BRCA1 (chromosome 17q) and BRCA2 (chromosome 13q) are tumour suppressor genes involved in transcriptional regulation and DNA repair (specifically homologous recombination repair of double-strand DNA breaks). ^[2]

BRCA genes are involved in transcriptional regulation and DNA repair; defective DNA repair may lead to excessive chromosomal damage, may increase radiosensitivity and may make cells more sensitive to chemotherapy. ^[2]

Why does losing BRCA function cause cancer?

• Normally, when your DNA gets a double-strand break (DSB), the BRCA1/BRCA2 proteins help recruit RAD51 to the break site for homologous recombination (HR) — the highest-fidelity repair mechanism.
• If BRCA is non-functional, cells must use error-prone repair pathways (e.g., non-homologous end joining, NHEJ), leading to accumulating genomic instability → progressive mutations in other genes → cancer.
• This follows Knudson's two-hit hypothesis: one allele is inherited as a germline mutation (first hit), and the second allele is lost somatically (second hit / loss of heterozygosity), resulting in complete loss of function.

BRCA mutations are autosomal dominant hereditary syndromes ^[2]

This means:

• 50% chance of passing the mutation to each offspring
• Both males and females can carry and transmit the mutation
• Penetrance is incomplete (not everyone with the mutation develops cancer, but their risk is vastly elevated above the general population)

BRCA gene mutations account for 30–70% of patients with hereditary breast/ovarian cancer and 5–10% of all breast and ovarian cancers ^[2]

This means the vast majority (90–95%) of breast/ovarian cancers are sporadic, not hereditary. But among the hereditary subset, BRCA1/2 are the dominant genes.

Three clinical syndrome presentations:

• Breast and ovarian cancer syndrome
• Site-specific breast cancer syndrome
• Site-specific ovarian cancer syndrome ^[2]

BRCA1 mutation carriers: 85% lifetime risk of breast cancer, 35–60% ovarian/fallopian tube/primary peritoneal cancer ^[2]

BRCA2 mutation carriers: 85% lifetime risk of breast cancer, 10–27% ovarian cancer, 6% male breast cancer ^[2]

Source: WHO Genetic Tumour Syndromes 2022 ^[2]

Both are large genes, encoding exceptionally large, highly charged proteins. No mutational hot-spots, although more than half of all mutations found in large exon 11. Up to 85% of BRCA1 mutations result in truncated protein. ^[2]

Why does this matter clinically?

• No hotspot → you cannot use a simple targeted assay; you need to sequence the entire gene (full gene sequencing)
• Truncating mutations (frameshift, nonsense) → produce a non-functional, truncated protein → clear pathogenicity
• Methods needed: Direct DNA sequencing, Next-generation sequencing (NGS), and MLPA analysis for detecting large genomic deletions and duplications that standard sequencing would miss ^[2]

"Mutation" = disease-causing change; "Polymorphism" = non-disease-causing change found at frequency ≥1%. Use term "sequence variation" to prevent confusion. ^[2]

The term "mutation" is used to mean disease causing change — "Deleterious mutation" — now called Germline Pathogenic Variant (GPV) ^[2]

This modern terminology shift matters: in clinical reports you'll see "germline pathogenic variant" rather than "mutation." A Variant of Unknown Significance (VUS) is a change whose clinical importance is unclear — this creates major counselling challenges.

Breast cancer diagnosed at an early age; Bilateral breast cancer; History of both breast and ovarian cancer; Presence of breast cancer in one or more male family members; Multiple cases of breast cancer in family; Both breast and ovarian cancer in family; Two or more primary types of BRCA1 or BRCA2 related cancers in a single family member ^[2]

Factors considered when assessing risk:

Number of affected relatives; Relationship of affected relatives; Age at diagnosis of breast cancer; Presence of ovarian cancer in family; Presence of associated malignancies ^[2]

Threshold for testing:

3 or more members of direct lineage with breast and/or ovarian cancer ^[2]

Expanded indication (regardless of family history):

Patients with TNBC (triple-negative breast cancer) and/or high grade serous ovarian carcinoma regardless of family history ^[2]

This table is directly from the WHO Classification of Tumours, 5th Edition, as presented on the lecture slide ^[2]:

