed on two consecutive days once a month

until maximal clearing has occurred This is followed by an

addi-tional 6 months of monthly therapy and then gradually tapered to

2 month intervals and eventually discontinued In the initial cohort

reported by Edelson et al (1987), the response rate in refractory

CTCL (most of whom had SS) was 73% and the median survival

was 60 months The side effects of ECP consist mostly of nausea

and vomiting associated with oral 8-MOP Newer formulations use

liquid 8-MOP that is administered directly into the photopheresis

machine, so patients do not have to ingest the psoralen The

pro-posed mechanism of action of ECP is twofold: (1) UVA irradiation

directly kills malignant T cells sensitized by 8-MOP, and (2) the

reinfused leukophoresis product stimulates a selective immune

re-sponse against the malignant cells Further studies have

retrospec-tively compared the outcomes of patients treated with or without

ECP, generally finding similar survivals Overall, ECP appears to

be effective only in a subset of patients with advanced-stage CTCL,

specifically SS with near normal CD8+ T cell counts, and a

rela-tively short duration of advanced disease However, these patients

are also more likely to respond to other immune-modifying

thera-pies and the value of ECP vis-à-vis other treatment modalities has

not been prospectively tested in clinical trials (Richardson et al.,

2003) Finally, due to the highly encouraging responses seen with

alemtuzumab in SS and due to the fact that ECP requires special

equipment, a significant time commitment, and lack of ubiquity at

medical centers, it is conceivable that monoclonal antibodies will

become more frequently used for this indication than ECP

Interferons

The most active single agent in the treatment of CTCL is IFN-α

Initial studies in heavily pretreated patients with CTCL, with doses

ranging from 36 million international units (MIU) per day to 50

MIU three times a week, showed objective response rates of 45–

65%, with complete response rates of 10–30% and response

dura-tion of 4.5–5.5 months (Olsen and Bunn, 1995) In patients with all

stages of CTCL who had not received prior systemic therapy, the

overall response rate was as high as 80% Enthusiasm for the high

response rate with IFN-α has been tempered by its significant

toxic-ity profile Adverse effects to IFN-α include malaise, fever, chills,

myalgias, depression, anorexia, and weight loss Although none of

these events are dose-limiting in isolation, their cumulative burden

is commonly severe and this fact often leads to substantial dose

modification or cessation of therapy Frequent laboratory

abnor-malities observed during therapy with IFN-α include dose-related

and reversible leukopenia and thrombocytopenia, abnormal liver

function tests, and, occasionally, hypothyroidism There is clearly

a dose–response effect with IFN-α in CTCL The current treatment

approach with IFN-α is to start at 3 MIU per day and increase as

tolerated, usually to a maximum of approximately 15 MIU per day

Lower doses of IFN-α have been successfully combined with other

treatment modalities with excellent results At Northwestern

Uni-versity, a study combining the use of IFN-α (3–12 MIU three times

a week) with PUVA in patients with all stages of MF/SS

demon-strated an overall response rate of 88%, with a complete response rate of 62% and median response duration of 28 months, establish-ing this combination as one of the most active therapies in CTCL

(Kuzel et al., 1995).

In addition to IFN-α, there is evidence that systemic or topical recombinant IFN-γ may have significant potential for the treatment

of CTCL, and that it may be better tolerated than IFN-α (Kaplan

et  al., 1990; Dummer et al., 2004) In addition to enhancing the

CTL function, IFN-γ suppresses excess Th2 cytokine production,

enhances CD40 expression, and primes abnormal dendritic cells for IL-12 production, particularly in response to CD40 ligation

retinoids

Retinoids are vitamin A derivatives and have been shown to have antiproliferative and differentiating effects in many hematologi-cal malignancies, including CTCL (Zhang and Duvic, 2003) Re-sponse rates ranging from 44% to 68% have been reported with

isotretinoin (13-cis-retinoic acid) in patients with Stage I–II disease (Fitzpatrick and Mellette 1986; Kessler et al., 1987) Due to its tera-

togenic properties with regard to teen pregnancies, this medication

is highly regulated, which limits its use, including the requirement for monthly physician evaluations and updates of the iPledge reg-istry The most common toxicities include drying of the skin and mucous membranes, conjunctivitis, fatigue, arthralgias, mental status changes, headaches, and elevated triglyceride levels More recently, bexarotene, a retinoid X receptor (RXR) selective agonist, was shown to have substantial clinical efficacy in both topical and oral formulations in patients with all stages of CTCL and was ap-

proved in 1999 by the FDA for this indication (Duvic et al., 2001)

In the pivotal trial, 94 patients with CTCL received oral tene at doses ranging from 300 to 650 mg/m2/day At a dose of 300 mg/m2/day, the response rate observed was 45–55%, depending on stage, with a median time to response of 180 days and a median duration of response of 10 months There was a nonstatistically significant trend toward a dose–response relationship, with greater complete response rate (13%), shorter time to response (59 days), and longer duration of response (13 months) at doses higher than

bexaro-300 mg/m2 However, dose-limiting toxicity (hypertriglyceridemia) prevented continuation of therapy at higher doses (650 mg/m2/day)

of bexarotene and the FDA-approved dosage is 300 mg/m2/day verse events included hypertriglyceridemia, hypercholesterolemia, central hypothyroidism, and leukopenia The activity of bexarotene

Ad-in the non-MF/SS-type of CTCL has not been Ad-investigated Ad-in clAd-ini-cal trials but responses in CD30-positive primary cutaneous ALCL and panniculitis-like T cell lymphoma have been reported.Bexarotene exerts a number of biological effects that may sensi-tize malignant T cells to other therapies At clinically relevant con-centrations, bexarotene upregulates both the p55 and p75 subunits

clini-of the interleukin-2 (IL-2) receptor in vitro, thus enhancing the

sus-ceptibility of T cell leukemia cells to denileukin diftitox (see munotoxins”) by five- to tenfold To determine whether this effect

“Im-could be reproduced in vivo, Foss et al (2005) completed a small

Phase I study of bexarotene (75–300 mg/day) and denileukin tox (18 μg/kg per day × 3 days every 21 days) in 14 patients with relapsed or refractory CTCL Overall response was 67%, with four complete responses Modulation of IL-2R expression on CTCL cells was observed at or above a bexarotene dose of 150 mg/day Leuko-penia and lymphopenia were observed in a small number of pa-tients, without clinical evidence of opportunistic infections Thus,

difti-the combination of denileukin diftitox and bexarotene was well

tol-erated and even low doses (150 mg/day) of bexarotene were capable

of upregulating CD25 expression (Foss et al., 2005).

