Biomarker Classification and Interpretation Guide

Overview

Biomarkers are measurable indicators of biological state or condition. In clinical decision support, biomarkers guide diagnosis, prognosis, treatment selection, and monitoring. This guide covers genomic, proteomic, and molecular biomarkers with emphasis on clinical actionability.

Biomarker Categories

Prognostic Biomarkers

Definition: Predict clinical outcome (survival, recurrence) regardless of treatment received

Examples by Disease

Cancer

• Ki-67 index: High proliferation (>20%) predicts worse outcome in breast cancer
• TP53 mutation: Poor prognosis across many cancer types
• Tumor stage/grade: TNM staging, histologic grade
• LDH elevation: Poor prognosis in melanoma, lymphoma
• AFP elevation: Poor prognosis in hepatocellular carcinoma

Cardiovascular

• NT-proBNP/BNP: Elevated levels predict mortality in heart failure
• Troponin: Predicts adverse events in ACS
• CRP: Inflammation marker, predicts cardiovascular events

Infectious Disease

• HIV viral load: Predicts disease progression if untreated
• HCV genotype: Predicts treatment duration needed

Application: Risk stratification, treatment intensity selection, clinical trial enrollment

Predictive Biomarkers

Definition: Identify patients likely to benefit (or not benefit) from specific therapy

Positive Predictive Biomarkers (Treatment Benefit)

Oncology - Targeted Therapy

• EGFR exon 19 del/L858R → EGFR TKIs: Response rate 60-70%, PFS 10-14 months
• ALK rearrangement → ALK inhibitors: ORR 70-90%, PFS 25-34 months
• HER2 amplification → Trastuzumab: Benefit only in HER2+ (IHC 3+ or FISH+)
• BRAF V600E → BRAF inhibitors: ORR 50%, PFS 6-7 months (melanoma)
• PD-L1 ≥50% → Pembrolizumab: ORR 45%, PFS 10 months vs 6 months (chemo)

Oncology - Immunotherapy

• MSI-H/dMMR → Anti-PD-1: ORR 40-60% across tumor types
• TMB-high → Immunotherapy: Investigational, some benefit signals
• PD-L1 expression → Anti-PD-1/PD-L1: Higher expression correlates with better response

Hematology

• BCR-ABL → Imatinib (CML): Complete cytogenetic response 80%
• CD20+ → Rituximab (lymphoma): Benefit only if CD20-expressing cells
• CD33+ → Gemtuzumab ozogamicin (AML): Benefit in CD33+ subset

Negative Predictive Biomarkers (Resistance/No Benefit)

• KRAS mutation → Anti-EGFR mAbs (CRC): No benefit, contraindicated
• EGFR T790M → 1st/2nd-gen TKIs: Resistance mechanism, use osimertinib
• RAS/RAF wild-type required → BRAF inhibitors (melanoma): Paradoxical MAPK activation

Diagnostic Biomarkers

Definition: Detect or confirm presence of disease

Infectious Disease

• PCR for pathogen DNA/RNA: SARS-CoV-2, HIV, HCV viral load
• Antibody titers: IgM (acute), IgG (prior exposure/immunity)
• Antigen tests: Rapid detection (strep, flu, COVID)

Autoimmune

• ANA: Screen for lupus, connective tissue disease
• Anti-CCP: Specific for rheumatoid arthritis
• Anti-dsDNA: Lupus, correlates with disease activity
• ANCA: Vasculitis (c-ANCA for GPA, p-ANCA for MPA)

Cancer

• PSA: Prostate cancer screening/monitoring
• CA 19-9: Pancreatic cancer, biliary obstruction
• CEA: Colorectal cancer monitoring
• AFP: Hepatocellular carcinoma, germ cell tumors

Pharmacodynamic Biomarkers

Definition: Assess treatment response or mechanism of action

Examples

• HbA1c: Glycemic control in diabetes (target <7% typically)
• LDL cholesterol: Statin efficacy (target <70 mg/dL in high-risk)
• Blood pressure: Antihypertensive efficacy (target <130/80 mmHg)
• Viral load suppression: Antiretroviral efficacy (target <20 copies/mL)
• INR: Warfarin anticoagulation monitoring (target 2-3 for most indications)

