STUDYING THE ASSOCIATION BETWEEN PREVALENCES OF CERTAIN BACTERIA IN TUMOR TISSUES AND KRAS, BRAF, AND HSP110 GENE MUTATIONS IN PATIENTS WITH COLORECTAL CANCER

MINISTRY OF EDUCATION MINSITRY OF NATIONAL AND TRAINING DEFENCEVIETNAM MILITARY MEDICAL UNIVERSITY VU SON GIANG STUDYING THE ASSOCIATION BETWEEN PREVALENCES OF CERTAIN BACTERIA IN TUMOR

MINISTRY OF EDUCATION MINSITRY OF NATIONAL AND TRAINING DEFENCE

VIETNAM MILITARY MEDICAL UNIVERSITY

VU SON GIANG

STUDYING THE ASSOCIATION BETWEEN PREVALENCES

OF CERTAIN BACTERIA IN TUMOR TISSUES AND KRAS,

BRAF, AND HSP110 GENE MUTATIONS IN PATIENTS

WITH COLORECTAL CANCER

Speciality: Bio-medical science Code: 9 72 01 01

SUMMARY OF MEDICAL DOCTORAL THESIS

HANOI - 2025

THE DISSERTATION WAS COMPLETED AT

VIETNAM MILITARY MEDICAL UNIVERSITY

The thesis was defended in front of the thesis review committee

at Vietnam military Medical University

The thesis can be founded in:

- National library

- Military Medical Academy’s library

INTRODUCTION Colorectal cancer (CRC) is a malignant disease that can occur at any

age and in both sexes In 2020, there were more than 1.9 million newCRC cases and 935,000 deaths worldwide, accounting forapproximately one-tenth of all cancer cases and deaths In Vietnam,annual statistics consistently show that CRC ranks among the top tencancers with the highest incidence and mortality rates, posing asignificant public health burden

Currently, gut microbiota has been identified as having an impact onintestinal homeostasis and a triggering factor in the development of theCRC microenvironment through various mechanisms Geneticmutations also play an important role in the pathogenesis of CRC

Therefore, We carried out the project named "Studying the association

between prevalences of certain bacteria in tumor tissues and KRAS, BRAF, and HSP110 gene mutations in patients with colorectal cancer".

Objectives:

1 To determine the prevalence of Fusobacterium nucleatum (F nucleatum), Bacteroides fragilis (B fragilis), Escherichia coli (E coli), Akkermansia muciniphila (A muciniphila), and KRAS, BRAF, and HSP110 gene mutations in CRC tissues

2 To evaluate the association between the infection rates of F nucleatum, B fragilis, E coli, A muciniphila and KRAS, BRAF, HSP110 gene mutations with the CRC anapathology.

Urgency of the study:

Research on gut microbiota and BRAF, KRAS and HSP110 gene

mutations in cancer tissues of CRC patients contributes to clarifyingthe roles of bacteria and genetic mutations in the pathogenesis ofCRC This research also holds potential applications in prognosis,targeted therapy, and prevention of CRC

Novel contributions of the thesis:

- Providing prevalences of F nucleatum, B fragilis, E coli, A muciniphila, and KRAS, BRAF, and HSP110 mutations in CRC tissues.

- Identifying association between the infection rates of F nucleatum, B fragilis, E coli, and A muciniphila and the KRAS, BRAF, HSP110 gene mutations with CRC anapathology.

Structure of the thesis:

The thesis comprises 144 pages, including: Introduction (2 pages),Chapter 1: Literature Review (37 pages), Chapter 2: Subjects andMethods (26 pages), Chapter 3: Results (43 pages), Chapter 4:Discussion (33 pages), Conclusion (2 pages), and Recommendations(1 page) The thesis includes 162 references (156 in English)

CHAPTER I: LITERATURE REVIEW

1.1 Colorectal cancer

1.1.1 Colorectal cancer status

- In the world: According to GLOBOCAN (2020), the estimatednumber of new CRC cases was 1,931,590 with an age-standardizedincidence rate of 19.5/100,000 persons/year The highest incidence rateswere estimated in Europe (30.4/100,000), followed by Oceania(29.8/100,000), North America (26.2/100,000), Asia (17.6/100,000),Latin America (16.6/100,000), the Eastern Mediterranean Region(EMRO) (9.1/100,000), and Africa (8.4/100,000)

- In Vietnam: In 2022, CRC ranked fourth in both incidence andmortality among the most common cancers, following liver, lung, andstomach cancers, with 16,835 new cases (accounting for 9.3%) and 8,454deaths (accounting for 7.0%) The incidence rate was 9.9% in males(ranked 4th) and 8.7% in females (ranked 3rd) among all cancer types

1.1.2 Risk factors

1.1.2.1 Modifiable risk factors: Alcohol and beer consumption;

smoking; obesity; sedentary lifestyle; diet; psychological stress.