Acitretin is the other systemic retinoid that is still available in

the United States It is not RXR specific nor FDA approved for this

indication, unlike bexarotene, but it has shown an overall response

of nearly 60% in small patient numbers (Cheeley et al., 2013) and

may be an option if a patient cannot tolerate the side effects of

bexarotene

Immunotoxins

Denileukin diftitox (DAB389IL-2, Ontak, Ligand Pharmaceuticals),

a genetically engineered fusion protein combining the

enzymati-cally active domain of diphtheria toxin and the full-length sequence

of interleukin-2 (IL-2), efficiently targets cells expressing the

high-affinity IL-2 receptor subunits that are highly expressed on the

ma-lignant T cells in the majority of cases of CTCL After binding to the

IL-2 receptor, denileukin diftitox undergoes endocytosis followed

by release of the diphtheria toxin, which results in arrest of protein

synthesis and, ultimately, apoptosis of T cells The initial Phase I

study with denileukin diftitox in 35 patients consisted of five daily

infusions every 3 weeks for up to six cycles at escalating doses,

yield-ing an overall response rate of 37% with 14% complete responses

Adverse effects of DAB389IL-2 included fever, chills, hypotension,

nausea, vomiting, and elevated transaminases In the following

piv-otal Phase III trial in heavily pretreated patients, two different doses

of denileukin diftitox (9 μg/kg and 18 μg/kg) were studied (Olsen

et al., 2001) The overall response rate was 29.5%,with 10% complete

responses and no difference between the two dose levels A mild,

self-limited form of vascular leak syndrome (VLS) is frequently

observed after the first cycle of denileukin diftitox, with lower

ex-tremity edema, hypoalbuminemia, and elevated serum creatinine

The frequency and intensity of VLS can be significantly reduced by

premedication with dexamethasone (4–8 mg orally) without

com-promising the efficacy of therapy Currently, denileukin difititox is

approved for use only in CD25-positive CTCL, as defined by

posi-tive staining by immunohistochemistry (IHC) on frozen or paraffin

embedded tissue However, the levels of CD25 expression sufficient

to confer sensitivity to denileukin diftitox are likely to be much

be-low the IHC threshold of detection, as suggested by the numerous

responses reported in CD25-negative CTCL in early clinical trials

The major mechanism of action of denileukin diftitox in CTCL

is believed to be direct killing of malignant T cells However, part

of its activity may also be immunologically mediated through the

elimination of CD4/CD25-positive regulatory T cells (Tregs), a

nor-mal subset of T cells with constitutive suppressive activity (Dannull

et al., 2005) Thus, denileukin diftitox, by depleting Treg cells, may

conceivably enhance normal residual antitumor responses in CTCL

patients Unfortunately, at the current time, this therapy is no longer

available in the United States

Monoclonal antibodies

A number of humanized monoclonal antibodies (mAbs) against

surface antigens expressed by normal and malignant T cells are

available for clinical use Alemtuzumab (Campath-1H) is a

hu-manized IgG1 mAb directed against CD52, a glycosylated peptide

antigen expressed on most malignant B and T cells Alemtuzumab

was developed primarily for the treatment of patients with B cell

chronic lymphocytic leukemia (B-CLL); however, it also showed

strong activity in T cell leukemias and lymphomas Lundin et al

(2003) prospectively studied alemtuzumab in 22 patients with vanced CTCL The overall response rate was 55%, with 32% com-plete responses, and median duration of response approaching

ad-12 months Impressively, Sézary cells were cleared from the blood in 86% of the patients Cytomegalovirus (CMV) reactivation occurred

in 18% of patients, but without clinical respiratory or nal syndromes Three patients had more significant infectious com-plications, such as generalized herpes simplex, aspergillosis, and mycobacterial pneumonia Larger studies have also corroborated

gastrointesti-an impressive benefit, particularly in patients with SS (de Masson

et al., 2014) Thus, alemtuzumab shows promising activity,

partic-ularly in patients with erythroderma and SS, but its use requires aggressive prophylactic therapy and surveillance for opportunistic infections It appears that alemtuzumab may become one of the drugs of choice for SS patients and further studies are in progress

In addition to alemtuzumab, a number of additional mAbs geting T cell markers such as CD2 and CD4 have been recently studied Preliminary results from a Phase I clinical trials of sipli-zumab (MEDI-507), a humanized anti-CD2 mAb, in patients with various types of T cell lymphoma have been presented Typical in-fusional adverse reactions occurred with the first dose and asymp-tomatic CMV reactivation was seen Treatment resulted in partial remission or stabilization of disease in several patients However, cases of EBV-induced lymphoproliferative disorders, some of them fatal, were observed and further development of the drug in CTCL was stopped

tar-Another mAb in early clinical development is zanolimumab

(Hu-MaxCD4) (Hagberg et al., 2005) Phase II data with this antibody in

47 CTCL patients relapsing after bexarotene showed response rates ranging from 22% to 75%, including some complete responses, de-pending on the dose (280, 560, or 980 mg) and the stage of disease

(Kim et al., 2007) More than 80% of the responses were reached

within 8 weeks and median response duration was more than 10 months Nine patients with SS were treated on this study at all dose levels, four of which obtained minor responses and one a partial response The observed responses in these patients were generally short-lived and depletion of CD4 T cells was limited, suggesting that this antibody may not be as effective as alemtuzumab in SS Although promising, the maker discontinued the Phase III trial due

to slow patient recruitment and limited market potential and it has not been further pursued

The mAb that has the greatest potential for reaching the CTCL market in the US at this time is mogamulizumab, a humanized glyco-engineered (defucosylated) antibody targeting the CC chem-okine receptor 4 (CCR4) The defucosylated Fc region enhances antibody-dependent cellular cytotoxicity (ADCC) CCR4 is mainly expressed on normal Tregs and type 2 helper T cells (TH2) CCR4

is highly expressed on adult T-cell leukemia/lymphoma (ATLL) cells and mogamulizumab is currently approved in Japan for the treatment of ATLL In addition, mogamulizumab depletes CCR4+

Tregs, potentially evoking antitumor immune responses (Ni et al.,

2015) Clinical trials with mogamulizumab have demonstrated ical efficacy and tolerability for the treatment of relapsed/refractory T-cell lymphomas, including CTCL A Phase I/II multicenter, dose-escalation study of mogamulizumab was conducted in relapsed

clin-patients with PTCL and CTCL (Duvic et al., 2010) Forty heavily

pretreated patients received at least four doses of KW-0761 at 0.1 mg/kg (n = 3), 0.3 mg/kg (n = 3) and 1 mg/kg (n = 34); 38 patients (23 with MF; 15 with SS) were evaluable for efficacy The objective

response rate (ORR) was 39%, with two patients achieving CR (5%),

and 13 achieving partial response (PR; 34%) With SS patients, 12 of

15 (80%) had a response in the blood, including seven (47%) CRs

SD was seen in 19 patients (50%), whilst PD was observed in four

patients (11%) As in the first study, the maximum tolerated dose

was not reached A Phase III randomized clinical trial comparing

mogamulizumab with the histone deacetylase inhibitor vorinostat

in patients with relapsed/refractory CTCL is currently recruiting

(ClinicalTrials.gov Identifier: NCT01728805)

histone deacetylase inhibitors (hDaCi)