Genomic Biomarkers

Mutation Analysis

Driver Mutations (Oncogenic)

• Activating mutations: Constitutive pathway activation (BRAF V600E, EGFR L858R)
• Inactivating mutations: Tumor suppressor loss (TP53, PTEN)
• Hotspot mutations: Recurrent positions (KRAS G12/G13, PIK3CA H1047R)
• Variant allele frequency (VAF): Clonality (VAF ≈50% clonal, <10% subclonal)

Resistance Mutations

• EGFR T790M: Resistance to 1st/2nd-gen TKIs (40-60% of cases)
• ALK G1202R, I1171N: Resistance to early ALK inhibitors
• ESR1 mutations: Resistance to aromatase inhibitors (breast cancer)
• RAS mutations: Acquired resistance to anti-EGFR therapy (CRC)

Mutation Detection Methods

• Tissue NGS: Comprehensive genomic profiling, 300-500 genes
• Liquid biopsy: ctDNA analysis, non-invasive, serial monitoring
• PCR-based assays: Targeted hotspot detection, FDA-approved companion diagnostics
• Allele-specific PCR: High sensitivity for known mutations (cobas EGFR test)

Copy Number Variations (CNV)

Amplifications

• HER2 (ERBB2): Breast, gastric cancer → trastuzumab, pertuzumab
  • Testing: IHC (0, 1+, 2+, 3+) → FISH if 2+ (HER2/CEP17 ratio ≥2.0)
• MET amplification: NSCLC resistance mechanism → crizotinib, capmatinib
  • Cut-point: Gene copy number ≥5, GCN/CEP7 ratio ≥2.0
• EGFR amplification: Glioblastoma, some NSCLC
• FGFR2 amplification: Gastric cancer → investigational FGFR inhibitors

Deletions

• PTEN loss: Common in many cancers, predicts PI3K pathway activation
• RB1 loss: Small cell transformation, poor prognosis
• CDKN2A/B deletion: Cell cycle dysregulation
• Homozygous deletion: Complete loss of both alleles (more significant)

Detection Methods

• FISH (Fluorescence In Situ Hybridization): HER2, ALK rearrangements
• NGS copy number calling: Depth of coverage analysis
• SNP array: Genome-wide CNV detection
• ddPCR: Quantitative copy number measurement

Gene Fusions and Rearrangements

Oncogenic Fusions

• ALK fusions (NSCLC): EML4-ALK most common (60%), 20+ partners
  • Detection: IHC (D5F3 antibody), FISH (break-apart probe), NGS/RNA-seq
• ROS1 fusions (NSCLC, glioblastoma): CD74-ROS1, SLC34A2-ROS1, others
• RET fusions (NSCLC, thyroid): KIF5B-RET, CCDC6-RET
• NTRK fusions (many tumor types, rare): ETV6-NTRK3, others
  • Pan-cancer: Larotrectinib, entrectinib approved across tumor types
• BCR-ABL (CML, ALL): t(9;22), Philadelphia chromosome

Fusion Partner Considerations

• Partner influences drug sensitivity (EML4-ALK variant 3 more sensitive)
• 5' vs 3' fusion affects detection methods
• Intron breakpoints vary (RNA-seq more comprehensive than DNA panels)

Detection Methods

• FISH break-apart probes: ALK, ROS1, RET
• IHC: ALK protein overexpression (screening), ROS1
• RT-PCR: Targeted fusion detection
• RNA-seq: Comprehensive fusion detection, identifies novel partners

Tumor Mutational Burden (TMB)

Definition: Number of somatic mutations per megabase of DNA

Classification

• TMB-high: ≥10 mutations/Mb (some definitions ≥20 mut/Mb)
• TMB-intermediate: 6-9 mutations/Mb
• TMB-low: <6 mutations/Mb

Clinical Application

• Predictive for immunotherapy: Higher TMB → more neoantigens → better immune response
• FDA approval: Pembrolizumab for TMB-H (≥10 mut/Mb) solid tumors (2020)
• Limitations: Not validated in all tumor types, assay variability

Tumor Types with Typically High TMB

• Melanoma (median 10-15 mut/Mb)
• NSCLC (especially smoking-associated, 8-12 mut/Mb)
• Urothelial carcinoma (8-10 mut/Mb)
• Microsatellite instable tumors (30-50 mut/Mb)