1.1.2.2 Non-modifiable Risk Factors: Age, gender, genetic factors,

family history of colorectal cancer, pelvic radiotherapy, and pre-existingmedical conditions (such as ulcerative colitis, Crohn’s disease, etc.)

1.1.3 Pathogenesis of CRC

1.1.3.1 Adenoma–Carcinoma progression: Most CRC arise from

precancerous polyps, including conventional adenomas or serratedadenomas Adenomas develop when there are abnormalities inmechanisms such as regulate DNA repair and cell proliferation Asmutated cells progress toward the colonic lumen, terminaldifferentiation and apoptosis are often disrupted, leading to theformation of adenomas

1.1.3.2 Chromosomal instability (CIN) Pathway: The CIN

pathway is observed in approximately 65%-70% of sporadiccolorectal tumors and is characterized by chromosomal alterationssuch as somatic copy number alterations (SCNAs) due to aneuploidy,deletions, insertions, amplifications, or loss of heterozygosity

1.1.3.3 Microsatellite instability (MSI) pathway: The MSI pathway

is the primary mechanism responsible for the hypermutated phenotype

Generally, mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) cause instability in microsatellite regions of DNA 1.1.3.4 Serrated neoplasia pathway: Serrated polyps are considered

precursors of approximately 30% of colorectal cancers This is aheterogeneous group of lesions characterized by a star-shaped cryptarchitecture, including benign hyperplastic polyps, sessile serratedadenomas (precancerous), and traditional serrated adenomas

1.1.3.5 Other pathways: Other mutations have been identified in genes such as ARID1A, SOX9, and FAM123B, as well as amplifications

of ERBB2 and IGF2 Recurrent chromosomal translocations include the fusion of NAV2 and TCF7L1 within the WNT signaling pathway.

1.1.3.6 KRAS, BRAF, and HSP110 gene mutations in CRC

* KRAS mutation: KRAS mutation are common, occurring in

approximately 40% of CRC These mutations lead to constitutive activation

of the KRAS protein, stimulating downstream signaling pathways involved

in cell proliferation and survival, ultimately contributing to tumor formation

KRAS plays a critical regulatory role in several cellular signaling pathways,

including PI3K-Akt and RAS-RAF-MAPK pathways (related to cellproliferation), and RAS-GEF signaling pathway

* BRAF mutation: BRAF functions as a regulator of the

MAPK/ERK signaling pathway The RAS/RAF/MEK/ERK pathwayacts as a signaling cascade linking extracellular stimuli to the cellnucleus Extracellular signals such as hormones, cytokines, and

various growth factors interact with their receptors to activate RAS family G-proteins Through adaptor proteins, recruits RAF proteins to the cell membrane, where they are activated BRAF signals through

MEK to activate ERK, initiating a series of biochemical processesincluding cell differentiation, proliferation, growth, and apoptosis

* HSP110 mutation: This gene encodes Heat Shock Protein 110

(HSP110) Overexpression of HSP110 has been observed in variouscancers, including melanoma, prolactin-secreting tumors, pituitaryadenomas, breast cancer, colorectal cancer, pancreatic cancer, thyroidcancer, and esophageal cancer HSP105 (as HSP110) is essential forWnt signaling; its depletion results in the accumulation of β-cateninand the transcription of Wnt target genes upon stimulation

1.2 Gut Microorganisms and CRC

1.2.1 The association between bacteria and CRC

1.2.1.1 E coli and CRC

E coli strains carrying the pks island (pks ⁺ E coli) can cause

somatic mutations leading to DNA damage and act as a carcinogenictrigger Chronic exposure of cells to pks ⁺ E coli results in a distinct

transcriptional signature characterized by base substitutions orinsertions/deletions in AT-rich DNA motifs, which have beenidentified in approximately 5% of metastatic CRC tumors.