The accessibility of nuclear chromatin to transcription factors and

RNA polymerase is regulated in part by the balance of

acetyla-tion of nucleosomal histones, which is the result of the

oppos-ing activities of histone deacetylases (HDACs) and histone acetyl

transferases (HATs) Deacetylation-induced silencing of tumor

suppressor genes, via HDAC, has been observed in a variety of

human cancers Restoration of gene expression through

inhibi-tion of histone deacetylainhibi-tion is a novel form of therapy that

se-lectively induces growth arrest, differentiation, and apoptosis in

malignant cells Several families of HDACi have been

character-ized Compounds such as suberoylanilide hydroxamic acid and

depsipeptide, now vorinostat and romidepsin, respectively, have

entered the clinical arena and shown promise in the treatment of

T cell lymphoma (Piekarz et al., 2004) In a Phase II study, 27

pa-tients with advanced CTCL were treated with vorinostat given as

a 4-hour infusion on days 1, 8, and 15 of a 28-day cycle (Piekarz et

al., 2005) Three patients with Sézary syndrome achieved a

com-plete response and five other patients had partial responses for

an overall response rate of 30% The median duration of response

is 18 months (range 6–48) A larger study of heavily pretreated

patients in advanced stages showed overall response rates of 34%

and 38% in a subgroup analysis of only advanced disease (Piekarz

et al., 2009, Whittaker et al., 2010) Overall, romidepsin was well

tolerated, with dose-related, but modest nausea, fatigue,

neutrope-nia, and thrombocytopenia However, a number of severe cardiac

adverse events have occurred in clinical trials with this agent and

monitoring during infusions is necessary Vorinostat is another

potent HDACi, which is prescribed orally at 400 mg daily and is

FDA approved for advanced primary CTCL Of 37 patients, 10

treated with various oral dosing regimens of vorinostat achieved

a partial response, including a decrease of lymphadenopathy

(Du-vic et al., 2005) In addition, nearly half of patients had relief from

CTCL-associated pruritus (Duvic et al., 2007) Unfortunately, it

had to be discontinued in several patients because of fatigue, rash,

anemia, neuropathy, thrombocytopenia, and weight loss The

follow-up Phase IIb study of heavily pretreated, advanced cases

of CTCL demonstrated an overall response of 29.7%, with high

frequencies of the same adverse events, but fortunately low-grade

(Olsen et al., 2007) Overall, HDACi appear to be very active in

CTCL and are effective second-line agents

antibody drug conjugates (aDC)

Brentuximab vedotin is an antibody drug cytotoxic conjugate

made up of an antibody to CD30 Fab′ domains conjugated with

monomethyl auristatin, an antimitotic agent receptor It is dosed

at 1.8 mg/kg every 21 days and is FDA approved in the treatment

of refractory Hodgkin lymphoma and systemic anaplastic large

cell lymphoma As far as CTCLis concerned, efficacy has been ported, including complete response, in patients with primary cu-

re-taneous anaplastic large cell lymphoma (Kaffenberger et al., 2013)

As CD30 positivity is a marker of transformed mycosis fungoides, results have also been seen in these cases, including four patients with Stage IV disease, one patient with complete resolution, and

two patients with a partial response (Mehra et al., 2015), which

allowed stem cell transplantation in one patient Interestingly, other report details a complete response and partial response in two separate patients with transformed disease, despite variable CD30-positive expression indicating testing for CD30 expres-sion may be insensitive and that there may be an effect in non-

an-transformed MF (Saintes et al., 2015) A Phase II study is ongoing

to determine the efficacy in cases of cutaneous lymphomas ropathy is an important adverse event, but there are also a growing number of reports of progressive multifocal leukencephalopathy

Neu-(Carson et al., 2014).

Studies are ongoing for the use of brentuximab vedotin in neous T-cell lymphomas; the initial reports appear very promising and although not FDA approved, the agent may be available for use

cuta-in refractory and compassionate cases

Cytotoxic chemotherapy

Systemic cytotoxic chemotherapy has generally been used for liation of CTCL patients with relapsed or refractory disease or for patients with advanced disease at presentation Methotrexate, glucocorticoids, cyclophosphamide, cisplatin, etoposide, bleomy-cin, doxorubicin, vincristine, and vinblastine have all been used as single agents or in combination, with complete response rates up

pal-to 30%, but very short remission durations and significant pal-ity, mostly due to myelosuppression (Rosen and Foss, 1995) Pred-nisone alone induces partial remissions in approximately 50% of patients and pulse dexamethasone is effective in obtaining sympto-matic relief in patients with severe SS However, flares are the rule once the dose is tapered and chronic steroid use is associated with significant morbidity

toxic-The purine analogs, such as 2′-deoxycoformycin (pentostatin),

2-chlorodeoxyadenosine (2-CDA), and fludarabine have profound effects on normal and malignant T cells Response rates in CTCL have been higher with 2-deoxycoformycin (54–100%) than with the latter two compounds, but no prospective comparative study has

been performed (Grever et al., 1983; Cummings et al., 1991; Kuzel

et al., 1996; Quaglino et al., 2004) Major toxicities of these drugs

are neurotoxicity and prolonged immunosuppression, leading to opportunistic infections

Trimetrexate (TMTX) is a lipophilic antifolate that enters cells by

passive diffusion and achieves intracellular concentrations 10–100 times greater than those of MTX, thus bypassing possible mecha-nisms of resistance In addition, TMTX is not polyglutamated and

is not retained in cells for prolonged periods, possibly accounting for its relatively low hepatotoxicity Based on the known clinical activity of MTX in CTCL, a Phase II study of TMTX (200 mg/m2

IV every 14 days up to 12 doses) in patients with relapsed CTCL was recently completed, showing a 45% response rate in heavily

pretreated patients and tolerable toxicity (Sarris et al., 2002)

Al-though this drug is probably significantly more active, the venous route of administration makes it unlikely to replace MTX Pralatrexate, another antifolate is now FDA approved for the treat-ment of peripheral T cell lymphoma It is an infusion given weekly, typically best-tolerated for MF patients at 15 mg/m2 for 3–4 weeks

intra-per cycle (Horwitz et al., 2012) In the PROPEL study, the

treat-ment was found to be effective for patients with transformed MF

with an objective response seen in 25% of patients by central review

and 58% according to the study investigator (Foss et al., 2012) It

has also been studied for relapsed and refractory CTCL and SS,

showing a 45% response rate in nontransformed cases (Horwitz

et al., 2012).

Several small studies have shown that gemcitabine

(2′,2′-difluoro-deoxycytidine, Gemzar), a pyrimidine analogue with

structural similarities to cytarabine, has significant activity in

various types of heavily pretreated T cell lymphoma, including

CTCL Marchi et al (2005) reported the results of a prospective

study of gemcitabine in 32 patients with systemically untreated

CTCL Gemcitabine was given on days 1, 8, and 15 of a 28-day

schedule at a dose of 1200 mg/m2 intravenously over 30 minutes

for a total of six cycles The overall response rate was 75% with

22% complete responses and median duration of response of 10

months

Overall, although these new agents appear to be promising, the

role of cytotoxic chemotherapy in the management of advanced

CTCL should be limited to patients who have failed other agents

with a more favorable immunomodulatory effect (ECP, HDACi,

retinoids, interferons)