Microsatellite Instability (MSI) and Mismatch Repair (MMR)

Classification

• MSI-high (MSI-H): Instability at ≥2 of 5 loci or ≥30% of markers
• MSI-low (MSI-L): Instability at <2 of 5 loci
• Microsatellite stable (MSS): No instability

Mismatch Repair Status

• dMMR (deficient): Loss of MLH1, MSH2, MSH6, or PMS2 by IHC
• pMMR (proficient): Intact expression of all four MMR proteins

Clinical Significance

• MSI-H/dMMR Tumors: 3-5% of most solid tumors, 15% of colorectal cancer
• Immunotherapy Sensitivity: ORR 30-60% to anti-PD-1 therapy
  • Pembrolizumab FDA-approved for MSI-H/dMMR solid tumors (2017)
  • Nivolumab ± ipilimumab approved
• Chemotherapy Resistance: MSI-H CRC does not benefit from 5-FU adjuvant therapy
• Lynch Syndrome: Germline MMR mutation if MSI-H + young age + family history

Testing Algorithm

Colorectal Cancer (all newly diagnosed):
1. IHC for MMR proteins (MLH1, MSH2, MSH6, PMS2)
   ├─ All intact → pMMR (MSS) → Standard chemotherapy if indicated
   │
   └─ Loss of one or more → dMMR (likely MSI-H)
      └─ Reflex MLH1 promoter hypermethylation test
         ├─ Methylated → Sporadic MSI-H, immunotherapy option
         └─ Unmethylated → Germline testing for Lynch syndrome

Expression Biomarkers

Immunohistochemistry (IHC)

PD-L1 Expression (Immune Checkpoint)

• Assays: 22C3 (FDA), 28-8, SP263, SP142 (some differences in scoring)
• Scoring: Tumor Proportion Score (TPS) = % tumor cells with membrane staining
  • TPS <1%: Low/negative
  • TPS 1-49%: Intermediate
  • TPS ≥50%: High
• Combined Positive Score (CPS): (PD-L1+ tumor + immune cells) / total tumor cells × 100
  • Used for some indications (e.g., CPS ≥10 for pembrolizumab in HNSCC)

Hormone Receptors (Breast Cancer)

• ER/PR Positivity: ≥1% nuclear staining by IHC (ASCO/CAP guidelines)
  • Allred Score 0-8 (proportion + intensity) - historical
  • H-score 0-300 (percentage at each intensity) - quantitative
• Clinical Cut-Points:
  • ER ≥1%: Endocrine therapy indicated
  • ER 1-10%: "Low positive," may have lower benefit
  • PR loss with ER+: Possible endocrine resistance

HER2 Testing (Breast/Gastric Cancer)

IHC Initial Test:
├─ 0 or 1+: HER2-negative (no further testing)
│
├─ 2+: Equivocal → Reflex FISH testing
│  ├─ FISH+ (HER2/CEP17 ratio ≥2.0 OR HER2 copies ≥6/cell) → HER2-positive
│  └─ FISH- → HER2-negative
│
└─ 3+: HER2-positive (no FISH needed)
   └─ Uniform intense complete membrane staining in >10% of tumor cells

HER2-positive: Trastuzumab-based therapy indicated
HER2-low (IHC 1+ or 2+/FISH-): Trastuzumab deruxtecan eligibility (2022)

RNA Expression Analysis

Gene Expression Signatures (Breast Cancer)

Oncotype DX (21-gene assay)

• Recurrence Score (RS): 0-100
  • RS <26: Low risk → Endocrine therapy alone (most patients)
  • RS 26-100: High risk → Chemotherapy + endocrine therapy
• Population: ER+/HER2-, node-negative or 1-3 positive nodes
• Evidence: TAILORx trial (N=10,273) validated RS <26 can omit chemo

MammaPrint (70-gene assay)

• Result: High risk vs Low risk (binary)
• Population: Early-stage breast cancer, ER+/HER2-
• Evidence: MINDACT trial validated low-risk can omit chemo

Prosigna (PAM50)

• Result: Risk of Recurrence (ROR) score + intrinsic subtype
• Subtypes: Luminal A, Luminal B, HER2-enriched, Basal-like
• Application: Post-menopausal, ER+, node-negative or 1-3 nodes

