scientific-skills/clinical-decision-support/references/biomarker_classification.md
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.
Definition: Predict clinical outcome (survival, recurrence) regardless of treatment received
Examples by Disease
Cancer
Cardiovascular
Infectious Disease
Application: Risk stratification, treatment intensity selection, clinical trial enrollment
Definition: Identify patients likely to benefit (or not benefit) from specific therapy
Positive Predictive Biomarkers (Treatment Benefit)
Oncology - Targeted Therapy
Oncology - Immunotherapy
Hematology
Negative Predictive Biomarkers (Resistance/No Benefit)
Definition: Detect or confirm presence of disease
Infectious Disease
Autoimmune
Cancer
Definition: Assess treatment response or mechanism of action
Examples
Driver Mutations (Oncogenic)
Resistance Mutations
Mutation Detection Methods
Amplifications
Deletions
Detection Methods
Oncogenic Fusions
Fusion Partner Considerations
Detection Methods
Definition: Number of somatic mutations per megabase of DNA
Classification
Clinical Application
Tumor Types with Typically High TMB
Classification
Mismatch Repair Status
Clinical Significance
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
PD-L1 Expression (Immune Checkpoint)
Hormone Receptors (Breast Cancer)
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)
Gene Expression Signatures (Breast Cancer)
Oncotype DX (21-gene assay)
MammaPrint (70-gene assay)
Prosigna (PAM50)
RNA-Seq for Fusion Detection
Verhaak 2010 Classification (4 subtypes)
Proneural Subtype
Neural Subtype
Classical Subtype
Mesenchymal Subtype
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.
PAM50-Based Classification
Luminal A
Luminal B
HER2-Enriched
Basal-Like
CMS1 (14%): MSI Immune
CMS2 (37%): Canonical
CMS3 (13%): Metabolic
CMS4 (23%): Mesenchymal
Required Testing (Label Indication)
Biomarker Drug(s) Indication Assay
EGFR exon 19 del/L858R Osimertinib NSCLC cobas EGFR v2, NGS
ALK rearrangement Alectinib, brigatinib NSCLC Vysis ALK FISH, IHC (D5F3)
BRAF V600E Vemurafenib, dabrafenib Melanoma, NSCLC THxID BRAF, cobas BRAF
HER2 amplification Trastuzumab, pertuzumab Breast, gastric HercepTest IHC, FISH
ROS1 rearrangement Crizotinib, entrectinib NSCLC FISH, NGS
PD-L1 ≥50% TPS Pembrolizumab (mono) NSCLC first-line 22C3 pharmDx
MSI-H/dMMR Pembrolizumab Any solid tumor IHC (MMR), PCR (MSI)
NTRK fusion Larotrectinib, entrectinib Pan-cancer FoundationOne CDx
BRCA1/2 mutations Olaparib, talazoparib Breast, ovarian, prostate BRACAnalysis CDx
Level 1: FDA-Approved
Level 2: Standard Care Off-Label
Level 3: Clinical Evidence
Level 4: Biological Evidence
Level R1-R2: Resistance
Evidence Levels
Clinical Significance Tiers
FoundationOne CDx
Guardant360 CDx (Liquid Biopsy)
Tempus xT
NSCLC (NCCN Guidelines)
Broad molecular profiling for all advanced NSCLC at diagnosis:
Required (FDA-approved therapies available):
✓ EGFR mutations (exons 18, 19, 20, 21)
✓ ALK rearrangement
✓ ROS1 rearrangement
✓ BRAF V600E
✓ MET exon 14 skipping
✓ RET rearrangements
✓ NTRK fusions
✓ KRAS G12C
✓ PD-L1 IHC
Recommended (to inform treatment strategy):
✓ Comprehensive NGS panel (captures all above + emerging targets)
✓ Consider liquid biopsy if tissue insufficient
At progression on targeted therapy:
✓ Repeat tissue biopsy or liquid biopsy for resistance mechanisms
✓ Examples: EGFR T790M, ALK resistance mutations, MET amplification
Metastatic Colorectal Cancer
Required before anti-EGFR therapy (cetuximab, panitumumab):
✓ RAS testing (KRAS exons 2, 3, 4; NRAS exons 2, 3, 4)
└─ RAS mutation → Do NOT use anti-EGFR therapy (resistance)
✓ BRAF V600E
└─ If BRAF V600E+ → Consider encorafenib + cetuximab + binimetinib
Recommended for all metastatic CRC:
✓ MSI/MMR testing (immunotherapy indication)