These three categories overlap but are NOT synonymous ^[2]:

Triple Negative ≠ Basal-like ≠ BRCA1

Basal-like tumours have a distinct genomic expression and protein profile, distinct morphology, and are often but not invariably Triple Negative (ER, PR, HER2 −ve). Found in 80–90% of BRCA1 mutation breast cancers and 15% of breast cancers with no family history. ^[2]

Histological features of basal-like breast cancer ^[2]:

• Grade 3 invasive carcinoma or metaplastic carcinoma
• Geographic necrosis
• Pushing borders of invasion
• Stromal lymphocytic response
• High mitotic count
• IHC: CK5/6 positive, ER negative, HER2 negative, p63 negative

Clinical behaviour of basal-like tumours ^[2]:

• Younger patients
• Frequently "interval cancers" with rapid progression (important: annual mammography may miss them)
• Heterogeneous — not all uniformly poor prognosis
• Visceral metastases to brain and lung, rather than lymph node (unlike luminal-type which metastasises to bone)
• Pathological complete response (pCR) to neoadjuvant chemotherapy (paradox: bad biology but good chemosensitivity)
• Not associated with locoregional relapse after conservative treatment

High grade serous adenocarcinoma — ovarian, tubal and peritoneal. Prophylactic oophorectomy in BRCA mutation carriers revealed high number of tubal neoplasms. Distal fallopian tube is an important source of pelvic serous cancer. ^[2]

Clinical implications:

• Recommendation to include removal of entire tubes at oophorectomy with thorough microscopic evaluation
• Visualisation of peritoneal surfaces with pelvic washings should be performed ^[2]

Why is this important? The paradigm has shifted: many "ovarian" high-grade serous carcinomas actually originate in the fimbriated end of the fallopian tube (serous tubal intraepithelial carcinoma, STIC). This is why prophylactic surgery must include the tubes, and pathology must section them thoroughly.

Families identified to have high risk should always start with "Index" testing — family member with diagnosis of breast or ovarian cancer. Once the germline mutation is identified for the index patient, then "carrier testing" can be offered to unaffected family members. ^[2]

Negative result useful only if a BRCA mutation has been identified in an affected first degree relative. ^[2]

Why index testing first?

If you test an unaffected person first and get a negative result, you learn nothing useful: the family might still harbour a BRCA mutation that this particular individual didn't inherit, OR the family's cancer predisposition might be caused by a different gene entirely. Only by finding the specific mutation in an affected family member can you then offer definitive carrier testing to relatives.

If no mutation can be found in any of the index patients, then BRCA testing should not be offered to unaffected relatives. ^[2]

12% of high risk families may have genomic rearrangements (these would be missed by standard sequencing → need MLPA) ^[2]

13% reported VUS — associated harm unknown ^[2]

Allelic heterogeneity: Different mutations within the same gene can confer different age-specific risks of breast/ovarian cancer ^[2]. This is why penetrance figures are given as ranges.

Penetrance — the probability of developing breast or ovarian cancer with a BRCA mutation. Incidence of diagnosis: Breast cancer 46% (BRCA1), 52% (BRCA2); Ovarian cancer 12% (BRCA1), 6% (BRCA2) ^[2]

Note: These incidence-based penetrance figures (from population studies) are lower than the lifetime risk figures (85%) quoted from high-risk family studies. The difference arises because high-risk families may have additional modifiers.

1. Interpretation of result; 2. Cancer risks; 3. Interventions offered; 4. Treatment response ^[2]

Mammography: Poor sensitivity 40% — no evidence of benefit for BRCA mutation carriers. Interval cancers — annual mammography may miss aggressive cancers. ^[2]

MRI: Higher sensitivity 81%, reduced specificity, PPV 53%. ^[2]

Transvaginal ultrasound for early detection of ovarian cancer: High false positive results. ^[2]

Combination of MRI, mammography, ultrasonography: 95% sensitivity. Effect on morbidity and mortality unclear. ^[2]

Why is mammography alone insufficient? BRCA1 cancers are often high-grade, rapidly growing "interval cancers" that arise between annual screens. The dense breast tissue in younger women also reduces mammographic sensitivity. MRI is far more sensitive but less specific (more false positives → more biopsies).

