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Procedure for Sending a Specimen for Immunohistochemistry (IHC)

1. Collection of the Specimen

• Obtain the biopsy using an appropriate technique (punch, incisional, excisional, shave, etc.) depending on the depth of the lesion and the clinical question.
• Handle the specimen with care using forceps to avoid crush artifact.

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2. Fixation — The Most Critical Step

Key distinction: IHC is performed on formalin-fixed, paraffin-embedded (FFPE) tissue — not fresh tissue, not frozen tissue, and not glutaraldehyde. Do not use Michel's medium (reserved for direct immunofluorescence) or saline for IHC specimens.

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3. Volume of Fixative

• The fixative volume should be approximately 10× the volume of the specimen to ensure adequate and even fixation.
• For small or thin specimens (e.g., punch biopsies): confirm the specimen is fully submerged in formalin — not adhering to the container walls or lid, which causes desiccation artifact.

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4. Container Labeling

• The specimen container must be pre-labeled with:
  • Patient's full name
  • Date of birth / hospital ID number
  • Date of collection
  • Biopsy site (anatomical location)
• If multiple biopsies are performed simultaneously, pre-label containers alphabetically with corresponding sites to prevent mix-ups.

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5. Completing the Requisition Form

• Patient demographics (name, age, sex, hospital/clinic ID)
• Clinical history and relevant diagnosis/differential
• Biopsy site and anatomical description
• Specific IHC markers requested (or "IHC as appropriate" if leaving to pathologist's discretion)
• Date of biopsy
• Clinician's name and contact
• Any prior pathology or relevant molecular testing

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6. Transport to the Laboratory

• Formalin-fixed specimens: can be transported at room temperature; no special cold-chain required.
• Fresh tissue (if the lab requires it — rare for IHC): transport on saline-moistened gauze and deliver promptly to a nearby laboratory.
• Frozen tissue (sent long-distance): ship on dry ice overnight.
• Establish a specimen tracking log within your practice to ensure results are received and acted upon.

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7. Laboratory Processing (for context)

1. Gross the specimen and place it in a cassette
2. Process through dehydration → clearing → paraffin embedding
3. Section at 4–5 µm on a microtome
4. Mount sections on charged (positively coated) glass slides
5. Perform antigen retrieval (heat-induced or enzymatic) — critical in FFPE tissue since formalin cross-links mask antigens
6. Apply primary antibody → secondary antibody → chromogen (usually immunoperoxidase/DAB, which gives a permanent brown reaction product visible under light microscopy)

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Key Pitfalls to Avoid

• Do NOT fix in glutaraldehyde (reserved for electron microscopy — destroys antigenicity for IHC)
• Do NOT fix in Michel's medium (reserved for direct immunofluorescence)
• Avoid over-fixation (>48 h in formalin can cause excessive cross-linking → poor antigen retrieval)
• Avoid under-fixation (< 6–8 h) or autolysis from delayed fixation
• Do NOT freeze specimens intended for routine IHC (unless requested by lab for frozen section IHC)
• Ensure specimen is not air-dried (desiccation artifact)

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Cost of Immunohistochemistry (IHC) in India

Per Marker / Per Block Pricing

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Practical Cost Breakdown

• A standard IHC panel for tumor diagnosis typically uses 3–6 markers. Total cost: ₹4,000 – ₹20,000 depending on the lab and number of markers.
• Some labs charge a base processing fee (block preparation, sectioning) of ₹500–₹1,500 separate from per-marker charges.
• Hormone receptor panel (ER, PR, HER2/neu) for breast cancer — a very common request — typically costs ₹3,000 – ₹8,000 for the full set.
• Ki-67 proliferation index alone: ₹800 – ₹2,500.

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Factors That Affect Cost

1. Number of markers — each additional antibody adds cost
2. Type of lab — government vs. private vs. super-specialty
3. City — metro cities (Mumbai, Delhi, Bengaluru) tend to be costlier than Tier 2/3 cities
4. Urgency — some labs charge extra for TAT (turnaround time) <48 h
5. Block availability — if the paraffin block must be retrieved from another centre, courier + retrieval fees apply
6. NABL/CAP accreditation — accredited labs may charge more but offer validated results

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Turnaround Time

• Routine: 3–7 working days
• Urgent: 24–48 hours (surcharge applies)

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Tips for Patients / Clinicians

• Always ask for a panel quote upfront, not just per-marker pricing.
• AIIMS and regional cancer institutes (TMC Mumbai, Kidwai Bengaluru, RGCI Delhi) offer significantly subsidised rates for referred patients.
• Many labs allow referral blocks — the FFPE block from one hospital can be sent to another lab for IHC without re-biopsy.

Note: Prices vary widely and change over time. Always confirm current rates directly with the laboratory before sending the specimen.