Investigational therapies

CTCL has been and remains a paradigm for the development of

new agents in T cell leukemia and lymphoma Among the

numer-ous therapies under study in early clinical trials, those that appear

to be most promising at the moment are: (1) monoclonal

antibod-ies (mAbs) against T cell surface markers, (2) immunomodulatory

agents, such as toll-like receptor (TLR) agonists and cytokines, and

(3) allogeneic hematopoietic stem cell transplantation

tLr agonists and cytokines

Toll-like receptors (TLRs) are transmembrane receptors expressed

on innate immune cells, such as monocytes, macrophages, and

dendritic cells that recognize conserved pathogen-associated

mo-lecular patterns, such as lipopolysaccharide (LPS) and CpG DNA

(McInturff et al., 2005) TLR activation triggers the expression of

immune response and cytokine genes, which are instrumental in

launching innate immune responses and influencing adaptive

im-munity TLRs have been identified as potential therapeutic targets

in infectious and inflammatory diseases and CpG

oligodeoxynu-cleotides (ODNs) have been studied as immune stimulatory agents

by virtue of TLR-9 activation of dendritic cells (Wysocka et al.,

2004) In vitro, CpG-ODNs activate dendritic cells, enhancing

IFN production and expression of costimulatory molecules,

ulti-mately augmenting T cell responses In vivo, CpG-ODNs lead to

strong antitumor responses in animal models Kim et al (2004)

completed a Phase I trial of subcutaneous CpG-ODNs (CPG-7909)

in refractory CTCL, demonstrating significant therapeutic efficacy,

including complete clinical responses Imiquimod, a potent TLR-7

agonist, which is FDA approved for the treatment of basal cell

carcinoma, actinic keratoses, and condyloma, also has substantial

activity as a topical agent in CTCL (Suchin et al., 2002; Dummer

et al., 2003) In addition to inducing IFN-α and TNF-α through

activation of TLR-7 on antigen-presenting cells (Hurwitz et al.,

2003), imiquimod can also directly induce apoptosis in a variety

of tumor cells (Schon and Schon, 2004) Newer members of this family have the capacity to activate TLR-8 in addition to TLR-7, resulting in broader dendritic cell activation and release of a more extensive array of cytokines Several clinical trials exploring the use of TLR agonists in CTCL, either alone or as part of a multimo-dality approach, are in progress

Interleukin-12 (IL-12) is a cytokine that enhances CTL and NK

cell function and induces Th1 cytokines (IFN-γ) Subcutaneous

ad-ministration of recombinant human IL-12 to a total of 32 patients with CTCL resulted in a response rate of approximately 50% (Rook

et al., 1999) Since malignant T cells often lack the IL-12β2 receptor,

the hypothesis is that the clinical responses were not due to a direct effect on malignant cells, but to the stimulation of residual normal CD8+ T cells Indeed, serial skin biopsies during treatment with IL-

12 revealed dense infiltrates of CD8-positive T cells that correlated with the time of response Although IL-12 appears to have signifi-cant potential as an immunomodulatory therapy in CTCL, at the present time it is not available from either industry or the NIH for further clinical studies

Unfortunately, although multiple therapies have been studied into early and late stage trials, the limited market has influenced many makers to discontinue expensive efficacy and safety studies

sib-visceral lesions (Molina et al., 2005) This effect is likely due to a

graft versus T cell lymphoma effect mediated by the host’s

reconsti-tuted immune system (Burt et al., 2000) However, only a minority

of CTCL patients is eligible for allogeneic HSCT or has an matched donor Furthermore, toxicity remains a major problem, with estimated treatment-related mortalities approaching 10–25% for allo-HSCT from sibling donors and 30–50% from unrelated donors Investigations of allo-HSCT following nonmyeloablative conditioning in T cell lymphoma and studies to identify, activate, and expand autologous effector T cells with antitumor activity offer the promise of extending the benefit of the graft versus T cell lymphoma effect to a larger population A recent retrospec-tive study has shown complete responses in 4 of 9 patients after allogeneic HSCT, with a follow-up of up to 36 months and recur-

HLA-rences in only 2 of 9 patients (Moritz et al., 2014) Polansky et al

(2015) have shown that in a large cohort of patients with Sézary syndrome, most patients that obtain a complete response, (13 of 18), did so with a HSCT However, a Cochrane review with the goal

of determining the optimal time to perform the HSCT was unable

to identify any studies that compared HSCT to conventional

thera-pies (Schlaak et al., 2013).

A greater understanding of the molecular biology and biology of CTCL has led to the identification of more relevant cel-lular targets of therapy and to the development of new drugs able to affect those targets Future challenges include the development of well-designed clinical trials to elucidate the optimal combination and timing of these therapies

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© 2016 John Wiley & Sons, Inc Published 2016 by John Wiley & Sons, Inc.

23

Molecular analysis in Cutaneous Lymphoid proliferation

Shabnam Momtahen, Cynthia Magro, and Carl Morrison

Introduction

The diagnosis of B and T cell lymphoproliferative disorders of the

skin is supported by the finding of clonally rearranged

immu-noglobulin (Ig) and T cell receptor (TCR) genes, whereas reactive

conditions are generally associated with a polyclonal status

Clon-ality analysis may therefore help to confirm the diagnosis in cases

where the clinical and/or pathological presentations are challenging

or inconclusive However, the identification of clonality is not the

sine que non of malignancy and its absence is not absolute proof of

benignancy Clonality results must be carefully incorporated with

the entire picture from both a clinical and a pathological perspective

Immunoglobulin gene rearrangement analysis can be performed

by either Southern blot analysis or polymerase chain reaction

(PCR)‐based techniques; however, the former is not readily

ap-plicable to the vast majority of skin biopsies Although Southern

blot used to be the gold standard for T cell clonality detection, it

has been gradually replaced by the PCR techniques, which have

superiority over the Southern blot technique due to enhanced

sen-sitivity, being less expensive, laborious, and time‐consuming

Fur-thermore, less DNA is needed to perform the test Not surprisingly,

older studies showed that clonality detected by the Southern blot

technique is associated with worse prognosis, likely reflective of the

relative tumor burden (Guitart et al., 2003) In contrast, using

cer-tain PCR‐based methodologies, an overall sensitivity between 70%

and 90% is achieved

An effective PCR‐based laboratory approach to both

immu-noglobulin heavy chain (IgH) and TCR gene rearrangements needs

to utilize a set of multiplex master mixes, all of which are run on

a thermocycler program targeting a different conserved region or

sequence for both loci In addition, there needs to be a sensitive and

reproducible method of product analysis

In 2003, the European BIOMED‐2 collaborative study group

from seven European countries developed and standardized PCR

protocols and PCR primer sets for detection of IgH and TCR gene

rearrangements, as well as guidelines for interpretation of the

re-sults This group tested a large number of different primers for both

IgH and TCR and also examined the different primer combinations

(van Dongen et al., 2003; von Krieken et al., 2003).

There was an effort in this study to keep the amplicon products

under 350 base pairs in size, making the design applicable to both

frozen and formalin‐fixed paraffin‐embedded (FFPE) tissues As a

result of this study and additional studies there was an increase in

the detection rate of clonal rearrangements (Sandberg et al., 2003;

van Dongen et al., 2003; Droese et al., 2004; Matthews et al., 2004; Hodges et al., 2005; Lassmann et al., 2005; McClure et al., 2005).

