QUESTION 2 (20 Marks)

Diagnosis of Small Round Cell Tumours (SRCTs)

Introduction

The Main Entities in the Differential Diagnosis

Diagnostic Approach

1. Morphology (H&E)

• Ewing sarcoma / PNET: Sheets of uniform small round blue cells with clear cytoplasm (glycogen); Homer-Wright rosettes in PNET
• Rhabdomyosarcoma: Cells with eosinophilic cytoplasm, "tadpole" or "strap" rhabdomyoblasts; alveolar pattern in ARMS
• Neuroblastoma: Homer-Wright pseudorosettes; neuropil background; variable schwannian stroma
• Lymphoblastic lymphoma: Convoluted/non-convoluted nuclei, "starry sky" pattern
• DSRCT: Nests of round cells surrounded by abundant desmoplastic stroma

2. Immunohistochemistry (IHC) - The Cornerstone

• CD99 strong membranous positivity is the hallmark of Ewing sarcoma but is not specific
• Co-expression of desmin + WT1 (C-terminal) + keratin (dot-like) in a desmoplastic background is virtually diagnostic of DSRCT
• Myogenin is more specific for RMS than MyoD1

3. Cytogenetics and Molecular Studies

4. Electron Microscopy (EM)

• RMS: Thick and thin filaments, Z-band material, primitive sarcomeres
• Neuroblastoma: Dense-core neurosecretory granules, neuritic processes
• Ewing sarcoma: Abundant glycogen particles, absence of specific organelles

5. Ancillary Techniques

• FISH: Now preferred for detecting chromosomal translocations
• RT-PCR: Detects gene fusion transcripts (very sensitive)
• Next-generation sequencing (NGS) / RNA sequencing: Particularly useful for cases with atypical fusions or when FISH/PCR are equivocal. RNA-seq can detect novel fusion partners
• Cytology and cell blocks: FNA with cell blocks allow IHC on limited material; useful in effusion cytology (see Han et al., Acta Cytol 2022, PMID: 34218227)

6. A Practical Diagnostic Algorithm

1. Perform H&E morphology assessment
2. First-line IHC panel: CD99, LCA, desmin, CD56/NSE, synaptophysin, chromogranin
3. If CD99+: add vimentin, NKX2.2 (Ewing-specific transcription factor) - strong NKX2.2 supports Ewing
4. If myogenic markers considered: MyoD1, myogenin, SMA
5. Confirm with FISH for EWSR1 rearrangement, PAX-FOXO1
6. If inconclusive: NGS/RNA-seq panel

QUESTION 3 (20 Marks)

Prognostic and Predictive Molecular Markers in Breast Carcinoma, with Emphasis on Recent HER2 Guidelines

Introduction

• Prognostic markers: predict natural history and outcome regardless of treatment
• Predictive markers: predict response (or lack of response) to a specific therapy

1. Estrogen Receptor (ER) and Progesterone Receptor (PR)

• ER: Assessed by IHC; positive if ≥1% of tumour cells stain (ASCO/CAP threshold)
• ER positivity is both prognostic (associated with lower grade, better survival) and predictive (predicts response to endocrine therapy: tamoxifen, aromatase inhibitors, fulvestrant)
• PR: Adds prognostic value; low/absent PR in an ER+ tumour suggests Luminal B biology and worse prognosis
• ER/PR status determines eligibility for CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) in metastatic HR+ disease

2. HER2 (Human Epidermal Growth Factor Receptor 2 / ErbB2 / c-erbB-2)

HER2 Testing Methods

• 0: No staining or faint/incomplete staining in ≤10% cells
• 1+: Faint/incomplete membranous staining in >10% cells
• 2+: Weak to moderate, complete membranous staining in >10% cells (equivocal - reflex ISH)
• 3+: Strong complete circumferential membranous staining in >10% cells (positive)

3. Recent HER2 Guidelines: The 2023 ASCO/CAP Update and the HER2-Low Revolution

The DESTINY-Breast04 Trial (Modi et al., NEJM 2022)

