Laboratory Diagnostic Tests of Cancer

1. Morphologic / Histologic Methods

A. Biopsy and Histologic Examination (H&E Staining)

• Tissue is removed (excisional biopsy, incisional biopsy, or core needle biopsy), fixed in formalin, sectioned, and stained with hematoxylin and eosin.
• Allows assessment of tumor architecture, cell morphology, and degree of anaplasia (loss of differentiation).
• Frozen section - freshly removed tissue is quick-frozen and sectioned for histology within minutes. Used intraoperatively to assess surgical margins or lymph node status. Standard permanent sections provide superior detail when critical decisions (e.g., amputation) are at stake.
• Clinical data (radiation exposure, fracture history, imaging findings) must accompany specimens - radiation changes can mimic cancer, healing fractures can mimic osteosarcoma.

B. Fine Needle Aspiration (FNA)

• A small-bore needle aspirates cells from palpable or image-guided masses.
• Cells are spread on a slide, stained, and examined cytologically.
• Widely used for breast, thyroid, lymph node, salivary gland, liver, pancreas, and pelvic lymph node lesions.
• Advantages: minimally invasive, rapid, avoids surgery.
• Limitations: small sample, potential sampling error; requires experienced hands.

C. Cytologic Smears (Papanicolaou/Pap Test)

• Exfoliated cells from surfaces or fluids are collected, smeared, fixed, and stained.
• Classic use: cervical carcinoma screening (Pap smear) - the best testament to the value of cytology in cancer control.
• Also used for: urine (bladder cancer), bronchial washings/brushings (lung cancer), sputum, CSF, pleural/peritoneal effusions, endometrial cells.
• Cancer cells show features of anaplasia: nuclear pleomorphism, increased N:C ratio, irregular chromatin, prominent nucleoli, abnormal mitoses.

2. Immunohistochemistry (IHC)

3. Tumor Markers (Serum / Biochemical Tests)

Important: Biochemical tumor markers lack the sensitivity and specificity for definitive cancer diagnosis on their own. Their main value is in monitoring response to therapy and detecting recurrence. With successful tumor resection, markers disappear; their reappearance almost always signals recurrence.

Major Tumor Markers

4. Flow Cytometry

• Rapidly and quantitatively measures multiple cell characteristics simultaneously using fluorescently labeled antibodies.
• Requires viable cells in suspension.
• Primarily used for hematologic malignancies: B-cell and T-cell lymphomas, leukemias (CLL, AML, ALL).
• Multiple antigens (CD markers) assessed simultaneously on individual cells - this is an advantage over IHC.
• Identifies cell lineage, differentiation stage, and aberrant antigen expression patterns that define specific leukemia/lymphoma subtypes.
• Also used for DNA ploidy analysis (aneuploidy suggests malignancy) and S-phase fraction (proliferative index).

5. Molecular Diagnostic Methods

A. Polymerase Chain Reaction (PCR)

• Lymphoma/leukemia diagnosis: Each B/T cell has unique antigen receptor gene rearrangements. PCR detects clonal (neoplastic) vs. polyclonal (reactive) lymphocyte populations.
• Specific translocations: BCR-ABL transcripts confirm CML diagnosis; JAK2 V617F mutations confirm polycythemia vera.
• Minimal residual disease (MRD) detection: Ultra-sensitive PCR detects residual BCR-ABL transcripts after CML treatment, guiding therapy decisions.

B. Fluorescence In Situ Hybridization (FISH)

• Detects specific chromosomal translocations, deletions, and amplifications.
• Examples:
  • t(11;22) in Ewing sarcoma
  • t(14;18) in follicular lymphoma
  • HER2 (ERBB2) gene amplification in breast cancer (guides trastuzumab therapy)
  • MYCN amplification in neuroblastoma (poor prognosis)
  • ALK rearrangements in lung adenocarcinoma and anaplastic large cell lymphoma

C. DNA Sequencing / Next Generation Sequencing (NGS)

• Sequencing of cancer genomes is now routine in many centers.
• Identifies point mutations in cancer driver genes: TP53, KRAS, BRAF, EGFR, PIK3CA, etc.
• Predicts prognosis (TP53 mutation → poor outcome in many cancers).
• Identifies druggable targets: EGFR mutations (erlotinib/gefitinib), BRAF V600E (vemurafenib), etc.

D. Liquid Biopsy

• Detection of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) in peripheral blood.
• Non-invasive; useful for monitoring treatment response and detecting early resistance.
• EGFR T790M mutation in plasma - guides second-line therapy in NSCLC.
• Stool DNA testing (APC, KRAS, RAS mutations + occult blood) - FDA-approved for colorectal cancer screening (Cologuard).

E. Microarray / Gene Expression Profiling

• Measures expression of thousands of genes simultaneously.
• Used for prognosis: Oncotype DX (21-gene assay) in breast cancer predicts recurrence risk and guides chemotherapy decisions; MammaPrint (70-gene signature).
• Identifies molecular subtypes of lymphoma (GCB vs. activated B-cell DLBCL).

6. Grading and Staging (Laboratory-Assisted)

• Grading (I-IV) assesses degree of differentiation and mitotic activity - done histologically.
• Staging (TNM system) assesses extent of spread - uses histology of biopsies (sentinel lymph node biopsy) and molecular markers.

7. Special Stains and Cytochemistry

8. Microbiology / Serology in Cancer Diagnosis

• HPV testing (PCR / hybrid capture): cervical cancer screening alongside Pap smear.
• H. pylori testing: gastric cancer / MALT lymphoma.
• EBV serology/PCR: EBV-associated Burkitt lymphoma, nasopharyngeal carcinoma, post-transplant lymphoproliferative disorder.
• HBV/HCV serology: hepatocellular carcinoma risk.

Summary: Which Test for Which Purpose