The basic approach for assessing clonality for both IgH and TCR gene rearrangements based on the European BIOMED‐2 collabora-tive study is multiplex PCRs using a set of primers and subsequent analysis of the products by fragment size analysis (base pair length)

A basic understanding of how these assays are designed and mented is best achieved by an examination of IgH and T cell recep-tor structure, PCR design, product detection, evaluation of results, utility, value and limitations of the assays

imple-Immunoglobulin and t cell receptor structure

IghThe human immunoglobulin heavy (IgH) gene is located on chro-mosome 14 at 14q32.3 and can be divided into variable (V), diver-sity (D), joining (J), and constant (C) gene segments The mature

B cell goes through a series of D to J and then DJ to V ments in the maturation process There are over 150 different vari-able heavy (VH) segments of which only about 50 are functional and commonly used All VH segments have a unique fragment of sequence referred to as the complementarity determining region (CDR), which is the site of frequent somatic mutations in the ger-minal center maturation process Since all VH segments were origi-nally derived from one ancestral gene, there is a large amount of identical sequence among the entire group In addition to the CDR, all VH segments contain three framework regions (FR1, FR2, and FR3) for which sequence is shared among the different groups The

rearrange-sequence that is present in all groups is referred to as the

consen-sus sequence The sequence that is shared among some, but not all,

VH segments is referred to as the family‐specific sequence Based

on this latter family‐specific sequence, the different functional VH segments can be divided into seven VH subgroups (VH1, VH2, VH3, VH4, VH5, VH6, and VH7) Rearrangement of any V to D and J will result in a complex rolling circle splice formation that will delete the intervening chromosomal material but leave any upstream V or downstream constant elements intact Thus, each mature B cell has a single productive V‐D‐J rearrangement that

is unique to that cell in both length and sequence If this one cell becomes a clonal population of cells, then one amplified product should be detected by any PCR‐based approach for the detection of clonality Two products can be detected if the initial rearrangement

was nonproductive (incomplete rearrangement) for one allele and

was followed by a productive (complete) rearrangement of the other

allele Incomplete rearrangements (D to J without V), as opposed to

complete rearrangements (V‐D‐J) are uncommon in lymphomas

but do occur with some frequency in B cell leukemias

The sensitivity of PCR for IgH is lower than TCR, which could

be attributed to the alterations in the DNA sequence such as

so-matic hypermutation of IgH variable region, which may result in

primer annealing failure As an alternative, immunohistochemistry

and in situ hybridization techniques can be used to assess kappa

and lambda Ig light‐chain expression on FFPE tissue (Magro et al.,

2003a, Subtil, 2011; Deonizio and Guitart, 2012)

tCr

TCRs are proteins that are expressed on the surface of T lymphocytes

The vast majority of TRCs are composed of α and β chains and the

minority is composed of γ and δ chains Like IgH, each of the TCR

chains is composed of distinct regions, including variable (V),

join-ing (J), diversity (D), and constant (C) Durjoin-ing T‐cell maturation, the

TCR genes undergo rearrangement In TCR‐γ chain assembly, a

gle V region from several V gene segments is positioned next to a

sin-gle J region that has also been selected from several J gene segments

to make a rearranged V/J gene region that is eventually expressed on

the TCR‐γ protein Furthermore, there would be deletion and

ran-dom insertion of nucleotides at the ends where the V and J segments

join, resulting in a unique V/N nucleotide/J structure, which belongs

to that particular T cell and all its clonal progeny (Wood et al., 1994;

Wood, 2001) TCR gene rearrangement analysis with PCR is usually

performed on the TCR‐γ gene located on chromosome 7 at 7p14; it

is preferred because of the simpler structure, with only 12 variable

segments compared with other TCR genes such as the TCR‐β gene,

which has over 60 variable segments Therefore, fewer sets of

prim-ers are needed to detect most of the possible gene rearrangements

In addition, the TCR‐γ gene is rearranged early in T‐cell

develop-ment both in α/β and in γ/δ T cells, and generally remains intact,

although the protein may no longer be expressed on mature T cells

(Wood, 2001; Kandolf Sekulovic et al., 2007; Chitgopeker and Sahni,

2014) However, the limited junctional diversity may result in a high

background amplification of rearrangements of reactive T cells In

addition, in a significant proportion of malignant proliferations, the

tumor clone might not be detected This gene is rearranged in most

immature as well as mature T cell neoplasms, even if the neoplastic

cells express the α/δ TCR rather than the γ/δ TCR on the cell surface.

pCr design for determination of clonality

The strategy behind any PCR‐based Ig receptor clonality assay is

to minimize the number of PCR reactions, while at the same time

covering greater than 95% of all possible rearrangements The

ap-proach to minimize the number of PCR assays for both IgH and

TCR is to utilize a panel of family‐specific primers that bind with

specificity to the majority of members of that family In addition,

multiple primers can be combined in one PCR tube in a process

referred to as multiplex PCR.

In recent years, detection of clonal TCR gene rearrangements has

been evaluated using a large number of PCR assays As mentioned

before, PCR analyses of the TCR‐γ genes are predominantly applied

in routine practice due to the relatively simple TCR‐γ locus

con-figuration, the large homology within the Vγ and Jγ gene segments,

and the limited number of required primers

Detection of pCr products for clonality

While the detection of clonality by TCR gene rearrangements was originally performed using Southern blot, as mentioned before, this technique is impractical because it requires large amounts of DNA that can only be extracted from fresh or frozen tissue specimens

In the last 15 years, procedures that separate DNA fragments according to their nucleotide sequence in addition to their size have supplanted the Southern blot assay The two PCR procedures to detect clonality are single‐stranded conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) The advantage of the SSCP method lies in its relative simplicity and rapidity, compared to the technically difficult DGGE

With respect to DGGE, this method involves the PCR‐based amplification of the TCR gene followed by its separation using de-naturing gradient gel electrophoresis In this methodology, there is fractionation of the same sized PCR products based on differences

in their nucleotide sequences There are few studies published lizing this technology in the evaluation of cutaneous lymphocytic infiltrates In 1999 we published a series employing this technology

uti-to evaluate atypical cutaneous lymphocytic infiltrates (Brady et al.,

1999) In this study we evaluated paraffin‐embedded biopsy mens from seven cutaneous T cell lymphomas (CTCL), one nodal

speci-T cell lymphoma, two cutaneous B cell lymphomas, three sitivity reactions, one tonsil, and 14 lymphomatoid hypersensitivity reactions In the case of nodal T cell lymphoma, in six of seven CTCL, one cutaneous B cell lymphoma, and 2 of 14 lymphomatoid hypersensitivity reactions a monoclonal TCR gene rearrangement pattern was observed B cell clonality (IgH rearrangement) was confined exclusively to cases of B cell lymphoma

hypersen-The common technique that has largely replaced the DGGE odology is single‐stranded conformational polymorphism analysis The technique involves producing a ribbon of 5–10 10 μm sections from paraffin‐embedded tissue, which is then deparaffinized and di-gested The sample is extracted and the DNA is precipitated from the aqueous phase by addition of sodium acetate and ethanol The PCR

meth-reaction is then performed using consensus primers to the Vγ and J

regions These primers generate a product of approximately 270 base pairs that spans the N region where V–J joining occurs Amplified products are heat denatured and placed immediately in an ice water bath for the formation of single‐stranded conformers Products are then resolved on a nondenaturing 5% polyacrylamide:bisacrylamide gel (29:1), 400 volts for a few hours at room temperature Gels are placed with X‐ray film for production of autoradiograms Interpre-tative criteria are: (1) polyclonal: a diffuse smear or multiple weak bands, and (2) monoclonal: sharp discrete reproducible bands