For ovarian cancer, no effective screening exists — transvaginal ultrasound has high false-positive rates and has not been shown to reduce mortality.

Selective estrogen modulators (SERMs) e.g. Tamoxifen, Raloxifene. Side effects: DVT, pulmonary emboli, endometrial cancer. Reduces relative risk for ER+ breast cancer BUT most breast cancers associated with BRCA1 mutations are ER−ve hence not prevented by Tamoxifen. ^[2]

Key exam point: Tamoxifen works by blocking estrogen receptor signalling. Since BRCA1 cancers are overwhelmingly ER-negative, tamoxifen is essentially useless for preventing BRCA1-associated breast cancer. It may have some role in BRCA2 carriers (whose cancers tend to be ER+).

Prophylactic bilateral mastectomy: 90% reduction in risk for breast cancer, but only 30% uptake. ^[2]

Prophylactic bilateral salpingo-oophorectomy (BSO): 85% reduction in ovarian cancer risk; 50% reduction in breast cancer risk and breast cancer survival in premenopausal women. ^[2]

Recommendation: Women with BRCA mutations should be offered risk-reducing salpingo-oophorectomy by age 40 years or when childbearing is complete. ^[2]

Why does BSO reduce breast cancer risk? Removing the ovaries eliminates the main source of endogenous estrogen in premenopausal women, effectively creating a surgical menopause. This reduces hormone-driven breast cancer risk (particularly relevant for BRCA2 carriers who get ER+ cancers).

Defective DNA repair results in better response to radiation and chemotherapeutic agents ^[2]

Sensitivity to DNA cross-linking agents: carboplatin, cisplatin, mitomycin C ^[2]

BRCA carriers more likely to achieve pCR (pathological complete response) to neoadjuvant chemotherapy. Platinum-based chemotherapy effective in BRCA1 carriers. ^[2]

First-principles explanation: DNA cross-linking agents (platinums) cause inter-strand crosslinks. Normal cells fix these via homologous recombination (which requires functional BRCA). BRCA-deficient tumour cells cannot repair these crosslinks → accumulate lethal DNA damage → cell death. This is why BRCA-mutant cancers are paradoxically more chemosensitive despite having aggressive biology.

If no chemotherapy for small sized, lymph node−ve BRCA carriers → worse prognosis, more significant risk of micrometastases ^[2]

This highlights that even "low-risk" BRCA cancers by conventional criteria may warrant chemotherapy because of the underlying biology.

Synthetic lethality restricted to tumour cells — Poly-ADP ribose polymerase (PARP) inhibitor ^[2]

Mechanism from first principles:

1. Cells have two main DSB repair pathways: Homologous Recombination (HR) and Base Excision Repair (BER)
2. PARP enzyme is critical for BER (single-strand break repair)
3. In BRCA-deficient cells, HR is already non-functional
4. If you inhibit PARP as well → both repair pathways are blocked → the cell accumulates DNA damage → cell death
5. This is "synthetic lethality" — neither loss of BRCA alone nor PARP inhibition alone kills the cell, but the combination is lethal
6. Normal cells still have functional BRCA/HR → they survive PARP inhibition → selectivity for tumour cells

^[3][4][5]

Per the 2021 SAQ ^[6] and AOS Pathology ^[7]:

• BRCA1/BRCA2 germline mutation testing in high-grade serous ovarian CA → determines eligibility for PARP inhibitors and identifies families for screening
• MMR/MSI testing in endometrial cancer → Lynch syndrome screening + immunotherapy eligibility
• These require genetic counselling and informed consent (for germline tests) — distinguishing feature from somatic-only tests ^[7]

HER2 IHC score 3+ = positive → start trastuzumab. IHC 2+ = equivocal → reflex FISH testing. This is relevant because BRCA-associated breast cancers are typically HER2-negative (both BRCA1 and BRCA2), so trastuzumab is generally NOT part of their treatment regimen. ^[8]

Stem: "Molecular genetic tests are increasingly applied in diagnosis and management of gynaecological malignancies. Which of the following conditions is MOST OFTEN associated with an increase in susceptibility to ovarian and endometrial carcinomas?"