• ~50% of all breast cancers that were classified as "HER2-negative" actually fall in the HER2-low category
• These patients, previously ineligible for HER2-targeted therapy, now have a proven targeted option

The New HER2 Classification (2023 ASCO/CAP + ESMO Consensus)

1. Accurate discrimination between IHC 0 and IHC 1+ is now clinically critical (previously clinically irrelevant)
2. Standardized pre-analytical and analytical procedures are mandatory
3. AI-assisted image analysis is emerging to support accurate low-spectrum HER2 scoring
4. Intratumoral heterogeneity is a challenge - HER2-low status may change between primary and metastatic sites

4. Ki-67 (Proliferation Index)

• Type: Prognostic (and partly predictive for chemotherapy benefit)
• Assessed by IHC using MIB-1 antibody; expressed as % of positive cells
• High Ki-67 (>20-30%) = Luminal B subtype, worse prognosis, benefits more from chemotherapy
• Also predicts response to neoadjuvant chemotherapy

5. Molecular Subtypes and Gene Expression Profiling

• Oncotype DX (21-gene recurrence score): Predicts recurrence risk and chemotherapy benefit in ER+/HER2- early breast cancer
• MammaPrint (70-gene signature): Predicts distant recurrence in early-stage breast cancer (MINDACT trial validated)
• Prosigna (PAM50): Assigns molecular subtype and provides ROR (Risk of Recurrence) score
• EndoPredict: Predicts late recurrence under endocrine therapy

6. Other Emerging Predictive Markers

Summary Table: Prognostic vs. Predictive in Breast Cancer

Key References

• Robbins, Cotran & Kumar Pathologic Basis of Disease - Chapter 23 (Breast)
• Fischer's Mastery of Surgery 8e - Breast Cancer Subtypes
• Current Surgical Therapy 14e - HER2-Low Breast Cancer
• Henry's Clinical Diagnosis and Management - Breast Cancer Molecular Markers
• Ivanova M et al. "Standardized pathology report for HER2 testing in compliance with 2023 ASCO/CAP updates and 2023 ESMO consensus statements on HER2-low breast cancer." Virchows Arch 2024 Jan. [PMID: 37770765]
• DESTINY-Breast04: Modi S et al. NEJM 2022;387:9-20

QUESTION 4 - Short Notes (40 Marks Total)

A. Molecular Classification of Endometrial Carcinoma

Background

The Four TCGA Molecular Subtypes

Subtype Details

1. POLE Ultra-Mutated (POLEmut)

• Mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE) - most commonly P286R, V411L
• Results in extremely high somatic mutation burden (ultramutated: >100 mutations/Mb)
• High tumour-infiltrating lymphocytes (TILs) - immune-hot tumour
• Despite often being grade 3 and with high-risk histological features, prognosis is EXCELLENT - this is the key paradox
• Now recognized in the 2023 ESGO/ESTRO/ESP guidelines to allow de-escalation of adjuvant therapy
• May respond well to immune checkpoint inhibitors (due to high mutational burden)

2. MMR-Deficient / MSI-High (MMRd)

• Loss of MMR proteins (MLH1, MSH2, MSH6, PMS2) detectable by IHC
• ~80% due to MLH1 promoter hypermethylation (sporadic); ~20% due to Lynch syndrome germline mutations
• All patients with MMRd EC should be screened for Lynch syndrome (germline testing)
• MSI-H tumours are eligible for pembrolizumab (anti-PD-1) - FDA approved in MSI-H solid tumours
• Dostarlimab is approved for MMRd recurrent/advanced EC (RUBY trial)

3. No Specific Molecular Profile (NSMP) / Copy Number Low

• Largest group; characterized by low copy number variation and absence of POLE mutations, MMR loss, or p53 abnormality
• Usually ER/PR positive, low-grade endometrioid
• Intermediate prognosis
• Hormone receptor positivity suggests benefit from endocrine therapy in advanced/metastatic disease

4. p53 Abnormal / Copy Number High (CNH)