In the study by Chen and coworkers using the single‐stranded DNA conformational assay, monoclonality and oligoclonality correlated strongly with a diagnosis of T cell dyscrasia with an overall specificity of 86%, being present in cases of CTCL and prelymphomatous conditions such as pityriasis lichenoides, pigmented purpuric dermatosis, and large plaque parapsoria-

sis (Chen et al., 2004) The cases that were monoclonal were

typically diagnostic of a more evolved stage of CTCL with gruent clinical evidence Polyclonality and oligoclonality were encountered in those CTCL cases where the histomorphol-ogy were equivocal, but the clinical history, including the ab-sence of an inciting immune dysregulatory drug trigger and the phenotypic profile suggested that a diagnosis of CTCL

con-was most likely (Chen et al., 2004) Oligoclonality con-was

par-ticularly characteristic for the prelymphomatous endogenous

T cell dyscrasias, including alopecia mucinosa, pityriasis

lichenoides, pigmented purpuric dermatosis, and large plaque

parapsoriasis, supporting the concept that the emergence of a

few dominant T cell clones is an event that may presage

pro-gression to lymphoma (Chen et al., 2004) There were, however,

a few drug‐associated lymphomatoid hypersensitivity reactions

that manifested a clonal and or oligoclonal pattern

A further modification of the SSCP is the capillary

electro-phoresis technique, which separates fragments according to

the unique size of the PCR products Capillary electrophoresis

of nucleic acids basically consists of two electrolyte chambers

linked by a thin capillary tube that is typically 50–100 μm in

di-ameter The typical setup for this technique is a single or

multi-ple tube capillary sequencer produced by many companies The

thin capillaries allow for excellent dissipation of heat and avoid

the problem of temperature gradients common in other methods

of electrophoresis As an electric field gradient is applied to the

medium within the capillary tube in continuity with each of the

electrolyte chambers, nucleic acids migrate through this field

Based on a complex group of factors, nucleic acids of similar

size generally migrate together Fluorescently labeled nucleic

acid PCR products are loaded in one end of the capillary tube

with a laser detection apparatus at the other end As the

prod-ucts migrate based on size and pass the laser, there is an

excita-tion of the fluorescent tag and the emission spectrum is gathered

by a charge‐coupled device If any one group of nucleic acids

is sufficiently present to reach the threshold of detection, this

information is electronically gathered and analyzed by a

soft-ware detection system and displayed on the computer screen

Known differentially labeled fluorescent‐tagged size standards

are run in conjunction with the test nucleic acid for calibration

of size One of the major strengths of fragment size analysis by

capillary electrophoresis is determination of the exact fragment

size in base pairs of the immunoglobulin or TCR rearrangement

produced by a clonal population of lymphocytes This can be

particularly useful in follow‐up or comparison of multiple

sam-ples from one patient

In one study performed to compare the sensitivity of PCR

anal-ysis of the TCR‐γ gene rearrangements using conventional

poly-acrylamide gel electrophoresis (PCR‐PAGE) versus fluorescent

capillary electrophoresis (PCR‐FCE), a total of 22 paraffin blocks

were analyzed There were 17 cases of mycosis fungoides (MF), 4

cases of non‐MF cutaneous T cell lymphoma (CTCL) and 1 case

of lymphoblastic leukemia The authors demonstrated complete

agreement between PCR‐PAGE and PCR‐FCE in 19 of the 22 cases,

with a concordance rate of 86.4% PCR‐FCE had a higher

sensi-tivity of 77.3%, compared to 63.6% for PCR‐PAGE, allowing the

detection of three additional cases of clonal T cell rearrangements

with equivocal or polyclonal bands, using the more conventional

PAGE technique PCR‐FCE also allowed the detection of similar

clones in specimens taken from different sites and at different times

in patients with MF However, similar clones from different

speci-mens can be achieved qualitatively in PCR‐PAGE by running and

comparing these on the same polyacrylamide gel block While both

PCR‐PAGE and PCR‐FCE are useful in detecting T cell clones in

CTCL, PCR‐FCE has the advantage of being a quantitative assay

where a precise base‐pair measurement is made of a dominant

clone and/or a few dominant clones This quantitative assessment

would allow one to follow disease progression in the

prelympho-matous dyscrasias to overt lymphoma, detect cutaneous relapses in

the setting of MF and identify incipient clones in early patch stage

MF and/or a prelymphomatous T cell dyscrasia; unfortunately, the complicated and expensive equipment limits its availability (Tang

et al., 2008) We were able to detect identical T cell clones between

biopsies over long periods of time in patients with indolent T cell dyscrasias such as pityriasis lichenoides and pigmented purpuric dermatosis using the PCR‐FCE technique, while the PCR‐PAGE technique typically reveals a polyclonal result, a point we cover in greater detail below

evaluation of results

Monoclonality in conventional PCR methods is defined by the ence of a discrete band after gel electrophoresis of the PCR product (see Figures 3.2, 3.3, 3.4, 3.8, and 3.9) If there are multiple bands, it indicates a polyclonal cell population

pres-With respect to the methodology using PCR‐FCE, due to the typical in‐frame (triplet) pattern of rearrangements in a typical polyclonal population of lymphocytes, the multiplex PCR design

of both IgH and TCR allows for a Gaussian distribution of ment sizes over a relatively small interval of product size (see Figure 3.5a) In a pure population of neoplastic lymphocytes, one expects and generally finds a single dominant peak (see Figure 3.5b) In many instances of clonal lymphoproliferative disor-ders, particularly cutaneous T cell lymphomas, there is a back-ground of polyclonal lymphocytes making the distinction of the true clonal process more difficult (see Figure 3.5c) Some of the more challenging evaluations occur when there is more than one peak in what appears to be a true clonal process, as shown in Figure 3.6a, where there are two dominant peaks and a complete absence of any polyclonal background There are several possi-bilities in this situation, all of which are difficult to prove with-out special techniques and a high‐quality DNA template for such procedures It is possible and most likely that this represents a complete rearrangement of both alleles, but the presence of two clonal populations of lymphocytes is not totally excluded An-other possibility is one true clonal population, but with an addi-tional relatively large peak caused by the primer annealing to the next downstream gene segment

frag-What is more common, particularly in evaluation of TCR, and of clinical concern is the definition and significance of an oligoclonal population or “restricted T cell repertoire.” A reasonable generally accepted definition of such a process is the presence of three or more rather dominant peaks in one multiplex assay (see Figure 3.6b) The difficulty with these cases is the possibility of pseudo-clonal results due to a paucity of lymphocytes or a poor‐quality DNA template, as can be seen in some formalin‐fixed paraffin‐em-bedded skin biopsies One reasonable way to exclude a poor‐qual-ity DNA template as a potential cause is simply to repeat the DNA amplification In cases with a poor‐quality DNA template, the frag-ment sizes detected will vary between each round of amplification

in the vast majority of cases Correlation with the histological tures is useful for excluding a paucity of lymphocytes as the cause Even more useful is comparison of multiple concurrent different skin biopsies from one patient, or multiple skin biopsies over an extended period of time (months to years) The restricted T cell repertoire shown in Figure 3.6b is from the same patient as the results shown in Figure 3.6a, but taken from a related site 2 years later

fea-the value and utility of molecular diagnostics

in primary cutaneous lymphomas

In ideal circumstances TCR clonality should be checked at the time

of diagnosis in the skin and blood The detection of a dominant

clone is important to confirm the diagnosis of lymphoma, as well as

to provide some prognostic guidance When early stage MF is being

considered in the differential diagnosis, detection of T cell clonality

could be viewed as potentially corroborative of a diagnosis of MF,

recognizing that clonality may be seen in reactive lesions such as

those of drug‐associated reversible T cell dyscrasia and other

prel-ymphomatous T cell dyscrasias, especially when using the PCR‐FCE

Multiple studies have been performed and revealed that TCR

gene clonal rearrangement analysis is a useful tool in the

diagno-sis of MF (Xu et al., 2010) In the study by Hsiao and coworkers,

TCR gamma clonality was positive in 53% of patch‐stage MF and

in 100% of cases of plaque‐ or tumor‐stage MF (Hsiao et al., 2007)