RNA-Seq for Fusion Detection

• Advantage: Detects novel fusion partners, quantifies expression
• Application: NTRK fusions (rare, many partners), RET fusions
• Limitation: Requires fresh/frozen tissue or good-quality FFPE RNA

Molecular Subtypes

Glioblastoma (GBM) Molecular Classification

Verhaak 2010 Classification (4 subtypes)

Proneural Subtype

• Characteristics: PDGFRA amplification, IDH1 mutations (secondary GBM), TP53 mutations
• Age: Younger patients typically
• Prognosis: Better prognosis (median OS 15-18 months)
• Treatment: May benefit from bevacizumab less than other subtypes

Neural Subtype

• Characteristics: Neuron markers (NEFL, GABRA1, SYT1, SLC12A5)
• Controversy: May represent normal brain contamination
• Prognosis: Intermediate
• Treatment: Standard temozolomide-based therapy

Classical Subtype

• Characteristics: EGFR amplification (97%), chromosome 7 gain, chromosome 10 loss
• Association: Lacks TP53, PDGFRA, NF1 mutations
• Prognosis: Intermediate
• Treatment: May benefit from EGFR inhibitors (investigational)

Mesenchymal Subtype

• Characteristics: NF1 mutations/deletions, high expression of mesenchymal markers (CHI3L1/YKL-40)
• Immune Features: Higher macrophage/microglia infiltration
• Subgroup: Mesenchymal-immune-active (high immune signature)
• Prognosis: Poor prognosis (median OS 12-13 months)
• Treatment: May respond better to anti-angiogenic therapy, immunotherapy investigational

Clinical Application

GBM Molecular Subtyping Report:

Patient Cohort: Mesenchymal-Immune-Active Subtype (n=15)

Molecular Features:
- NF1 alterations: 73% (11/15)
- High YKL-40 expression: 100% (15/15)
- Immune gene signature: Elevated (median z-score +2.3)
- CD163+ macrophages: High density (median 180/mm²)

Treatment Implications:
- Standard therapy: Temozolomide-based (Stupp protocol)
- Consider: Bevacizumab for recurrent disease (may have enhanced benefit)
- Clinical trial: Immune checkpoint inhibitors ± anti-angiogenic therapy
- Prognosis: Median OS 12-14 months (worse than proneural)

Recommendation:
Enroll in combination immunotherapy trial if eligible, otherwise standard therapy
with early consideration of bevacizumab at progression.

Breast Cancer Intrinsic Subtypes

PAM50-Based Classification

Luminal A

• Characteristics: ER+, HER2-, low proliferation (Ki-67 <20%)
• Gene signature: High ER-related genes, low proliferation genes
• Prognosis: Best prognosis, low recurrence risk
• Treatment: Endocrine therapy alone usually sufficient
• Chemotherapy: Rarely needed unless high-risk features

Luminal B

• Characteristics: ER+, HER2- or HER2+, high proliferation (Ki-67 ≥20%)
• Subtypes: Luminal B (HER2-) and Luminal B (HER2+)
• Prognosis: Intermediate prognosis
• Treatment: Chemotherapy + endocrine therapy; add trastuzumab if HER2+

HER2-Enriched

• Characteristics: HER2+, ER-, PR-
• Gene signature: High HER2 and proliferation genes, low ER genes
• Prognosis: Poor if untreated, good with HER2-targeted therapy
• Treatment: Chemotherapy + trastuzumab + pertuzumab

Basal-Like

• Characteristics: ER-, PR-, HER2- (triple-negative), high proliferation
• Gene signature: Basal cytokeratins (CK5/6, CK17), EGFR
• Overlap: 80% concordance with TNBC, but not identical
• Prognosis: Aggressive, high early recurrence risk
• Treatment: Chemotherapy (platinum, anthracycline), PARP inhibitors if BRCA-mutated
• Immunotherapy: PD-L1+ may benefit from pembrolizumab + chemotherapy

Colorectal Cancer Consensus Molecular Subtypes (CMS)

CMS1 (14%): MSI Immune

• Features: MSI-high, BRAF mutations, strong immune activation
• Prognosis: Poor survival after relapse despite immune infiltration
• Treatment: Immunotherapy highly effe