• TP53 mutations are the defining feature; detected by IHC (aberrant/null p53 expression)
• High somatic copy number alterations (SCNA), chromosomal instability
• Biologically similar to high-grade serous ovarian cancer
• Worst prognosis - highest risk of recurrence and distant metastasis
• May benefit from platinum-based chemotherapy (similar to HGSOC)
• Emerging use of PARP inhibitors and bevacizumab

Practical Implementation - The ProMisE Algorithm

Step 1: POLE sequencing (hotspot mutations in exons 9, 13, 14)
   → POLEmut → POLE ultra-mutated subtype

Step 2: MMR IHC (MLH1, PMS2, MSH2, MSH6)
   → Any MMR loss → MMRd subtype

Step 3: p53 IHC (TP53 aberrant expression)
   → Aberrant p53 → p53abn subtype

Step 4: All negative
   → NSMP subtype

Clinical Relevance (2023 ESGO/ESTRO/ESP Guidelines Integration)

• POLEmut: De-escalate therapy - may omit adjuvant radiotherapy even for grade 3 tumours
• MMRd: Escalate immunotherapy; Lynch syndrome counselling
• p53abn: Escalate adjuvant chemotherapy + radiotherapy
• NSMP: Standard risk stratification based on histological features

B. Recent Advances in Classification of MDS (Myelodysplastic Syndromes/Neoplasms)

Background

The Two 2022 Classification Systems

WHO 5th Edition (2022) - MDS Entities

Major Categories:

Key Advances in 2022 vs. Prior WHO 2016 Classification

1. Genetics Now Drive Classification

• SF3B1 mutations are sufficient to diagnose MDS-SF3B1 without requiring ≥15% ring sideroblasts (old threshold)
• TP53 mutations: Biallelic TP53 mutations (or mono-allelic with VAF >10%) now define a high-risk entity with very poor prognosis
• Spliceosome mutations (SF3B1, SRSF2, U2AF1, ZRSR2) - inform prognosis
• Epigenetic mutations (TET2, DNMT3A, IDH1/2, EZH2, ASXL1) - frequently mutated in MDS

2. "Clonal Cytopenia of Undetermined Significance" (CCUS)

• New pre-malignant entity: cytopenia + somatic mutation but no dysplasia diagnostic of MDS
• Distinguishes from CHIP (clonal haematopoiesis of indeterminate potential - no cytopenia)
• Higher risk of progression to overt MDS/AML than CHIP

3. Blast Count Changes

• The distinction between AML (≥20% blasts) and MDS-IB2 (10-19%) was refined
• MDS with Auer rods at any blast percentage is classified as MDS-IB2 (regardless of blast count if <10%)

4. Hypoplastic MDS

• Now a distinct entity (MDS-h); recognizing overlap with immune-mediated aplastic anaemia
• Important therapeutic implication: may respond to immunosuppressive therapy

5. Revised IPSS-M (Molecular IPSS)

• 31 gene mutations in addition to traditional IPSS-R parameters (cytopenias, blast %, cytogenetics)
• Provides more accurate risk stratification than IPSS-R alone
• Categories: Very Low, Low, Moderate-Low, Moderate-High, High, Very High

6. Therapeutic Implications of New Classification

• MDS-SF3B1: Luspatercept (activin receptor ligand trap) - FDA approved; reduces transfusion burden
• MDS-5q: Lenalidomide remains standard
• High-risk MDS (IB1/IB2): Hypomethylating agents (azacitidine, decitabine); allo-HSCT
• TP53-mutant MDS: APR-246 (eprenetapopt) - in trials; poor prognosis
• IDH1/IDH2 mutant: Ivosidenib/enasidenib - targeted therapy

C. PD-1/PD-L1 Axis and Its Score in Immunotherapy

The PD-1/PD-L1 Pathway - Biology

• A type I transmembrane receptor belonging to the CD28 superfamily
• Expressed on activated T cells, B cells, NK cells, monocytes, and dendritic cells
• Acts as an inhibitory checkpoint receptor - its natural physiological role is to prevent autoimmunity by dampening T-cell activity