E coli relates to BRAF, KRAS, and HSP110 mutations: pks ⁺ E coli induces mutations in regulatory genes and chromatin modulators, including APC, KRAS, and PIK3C APC and BRAF mutations are

more likely to be triggered by colibactin However, further research

is necessary to fully understand the multifaceted role of colibactin ininitiating and driving gene mutations involved in cancer progression

1.2.1.2 B fragilis and CRC

Tumorigenesis is closely linked to the immune system The

Bacteroides fragilis toxin (BFT) triggers a multistep inflammatory

cascade promoting cancer development, requiring IL-17R, NF-κB, andSTAT3 signaling in colonic epithelial cells Additionally, BFT toxicitydirectly activates transcriptional pathways associated with cellproliferation and stemness characteristics seen in various cancers BFTfacilitates E-cadherin degradation, thereby activating the WNT/β-cateninsignaling pathway, which directly promotes cell proliferation

B fragilis and its relation to BRAF, KRAS, and HSP110 mutations: In MinApc716/+ mice infected with B fragilis, tumors

were observed in the mid-colon In BRAFV600E^Lgr5CreMin(BLM) mice, tumors resembled human BRAFV600E-associatedtumors Immunohistochemical analysis revealed clear expression of

KRAS and BRAF in tumors with high B fragilis colonization 1.2.1.3 F nucleatum and CRC

F nucleatum has been identified as a potential carcinogenic agent and is found in high abundance in colorectal tumor tissues F nucleatum promotes tumor growth in ApcMin/+ mice The underlying mechanisms

of tumorigenesis involving F nucleatum include the production of

virulence factors such as Fusobacterium adhesin A (FadA) and

fibroblast activation protein 2 (Fap2), activation of β-catenin signalingpathways in cancer cells to promote tumor growth, suppression of theimmune microenvironment, reduced antitumor immunity, andpromotion of IL-17-related carcinogenesis.

F nucleatum and its association with BRAF, KRAS, and HSP110

mutations: Several studies have shown that CRC with MSI-H, BRAFmutations, or high CpG island methylator phenotype (CIMP-high) is

associated with F nucleatum infection Other studies have also reported

a link between F nucleatum infection and KRAS mutations in CRC MSI-H, MLH1 hypermethylation, BRAF and KRAS mutations were independently associated with F nucleatum infection.

1.2.1.4 A muciniphila and CRC

A muciniphila: Baxter et al (2014) reported a positive correlation between increased tumor burden and the presence of Akkermansia A muciniphila may influence inflammation and epithelial cell

proliferation Regarding its association with CRC, Wang et al (2022)

demonstrated that A muciniphila could induce CRC in mice by

promoting inflammation and the growth of intestinal epithelial cells

1.2.2 Co-infection of Bacteria in CRC

Considering four bacterial species, each can contribute to CRCprogression through different mechanisms It is possible that thesebacteria may act synergistically to promote CRC development.Furthermore, biofilms commonly found in the colons of CRC patientsoften contain these bacteria, providing further evidence of theirinteraction Dejea et al (2018) presented the first evidence suggesting apotential synergistic role between this bactarial in CRC progression

1.3 Current research on the association between gut

microbiota and KRAS, BRAF, HSP110 mutations in CRC

- In the world: Aref Shariati et al (2021) conducted a study on the

association between CRC and F nucleatum and B fragilis in Iranian

patients Jihoon E Joo et al (2024) investigated the clinical and

pathological features of CRC tumors associated with Escherichia coli

(both pks and pks strains), ⁺ ⁻ B fragilis, and F nucleatum.

- In Vietnam: The study by Pham Van Dung et al (2024) reported

that 34.1% of CRC patients had KRAS/NRAS mutations, while no patients carried BRAF mutations Nguyen Trong Hoa et al (2021) analyzed KRAS, NRAS, and BRAF mutations in 76 cases of metastatic

CRC diagnosed and treated at the 108 Military Central Hospital

CHAPTER 2: SUBJECTS AND METHODS

2.1 Participants

The participants included patients with a confirmed diagnosis ofCRC The control group consisted of patients diagnosed of benigncolorectal polyps, treated at the Department of Gastroenterology, 175Military Hospital

2.1.1 Inclusion Criteria

- CRC Group: Participants aged ≥18 years with a confirmeddiagnosis of CRC Patients must have sufficient paraffin-embeddedtissue blocks and H&E-stained slides available They must not haveundergone chemotherapy or radiotherapy, and must not have usedantibiotics, immunosuppressive agents, or probiotics for at least onemonth prior to sample collection Patients consented to participate inthe study The tissue samples had adequate size and collected understerile conditions to prevent contamination

- Control Group: Participants aged ≥18 years with a diagnosis of

benign colorectal polyps Patients must not have used antibiotics,immunosuppressants, or probiotics for at least one month prior tosample collection Sufficient paraffin-embedded tissue blocks andH&E-stained slides must be available Tissue samples were collected

following proper procedures to avoid contamination Patientsconsented to participate in the study.