✓ HER2 amplification (investigational trastuzumab-based therapy if RAS/BRAF WT)
✓ NTRK fusions (rare, <1%, but actionable)
Left-sided vs Right-sided:
- Left-sided (descending, sigmoid, rectum): Better prognosis, anti-EGFR more effective
- Right-sided (cecum, ascending): Worse prognosis, anti-EGFR less effective, consider bevacizumab
Melanoma
All advanced melanoma:
✓ BRAF V600 mutation (30-50% of cutaneous melanoma)
└─ If BRAF V600E/K → Dabrafenib + trametinib or vemurafenib + cobimetinib
✓ NRAS mutation (20-30%)
└─ No targeted therapy approved, consider MEK inhibitor trials
✓ KIT mutations (mucosal, acral, chronic sun-damaged melanoma)
└─ If KIT exon 11 or 13 mutation → Imatinib (off-label)
✓ PD-L1 (optional, not required for immunotherapy eligibility)
Note: Uveal melanoma has different biology (GNAQ, GNA11 mutations)
Methods for Cut-Point Determination
Data-Driven Approaches
Biologically Informed
Clinically Defined
PD-L1 Example
Continuous Analysis Advantages
Categorical Analysis Advantages
Best Practice: Report both continuous and categorical analyses
Indications for Germline Testing
Common Hereditary Cancer Syndromes
Genetic Counseling
Tumor Tissue Testing
Distinguishing Germline from Somatic
MOLECULAR PROFILING REPORT
Patient: [De-identified ID]
Tumor Type: Non-Small Cell Lung Adenocarcinoma
Specimen: Lung biopsy (left upper lobe)
Testing Date: [Date]
Report Date: [Date]
METHODOLOGY
- Assay: FoundationOne CDx (comprehensive genomic profiling)
- Specimen Type: Formalin-fixed paraffin-embedded (FFPE)
- Tumor Content: 40% (adequate for testing)
RESULTS SUMMARY
Biomarkers Detected: 4
- 1 FDA-approved therapy target
- 1 prognostic biomarker
- 2 variants of uncertain significance
ACTIONABLE FINDINGS
Tier 1: FDA-Approved Targeted Therapy Available
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
EGFR Exon 19 Deletion (p.E746_A750del)
Variant Allele Frequency: 42%
Clinical Significance: Sensitizing mutation
FDA-Approved Therapy: Osimertinib (Tagrisso) 80 mg daily
Evidence: FLAURA trial - median PFS 18.9 vs 10.2 months (HR 0.46, p<0.001)
Guideline: NCCN Category 1 preferred first-line
Recommendation: Strong recommendation for EGFR TKI therapy (GRADE 1A)
Tier 2: Prognostic Biomarker
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
TP53 Mutation (p.R273H)
Variant Allele Frequency: 85%
Clinical Significance: Poor prognostic marker, no targeted therapy
Implication: Associated with worse survival, does not impact first-line treatment selection
BIOMARKERS ASSESSED - NEGATIVE
- ALK rearrangement: Not detected
- ROS1 rearrangement: Not detected
- BRAF V600E: Not detected
- MET exon 14 skipping: Not detected
- RET rearrangement: Not detected
- KRAS mutation: Not detected
- PD-L1 IHC: Separate report (TPS 30%)
TUMOR MUTATIONAL BURDEN: 8 mutations/Mb (Intermediate)
- Interpretation: Below threshold for TMB-high designation (≥10 mut/Mb)
- Clinical relevance: May still benefit from immunotherapy combinations
MICROSATELLITE STATUS: Stable (MSS)
CLINICAL RECOMMENDATIONS
Primary Recommendation:
First-line therapy with osimertinib 80 mg PO daily until progression or unacceptable toxicity.
Monitoring:
- CT imaging every 6 weeks for first 12 weeks, then every 9 weeks
- At progression, repeat tissue or liquid biopsy for resistance mechanisms (T790M, C797S, MET amplification)
Alternative Options:
- Clinical trial enrollment for novel EGFR TKI combinations
- Erlotinib or afatinib (second-line for osimertinib if used first-line)
References:
1. Soria JC, et al. Osimertinib in Untreated EGFR-Mutated Advanced NSCLC. NEJM 2018.
2. NCCN Guidelines for Non-Small Cell Lung Cancer v4.2024.
Report Prepared By: [Lab Name]
Medical Director: [Name, MD, PhD]
CLIA #: [Number] | CAP #: [Number]