• Expressed on tumour cells, tumour-infiltrating immune cells, and normal tissues
• Also expressed constitutively on many normal tissues to protect against autoimmunity

• Second ligand for PD-1; primarily expressed on dendritic cells and macrophages

Mechanism of the PD-1/PD-L1 Axis

Normal Physiological Role:

Tumour Immune Evasion:

Molecular Signalling (When PD-1 is Ligated):

PD-1 + PD-L1 binding
       ↓
Phosphorylation of ITSM motif in PD-1 cytoplasmic tail
       ↓
Recruitment of SHP-1/SHP-2 phosphatases
       ↓
Dephosphorylation of CD28 and ZAP-70
       ↓
Inhibition of PI3K/Akt and RAS/ERK pathways
       ↓
T cell exhaustion: ↓ IL-2, IFN-γ, TNF-α production
                   ↓ T cell proliferation
                   ↓ Cytotoxic activity

Checkpoint Inhibitors Targeting PD-1/PD-L1

PD-L1 Scoring in Immunotherapy

1. Tumour Proportion Score (TPS)

• Definition: Percentage of viable tumour cells showing partial or complete membrane staining for PD-L1, regardless of staining intensity
• Formula: TPS = (number of PD-L1+ tumour cells / total viable tumour cells) × 100
• Used in: NSCLC (22C3 assay - Pembrolizumab); also HNSCC, cervical, gastric cancer
• Thresholds in NSCLC:
  • TPS ≥50%: Pembrolizumab monotherapy first-line (KEYNOTE-024)
  • TPS 1-49%: Pembrolizumab + chemotherapy (KEYNOTE-189)
  • TPS <1%: Pembrolizumab not recommended as monotherapy

2. Combined Positive Score (CPS)

• Definition: Number of PD-L1 staining cells (tumour cells + lymphocytes + macrophages) divided by total viable tumour cells, multiplied by 100
• Formula: CPS = (PD-L1+ tumour cells + PD-L1+ lymphocytes + PD-L1+ macrophages) / total viable tumour cells × 100
• Maximum possible CPS = unlimited (can exceed 100)
• Used in:
  • Gastric/GEJ cancer: CPS ≥1 for nivolumab (CheckMate-649); CPS ≥5 for pembrolizumab (KEYNOTE-590)
  • Cervical cancer: CPS ≥1 for pembrolizumab (KEYNOTE-826)
  • TNBC: CPS ≥10 for pembrolizumab (KEYNOTE-522)
  • HNSCC: CPS ≥1 for pembrolizumab (KEYNOTE-048)
  • Endometrial cancer (MMR-proficient): CPS ≥10 for pembrolizumab + lenvatinib (KEYNOTE-146)
  • Esophageal SCC: CPS ≥10 for pembrolizumab

3. Tumour Area Positivity (TAP) / IC Score

• IC score (Immune Cell score): Percentage of tumour-infiltrating immune cells staining for PD-L1
• Used with atezolizumab in urothelial carcinoma (SP142 assay) and TNBC (IMpassion130)
• IC 1%: Cut-off for atezolizumab benefit in TNBC

Companion Diagnostic Assays - Not Interchangeable

Predictive vs. Prognostic Value of PD-L1

• PD-L1 is primarily a predictive biomarker (predicts response to anti-PD-1/PD-L1 therapy)
• It has limited prognostic value on its own
• Important limitations: PD-L1 expression is heterogeneous (intratumoral and intertumoral), changes with therapy, differs between primary and metastatic sites, and does not always predict response - some PD-L1-negative tumours still respond to immunotherapy (especially MSI-H tumours)

D. Role of Quality Control (QC) in the Hematology Laboratory

Definition

Why QC is Critical in Haematology

• Unnecessary/missed transfusions
• Missed leukaemia diagnoses
• Incorrect chemotherapy dosing
• Sepsis under/over-treatment