The prognostic and diagnostic value of the detection of circulating

clonal T cells in peripheral blood has been debated Detection of

circulating clonal T cells is uncommon among conventional patch

and plaque‐stage MF patients (12.5%), but more common in

eryth-rodermic MF patients (42%) Defining a circulating T cell clone

may help in distinguishing a dominant clone from the innocent

cy-totoxic T cell clones, which are occasionally detected in the

periph-eral blood of elderly patients (Delfau‐Larue et al., 2000; Dereure

et al., 2003).

To evaluate the reliability and applicability of Biomed‐2 methods

in archival tissue of CTCL, Lukowsky and coworkers applied the

Bi-omed‐2 TCR‐γ and TCR‐β assay to 107 archival paraffin‐embedded

tissue samples of 84 CTCL patients, 3 systemic T cell lymphoma

patients and 20 controls The Biomed‐2 TCR‐γ PCR revealed 81% clonality, while the Biomed‐2 TCRβ revealed 78% clonality in CTCL samples They found clonal TCRβ rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed‐2 TCR‐γ PCR

By combining all Biomed‐2 assays, one or more clonal ments were detected in 87% of CTCL and in all 3 systemic T cell

rearrange-lymphomas (Lukowsky et al., 2012).

In another study by Dr Plaza and his colleagues from Ohio State University, FFPE skin biopsies from 80 patients containing a T cell

dominant lymphocytic infiltrate were analyzed for TCR‐β gene

rear-rangement The findings indicated that monoclonality is a reliable characteristic of CTCL, with polyclonality being very infrequent However, some cases of drug‐associated lymphomatoid hypersensi-tivity, collagen vascular disease and the various cutaneous lymphoid dyscrasias (i.e pityriasis lichenoides chronica (PLC), pigmented purpuric dermatosis (PPD), and atypical lymphocytic lobular pan-niculitis) could also manifest restricted molecular profiles in the

context of an oligoclonal or monoclonal process (Plaza et al., 2008).

The use of dual TCR‐PCR analysis (T‐cell clonality analysis in opsy specimens from two different sites) has been suggested to pro-vide specificity in distinguishing MF from inflammatory conditions

In the study by Dabiri and coworkers, the authors suggested the

use of dual TCR‐PCR analysis as an adjunct to assist in

distinguish-ing granulomatous inflammatory reactions from granulomatous T

cell lymphoma, including granulomatous MF (Dabiri et al., 2011)

While such studies emphasize an apparent specificity of a common

clone preserved over time and present at different sites being

al-most a sine qua non of malignancy, we have had the opportunity

to examine several cases of prelymphomatous T cell dyscrasia, as

well as drug‐associated reversible T cell dyscrasia; using the PCR‐

FCE TCRβ assay, one dominant or a few prominent T cell clones

were commonly found in biopsies procured at different times and/

or representing different sites from the same patient in these

afore-said scenarios Using the more sensitive capillary gel

electrophore-sis technique, clonality is quite common in the setting of various

prelymphomatous T cell dyscrasias For example, in a study

stratify-ing pityriasis lichenoides accordstratify-ing to its phenotypic and

molecu-lar profile, 50% of cases showed a monoclonal and or oligoclonal

result, with two of these 24 cases progressing to MF The same

T cell dominant clonotypes were identified in cases with multiple

biopsies, indicative of a true monoclonal and/or oligoclonal pattern

(Magro et al 2007a) (see Figures 3.10 and 3.11) In the same vein,

clonality using the same highly sensitive TCRβ capillary gel

electro-phoresis technique was seen in 50% of cases of pigmented purpuric

dermatosis Roughly 50% of these patients went on to develop MF;

however, 50% did not demonstrate progressive disease, despite the

identification of clonality The polyclonal cases did not exhibit any

disease progression (Magro et al., 2007b) (see Figure 3.12) The

au-thors concluded that stratification of cases of pigmentary purpura

might be helpful prognostically and in devising a treatment plan

Another approach that has been suggested to improve

sensitiv-ity without sacrificing specificsensitiv-ity is the use of microdissection to

more readily identify a clonal population of lymphocytes In a case

reported by Hoffman and coworkers, the patient was a 47‐year‐old

female with a 10‐year history of discoid lupus erythematous who

presented with tender, enlarging subcutaneous nodules for a

pe-riod of two months The histopathologic features of the skin

bi-opsy along with immunophenotypic assessment were sufficiently

atypical to raise concern for an evolving panniculitis‐like T cell

lymphoma; however, initial TCR (β and γ) and B cell gene

rear-rangement studies revealed polyclonality Subsequently the TCR‐γ

gene rearrangement study was repeated after isolating the most

cytologically atypical portion of the infiltrate by manual

microdis-section This procedure led to the isolation of a monoclonal T cell

population (Hoffman et al., 2012).

To investigate the value of molecular techniques in the

diagno-sis of primary cutaneous B cell lymphoma (PCBCL), Felcht and

coworkers evaluated 18 patients with a well‐defined PCBCL and 9

patients with benign lymphocytic infiltrate, using BIOMED‐2

Con-certed Action BMH4 CT98‐3936 protocol In patients with PCBCL,

5 of 18 (28%) were given a diagnosis of primary cutaneous follicle

center lymphoma (PCFCL), 7 of 18 (39%) had primary cutaneous

marginal zone lymphoma (PCMZL), and 6 of 18 patients (33%) had

been diagnosed as primary cutaneous large B cell lymphoma, leg

type Monoclonality was detected in 88% of PCBCL cases and 22%

of benign lymphocytic infiltrates Identical skin, blood, and marrow

clones could be detected in leg type lymphoma cases emphasizing

the value of this technique for staging In primary cutaneous

fol-licle center/marginal zone B‐cell lymphoma, molecular analyses

are not needed for routine use, but may help differentiate benign

lymphocytic infiltrates from primary cutaneous follicle

center/mar-ginal zone B‐cell lymphoma They also suggested that each sample

should be analyzed at least twice to assess the possibility of

pseu-domonoclonality (Felcht et al., 2011).