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IHC Markers for Endometrial and Cervical Carcinoma

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A. ENDOMETRIAL CARCINOMA

1. Diagnostic / Subtyping Panel

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2. Mismatch Repair (MMR) Panel — Mandatory in Endometrial Carcinoma

Loss of any one marker = mismatch repair deficient (dMMR) → warrants reflex microsatellite instability (MSI) testing and germline counselling

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3. POLE / Molecular Subtyping (TCGA-based)

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B. CERVICAL CARCINOMA

1. Distinguishing Squamous Cell Carcinoma vs. Adenocarcinoma vs. Endometrial Primary

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2. Cervical HPV-Related Markers

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3. Neuroendocrine Carcinoma of Cervix (rare, HPV+)

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Quick Summary: Endometrial vs. Endocervical — The Core Panel

The vimentin/ER/p16 combination is the most practical first-line panel to distinguish endometrial from endocervical primary in biopsy material.

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Uses of IHC in Endometrial Carcinoma

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1. Histological Subtyping (Type I vs. Type II)

Serous carcinoma has >90% TP53 mutations — IHC for p53 is the most reliable surrogate. Diffuse strong or completely absent (null) p53 staining = mutant p53 = aggressive behaviour and worse prognosis. — Goldman-Cecil Medicine, Goldman-Cecil Medicine

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2. Distinguishing Endometrial from Endocervical Primary

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3. Lynch Syndrome Screening (MMR IHC) — Now Recommended Universally

"Immunohistochemistry is frequently used because of the lower cost of this testing... All women who are diagnosed with endometrial cancer should undergo systematic clinical screening for Lynch syndrome and/or molecular screening." — Goldman-Cecil Medicine, Endometrial Cancer chapter

• ~20–40% of endometrioid carcinomas are dMMR (mismatch repair deficient)
• dMMR tumours have ~1700 mutations on average vs. 70 in typical cancers → generate neoantigens → recognised by the immune system
• dMMR = predictive biomarker for pembrolizumab (anti-PD-1) and dostarlimab — both approved for dMMR endometrial carcinoma
• Loss of MLH1 in the absence of germline mutation → MLH1 promoter hypermethylation (sporadic)
• Loss of MSH2 or MSH6 → suspect germline Lynch syndrome → refer for genetic counselling
• Lynch syndrome women have 40% lifetime risk of endometrial cancer and 10% risk of ovarian cancer

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4. TCGA Molecular Subgrouping (via IHC Surrogates)

"Suggested schema for molecular classification of endometrial cancer using sequencing and immunohistochemistry results to segregate patients into the molecular subtypes defined by The Cancer Genome Atlas (TCGA)." — Goldman-Cecil Medicine, Fig. 184-1

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5. Hormone Receptor Status — Guiding Hormonal Therapy

• ER / PR positivity → patient may benefit from progestins (medroxyprogesterone acetate) or aromatase inhibitors
• Used particularly in patients unfit for surgery or with recurrent/metastatic disease
• Type I tumors: ER/PR positive → better response to hormonal therapy and better overall prognosis
• Type II tumors: ER/PR negative → hormonal therapy not useful

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6. HER2/neu (ERBB2) — Targeted Therapy Selection

• HER2 overexpression (3+ by IHC or amplified by FISH) is seen in ~25–30% of serous endometrial carcinomas
• Trastuzumab (anti-HER2) combined with chemotherapy has shown improved survival in HER2-positive uterine serous carcinoma
• Type II endometrial cancers are characterized at the molecular level by ERBB2 overexpression alongside p53 mutations

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Summary Table

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Is IHC Necessary in FIGO Stage IA Endometrial Carcinoma?

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What is FIGO Stage IA?

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Why IHC is Still Indicated in Stage IA

1. Universal MMR IHC — Recommended for ALL Endometrial Carcinomas, Including Stage IA

"All women who are diagnosed with endometrial cancer should undergo systematic clinical screening for Lynch syndrome and/or molecular screening. Immunohistochemistry is frequently used because of the lower cost of this testing." — Goldman-Cecil Medicine

• Lynch syndrome affects 40% lifetime risk of endometrial cancer in mutation carriers
• Lynch syndrome is not predicted by stage — it can present as Stage IA, low-grade tumour
• Identifying a Lynch syndrome patient through Stage IA testing protects the patient (surveillance for colorectal, ovarian, urinary tract cancers) and her family (cascade testing)
• dMMR also identifies patients eligible for pembrolizumab / dostarlimab if they recur or have advanced disease
• In Sweden, England, and Australia, universal IHC testing of all endometrial cancers is now standard practice [PMID: 39175077, PMID: 39433398]

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2. Molecular Subtyping (p53 IHC) — Prognostic Even in Stage IA

A Stage IA tumour that appears "low risk" morphologically may carry a p53 mutation (serous-like), which significantly changes management and prognosis. IHC for p53 unmasks this.