Types of QC in the Hematology Laboratory

1. Internal Quality Control (IQC)

• Whole blood controls at 3 levels: low (anaemia range), normal, and high (polycythaemia/thrombocytosis range)
• Run at the beginning of each shift, after instrument maintenance, after reagent change, and after calibration
• Commercially stabilized blood cells (e.g., Bio-Rad Liquichek, Sysmex XN-series controls)

• Graphical representation of QC results over time plotted against mean ± SD limits
• Allow visual detection of trends, shifts, and random errors
• Trend: Progressive drift in one direction over ≥5 consecutive points
• Shift: Abrupt change to a new mean level

• Used for CBC parameters; calculates moving average of patient RBC indices (MCV, MCH, MCHB) over batches of 20 patients
• Flags instrument drift without using external controls
• Particularly useful for MCV, MCH, MCHB - these are physically stable indices

2. External Quality Assessment (EQA) / Proficiency Testing

• PT/EQA schemes (e.g., UKNEQAS for haematology, CAP proficiency testing, EQAS)
• Unknown samples distributed to participating laboratories; results compared to peer group
• Assesses accuracy (agreement with peer laboratories and assigned target values)
• Identifies systematic biases not detected by IQC
• Mandatory for accreditation (ISO 15189, CAP, NABL)

3. Delta Checks (Patient-Based QC)

• Compares current patient result with the same patient's previous result
• Alerts generated when change exceeds a defined delta limit
• Useful for detecting specimen labelling errors (wrong patient), hemolysis, and dilution errors
• Example: Hb dropping by >3 g/dL without clinical explanation triggers a delta check flag

4. Morphological QC - Blood Film Review

• Mandatory peripheral blood film review when CBC flags are triggered (blast flags, variant lymphocyte flags, platelet clumping alerts, immature granulocyte flags)
• ICSH guidelines define criteria for mandatory blood film review
• Internal QC for morphology: use of documented reference images, proficiency testing slides, and standardized reporting terminology

QC for Specific Hematology Instruments

Automated Haematology Analyzers (e.g., Sysmex XN, Beckman DxH, Abbott CELL-DYN)

• CBC parameters: Hb, RBC, WBC, PLT, MCV, MCH, MCHB, HCT, RDW
• Differential counts: 5-part or 3-part WBC differential
• Reticulocyte counts
• Extended parameters: IPF (Immature Platelet Fraction), RET-He, IG%

• Fresh whole blood calibrators traceable to international reference methods (ICSH/CLSI standards)
• Hb calibrated using the cyanmethaemoglobin reference method (HiCN)
• RBC/PLT calibrated using reference photometric methods

Coagulation Analyzers (PT, APTT, Fibrinogen, D-dimer)

• Daily IQC with commercial plasma controls at normal and abnormal levels
• INR calibration using International Sensitivity Index (ISI) of the thromboplastin reagent
• Special QC for anti-Xa levels in LMWH monitoring

Point-of-Care Testing (POCT) - e.g., bedside Hb, INR, ABG analyzers

• Requires its own QC program separate from the central laboratory
• Electronic QC (e-QC) and liquid QC
• Staff competency assessment essential

QC Documentation and Regulatory Requirements

Six Sigma in the Hematology Laboratory

• Sigma metric = (TEa - bias) / CV
  • TEa = Total Allowable Error (from CLIA, biological variation databases)
  • Bias = from EQA or method comparison
  • CV = coefficient of variation from IQC

Key References

• Kumari A et al. "Molecular Classification of Endometrial Carcinomas: Review and Recent Updates." Cancers 2025. [PMID: 41514564]
• Xiao W et al. "A practical approach on the classifications of myeloid neoplasms and acute leukemia: WHO and ICC." J Hematol Oncol 2024. [PMID: 39075565]
• Ning Y et al. "Cytogenetics and molecular genetics of myelodysplastic neoplasms." Best Pract Res Clin Haematol 2023. [PMID: 38092472]
• Sabiston Textbook of Surgery (Section: Immunotherapy) - PD-L1 CPS scoring
• WHO Classification of Haematolymphoid Tumours (5th edition), 2022
• TCGA Research Network. Integrated genomic characterization of endometrial carcinoma. Nature. 2013