Limitations of clonality assessment by pCr

There are certain limitations to this testing that should always be remembered in the evaluation of a clinical specimen First is the issue regarding sensitivity, or the ability to detect a clonal popula-tion of lymphocytes in a polyclonal background The BIOMED‐2 study group has shown convincingly that these assays as designed have a sensitivity of 1–5%; in other words, 1–5 clonal lymphocytes can be detected in a population of 100 lymphocytes in which the remaining lymphocytes are benign A caveat to this is that many of the specimens in this study were lymph nodes with an abundance

of lymphocytes for DNA isolation and amplification In our ion, and one given with a considerable degree of experience with formalin‐fixed paraffin‐embedded skin biopsies, the lower limit of sensitivity in such samples is somewhere between 10% and 15%

opin-A second issue that was previously mentioned is that clonality does not necessarily equate with malignancy Pseudoclonality in PCR‐based assays is difficult to recognize A highly sensitive PCR assay may detect an amplification of very few IgH or TCR gene rearrangements

in a limited number of B or T cells in the tissue sample In fact, to avoid the problem of a pseudoclonal result, it is likely wise not to submit cell‐poor skin biopsies for molecular analysis For example, if a cell‐poor interface dermatitis is examined from a molecular perspective,

it would not be unusual for a T cell clone to be detected, reflective of amplification of the few T cells present on the biopsy as opposed to implying a neoplastic event An oligoclonal T cell PCR result is not an uncommon finding in a biopsy sample of a B cell lymphoma with high tumor load and reactive T cells in the background This is particularly

seen with TCRγ genes as the PCR targets Repeating the PCR analyses

followed by mixing of the PCR products will clarify whether the clonal PCR products are in fact derived from different lymphocytes Thus, it

is important to correlate the molecular results with the cal and clinical findings and not to evaluate the results of any clonality assay in a vacuum An important source of false positives is cross‐contamination of specimens by clonal DNA from positive controls (Chitgopeker and Sahni, 2014) While B cell clonality is unusual in the realm of benign lymphocytic infiltrates (Magro and Schaefer, 2008),

histopathologi-we have seen and documented T cell clonality in the following settings

(Magro et al., 2001; Magro et al., 2002; Magro et al., 2003b; Chen et al., 2004; Plaza et al., 2008):

1 Any of the prelymphomatous T cell dyscrasias, including mented purpuric dermatosis, pityriasis lichenoides chronica, and atypical lymphocytic lobular panniculitis with clone presentation between biopsies, over time and in cases progressing to MF

pig-2 The drug‐associated reversible T cell dyscrasias, including the interstitial granulomatous drug reaction where common T cell clones in the skin biopsy specimen and peripheral blood have been documented

3 As part of an exuberant T cell response in the setting of B cell lymphoma

4 Lupus erythematosus profundus

5 Lichen sclerosus et atrophicus

It is important to remember that conditions associated with trogenic and or endogenous immune dysregulation can lead to overzealous T cell responses, which from a molecular perspective will manifest as one of clonality or oligoclonality Our detailed experience assessing clonality in various cutaneous T‐cell infil-trates utilizing the PCR‐FCE technique was outlined in our paper

ia-(Plaza et al., 2008), which provides an account of the potential

needs to be based on the immunophenotypic features of the case

A fourth issue that was previously discussed is the pseudoclonality and oligoclonal processes in a lymphocyte‐poor specimen

Amplification of the TCR‐γ gene of selected T lymphocytes may

be also affected due to factors such as tissue thickness, cell size ations or the degradation of DNA in the tissue processing Other reasons include poor tissue sampling (i.e., small number of malig-nant T cells in the skin sample) In addition, TCR gene deletion

vari-is possible in the later stages of the dvari-isease, which may result in

non‐amplification of TCR‐γ gene sequences as the cell undergoes malignant transformation (Kandolf Sekulovic et al., 2007).

As for B cell clonality, we feel that this finding typically correlates with a diagnosis of B cell malignancy Pseudolymphomas exhibiting

B cell hyperplasia do not typically exhibit B cell clonality by either the PAGE or CPE techniques There are rare reported cases of B cell clonality in a reactive setting, but, in general, B cell clonality does suggest B cell neoplasia

The final caveat is that failure to identify clonality in skin biopsy specimens that are otherwise diagnostic of cutaneous T cell or B cell lymphoma should not specifically influence or alter the final diagnosis If all other aspects of the clinical presentation, morpho-logic findings, and phenotypic profile point toward categorization

as a lymphoma, a negative clonal result should not be an absolute determinant in changing the diagnosis to a benign inflammatory process

spectrum of molecular profiles that one can encounter in a

di-verse spectrum of cutaneous T cell infiltrates In the setting of MF,

the PCR‐FCE results disclosed a monoclonal and/or oligoclonal

result in >90% of cases Interestingly, in almost a quarter of the

cases, 4 dominant T cell clones were seen while three cases had 3

dominant clones and two had 2 dominant clones There was clone

preservation between the biopsies, despite the presence of more

than one dominant clone In the cutaneous lymphoid dyscrasia

category represented primarily by large plaque parapsoriasis,

pig-mented purpuric dermatosis, pityriasis lichenoides and alopecia

mucinosa, approximately one‐third of the cases showed a

mono-clonal result In all cases there was a polymono-clonal background and

the number of dominant clones ranged from one to a few, similar

to the findings seen in the setting of MF In the drug‐associated

T cell dyscrasia setting we studied patients who had different

bi-opsies that showed the same dominant clones (1 dominant T cell

clone in the two cases) The drugs involved in these cases were

nifedipine and lisinopril, neurontin, IDEC, rituximab, statin and

hydrochlorothiazide In all of these cases, clinical history and

im-munophenotype favored drug‐associated reversible T cell

dyscra-sia (Plaza et al., 2008).

A third issue is that IgH and TCR gene rearrangements are not

necessarily markers for B or T cell lineage determination,

respec-tively Dual rearrangements (lineage infidelity) can be observed in

both B and T cell lymphomas, and the final determination of lineage

(a)

(b) Peaks 196 & 188

Peaks 302

Two dominantpeaks 260 & 271 bp

Figure 3.6 Gene scanning images

Case vignettes

Case vignette 1

An 85‐year‐old male presented with a violaceous plaque involving the forehead; his past medical history was remarkable for chronic lymphocytic leukemia A biopsy of the skin plaque showed diagnostic features of follicular mycosis fungoides with concomitant chronic lymphocytic leukemia The subsequent molecular analysis of the same biopsy confirmed the presence of both B and T cell monoclonal populations demonstrating IgH and TCR gamma gene rearrangement, reflecting the dual T and B cell malignancy coexisting in the pa-tient’s skin (Figures 3.1, 3.2, 3.3, and 3.4)

Molecular gel and diagnostic interpretation provided by Molecular Hematopathology Laboratory, Department of Pathology and ratory Medicine, Weill Cornell Medicine

Labo-Figure 3.1 Violaceous plaque involving the forehead

IgH gene rearrangement

Skin biops

yMonoclonal contr

ol

Polyclonal contr

ol

No templat

e control

Figure 3.2 IgH gene rearrangement A distinct band is seen, suggesting the

presence of monoclonal B cell population

TCR gamma tube B

Skin biops

yMonoclonal contr

e control

Figure 3.4 TCR gamma tube B A distinct band is seen, indicative of a monoclonal T cell population.

TCR gamma tube A

Skin biops

yMonoclonal contr

e control

Figure 3.3 TCR gamma tube A A distinct band is seen, indicative of a monoclonal T cell population.

Case vignette 1 (Continued)