2.1.2 Exclusion Criteria

- CRC Group: Patients with other concurrent malignancies, a

history of chronic inflammatory bowel disease, or chronic colitis.Patients who decline to participate or withdraw from the study willalso be excluded

- Control Group: Patients with colorectal polyps larger than 0.5 cm in

diameter or with histopathological findings showing cellular dysplasia

2.2 Study duration and location

- Study period: From May 2023 to December 2024

- Study location: Department of Biochemistry, Department of Pathology,

and Center for Oncology and Nuclear Medicine, 175 Military Hospital

2.3 Research methods

2.3.1 Study design: A cross-sectional descriptive study with a

comparative control group

2.3.2 Sample size and sampling method

2.3.2.1 Sample size formula:

2.3.3 Materials and Equipment Used in the Study

2.3.3.1 Primers and Probes: Primers and probes were used to detect Bacteroides fragilis, Fusobacterium nucleatum, Akkermansia muciniphila, and Escherichia coli These were designed based on gene sequences encoding toxins or oncogene activators, such as the bft-1,

nusG, RluA, and eae genes The SLCO2A1 gene was used as an internal

t Plasmid Insert Size (bp) Cloning Vector Purpose of Use

p.Bft-1 100 pGEM-T Detection of B fragilis via bft-1gene sequence

p.nusG 108 pGEM-T Detection of F nucleatum via nusG gene sequence

p.RluA 104 pGEM-T Detection of A muciniphila via RluA gene sequence

p.eae 95 pGEM-T Detection of E coli via eae genesequence

p.SLCO2A1 115 pJET1.2 Internal control using SLCO2A1gene sequence

2.4.2 Research Indicators and Evaluation Criteria

- Patient characteristics: Age, gender, BMI, and tumor histopathology

- Assessment of bacterial prevalence: Determine the presence

rates of F nucleatum, B fragilis, E coli, and A muciniphila, as well

as mutations in the KRAS, BRAF, and HSP110 genes in CRC tissues.

- Evaluate associations: Analyze the relationship between

bacterial infections and gene mutations with the histopathologicalcharacteristics of CRC

2.4.3 Research Techniques

2.4.3.1 Histopathological analysis of tumor and colorectal polyp tissues: Conducted at the Department of Pathology, 175 Military Hospital 2.4.3.2 Extraction of total DNA from paraffin-embedded tissue samples: DNA was extracted using the PureLink Genomic DNA Kit

(Invitrogen) following the standard protocol for gDNA isolation

2.4.3.3 DNA quality and concentration assessment after extraction: DNA concentration and purity were measured by

spectrophotometry using the Biophotometer D30 (Eppendorf,Germany), with detection range from 2.5 to 1500 ng

2.4.3.4 Electrophoresis method: Used to separate DNA fragments

based on size and structure under a direct current electric field

2.4.3.5 Real-time PCR protocol for detection of B fragilis, F nucleatum, A muciniphila, and E coli:

Table 2.3 Primer/Probe Sequences Used in the Study

Target Primer/Probe Sequence (5'-3') Amplicon Size (bp)

ΔCt value was calculated using the formula: ΔCt= Ctgen vi khuẩn-Ct gen nội kiểm

Specifically: ΔCt= Ct Bft-1 - Ct SLCO2A1 ; ΔCt= Ct nusG - Ct SLCO2A1 ΔCt= Ct RluA - Ct SLCO2A1; ΔCt= Ct eae - Ct SLCO2A1

The bacterial load of B fragilis, F nucleatum, A muciniphila, and

E coli was calculated using the formula: Bacterial load = 2-ΔCt

2.4.3.6 Procedure of determining BRAF, KRAS and HSP110 gene mutations using realtime PCR

- BRAF mutation detection was performed using the AmoyDx® BRAF V600 Mutations Detection Kit.

- KRAS mutation detection was conducted using the AmoyDx® KRAS/NRAS Mutations Detection Kit.

- HSP110 mutation detection was performed using qRT-PCR with the following primers and probes: Forward primer (HSP110F): 5′- AGATATTAGCACAACACTCAATGC-3′; Reverse primer (HSP110R): 5′-

GAGCAGCATGGTTTCGACTAA-3′; Probe R1 (mutant-specific, labeled): 5′-FAM-AGTATTGCACACTGTAATGCACATCCTCTG-3′; ProbeR2 (HEX-labeled): 5′-HEX-CTCATGAACACTGTAATGCACATCCTCTG-3′

FAM-2.5 Data Analysis

All data were analyzed using SPSS 26.0 Graphs and statisticalvisualizations were generated and analyzed using GraphPad Prism 9.3.0

CHAPTER 3: RESEARCH RESULTS

Table 3.1 Distribution of Age, Gender, and BMI of the Participants Variables CRC (n=149) Polyp (n=109)