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3. Histological Confirmation / Subtype Clarification

• Confirm endometrioid vs. serous vs. clear cell when morphology is equivocal
• Exclude a metastasis to the uterus (breast, colon, ovary)
• Identify carcinosarcoma (aggressive, requires IHC to characterise both components)

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4. When IHC May Be Omitted in Stage IA

• Unequivocally Grade 1 endometrioid on H&E
• Clinically/surgically confirmed Stage IA
• Detailed family history negative for Lynch syndrome criteria

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Summary

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Is PET Scan Necessary for Stage I Endometrial Carcinoma?

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What the Guidelines Say

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Why PET is NOT Routinely Indicated in Stage I

1. Low pre-test probability of distant metastasis

• Stage I disease is, by definition, confined to the uterus
• In Stage IA, Grade 1–2 endometrioid carcinoma, the risk of lymph node metastasis is only ~3–5%
• PET/CT adds little clinical value when metastatic disease is unlikely

2. Low sensitivity for small/microscopic nodal disease

• PET/CT detects lymph node metastases only when nodes are FDG-avid and ≥~8–10 mm
• Micrometastases (the relevant risk in Stage I) are missed by PET
• Sensitivity for nodal disease in early endometrial cancer: ~50–70% — insufficient to replace surgical staging
• Sentinel lymph node (SLN) biopsy at the time of surgery is superior to PET for nodal assessment

3. MRI is the investigation of choice for local staging

• Pelvic MRI (with DWI — diffusion-weighted imaging) is the standard preoperative investigation
• MRI accurately assesses:
  • Depth of myometrial invasion (differentiates Stage IA from IB)
  • Cervical stromal invasion (Stage II)
  • Parametrial extension
• This directly guides surgical planning — PET cannot replace MRI for local staging

4. Cost, radiation, and false positives

• PET/CT involves significant radiation exposure and cost
• False-positive FDG uptake in benign uterine pathology (fibroids, adenomyosis, post-biopsy inflammation) is common
• Unnecessary PET leads to further investigations and patient anxiety

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When PET/CT IS Indicated in Endometrial Carcinoma

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Recommended Preoperative Workup for Stage I

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Summary

PET/CT is not a standard or necessary investigation for Stage I endometrial carcinoma. It does not improve surgical planning, cannot reliably detect micrometastases, and adds cost and radiation without benefit. Pelvic MRI is the recommended staging modality. PET/CT is reserved for suspected recurrence, advanced disease, or high-risk histotypes where distant metastatic workup is clinically indicated.

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When to Do PET Scan Post-Surgery in Stage I Endometrial Carcinoma

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Standard Post-Surgical Surveillance for Stage I (No PET)

Imaging (CT, MRI, PET) is not part of routine surveillance in asymptomatic Stage I patients — it has not been shown to improve survival in this setting.

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Specific Triggers to Order PET Post-Surgery in Stage I

1. Symptoms Suggestive of Recurrence

• Vaginal bleeding or discharge post-hysterectomy
• Pelvic/abdominal pain
• New urinary or bowel symptoms
• Unexplained weight loss, fatigue, or anorexia

2. Abnormal Clinical Findings

• Palpable vaginal vault mass or nodule on examination
• Abnormal cytology on vault smear
• Rising CA-125 (if monitored)

3. Indeterminate Findings on Routine CT or MRI

• A lesion on CT/MRI that is equivocal — PET/CT helps confirm whether it is metabolically active (recurrent disease) vs. post-surgical change

4. Upstaging After Final Histopathology

• If final pathology upstages the tumour (e.g., previously thought Stage I but LVSI+, high grade, or positive margins found) → PET/CT appropriate to rule out residual/nodal disease before adjuvant therapy

5. Suspected Isolated Recurrence Being Considered for Salvage Treatment

• Before salvage radiotherapy or surgery for presumed isolated vaginal/pelvic recurrence — PET/CT is essential to exclude distant metastases first

• "A search for distant recurrences is obligatory, as patients with metastatic disease are best treated with chemotherapy." — Berek & Novak's Gynecology

6. dMMR / MSI-H Tumour Progressing — Before Immunotherapy

• If the patient is being considered for pembrolizumab / dostarlimab for recurrent dMMR disease — PET/CT documents extent of disease at baseline

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Why Routine Post-Operative PET is NOT Done in Stage I

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Recurrence Risk Profile — Stage I at a Glance

High-grade Stage I (G3, serous, carcinosarcoma) patients warrant closer imaging follow-up — CT chest/abdomen at 6–12 months is often done; PET if symptoms arise.

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Summary