A. Ataxia telangiectasia B. DNA mismatch-repair / Lynch syndrome C. Familial adenomatous polyposis D. Peutz-Jeghers syndrome

Answer: B. DNA mismatch repair / Lynch syndrome

Rationale: Lynch syndrome (MMR gene defects) is the most common hereditary cause of both ovarian and endometrial carcinoma together. BRCA gives ovarian but NOT endometrial cancer (only "possible" association). FAP is colorectal. Ataxia telangiectasia is associated with lymphoma/leukaemia. Peutz-Jeghers gives GI and sex cord tumours. The discriminator is "both ovarian AND endometrial."

Stem: "A 68-year-old male has melanoma. The BRAF gene mutation status can be used as a biomarker to predict the treatment response to vemurafenib. Which assay is MOST USEFUL to detect BRAF mutations in the tumour biopsy?"

A. DNA Sequencing B. Fluorescence in-situ hybridisation C. Single nucleotide polymorphism microarray D. Southern blot

Answer: A. DNA Sequencing

Rationale: BRAF mutations are point mutations (e.g., V600E). DNA sequencing (Sanger or NGS) is the gold standard for detecting specific point mutations. FISH detects structural rearrangements/amplifications. SNP microarray detects copy number changes. Southern blot detects large genomic changes. This parallels the BRCA testing principle: sequencing for point mutations, MLPA for large deletions.

Stem: "With advances of knowledge in carcinogenesis and development of molecular diagnostic tools, molecular genetic testing has been more frequently applied in the diagnosis and management of malignancies. (a) Please list applications in gynaecological cancers. (6 marks) (b) Please list specific tests used in the above applications. (4 marks)"

Markscheme answer: (a) Applications:

1. Identifying genetic susceptibility/hereditary cancer syndromes (e.g., BRCA germline testing for HBOC, MMR for Lynch)
2. Diagnosis (e.g., somatic FOXL2 in adult granulosa cell tumour)
3. Prognosis prediction
4. Guiding targeted therapy (e.g., PARP inhibitor for BRCA-mutant ovarian cancer; immunotherapy for MSI-H endometrial cancer)
5. Screening (e.g., HPV testing for cervical cancer)
6. Monitoring treatment response / recurrence

(b) Specific tests:

1. NGS / Sanger sequencing (BRCA, MMR genes)
2. IHC for MMR proteins (MLH1, MSH2, MSH6, PMS2)
3. PCR for MSI
4. FISH (HER2 in breast cancer)
5. HPV DNA testing (PCR-based)

Stem: "A 58-year-old lady had a modified radical mastectomy done for a 5 cm breast mass. Pathology reported a grade 3 invasive ductal carcinoma with lymph node metastases. Immunohistochemistry for oestrogen and progesterone receptors was negative. HER2 immunohistochemistry was positive (score 3+). What is the next step that should be taken?"

A. Perform HER2 FISH test on the primary tumour B. Start the patient on adjuvant tamoxifen C. Start the patient on conventional adjuvant chemotherapy D. Start the patient on Trastuzumab (Herceptin) treatment

Answer: D. Start the patient on Trastuzumab (Herceptin) treatment

Rationale: HER2 IHC 3+ = definitive positive → no need for FISH (FISH is for equivocal 2+ results). ER/PR negative → tamoxifen useless. HER2-positive → trastuzumab is standard of care. Conventional chemo alone without trastuzumab would be suboptimal. Note: this patient is ER−/PR−/HER2+ — NOT triple negative. Triple negative = ER−/PR−/HER2−.

Stem: "A 50-year-old lady presented with a 2-month history of enlarging left breast lump... confirmed to be a triple-negative breast cancer... (c) After repeated investigation, the lesion was confirmed to be a triple-negative breast cancer and a repeated axillary ultrasonography and lymph node biopsy showed nodal involvement, please describe your next step of investigation. (1 mark)"

Answer: Staging investigations (CT chest/abdomen/pelvis or PET-CT) to assess for distant metastases before planning neoadjuvant chemotherapy. Consider BRCA germline testing given triple-negative breast cancer (per lecture, TNBC is an indication for BRCA testing regardless of family history).