bio-clinical-databases-msi-detection

## Version Compatibility Reference examples tested with: MSIsensor-pro 1.2+, MSIsensor 0.6+, MANTIS 1.0.5+, samtools 1.19+, mSINGS 5.6+, pandas 2.2+, cyvcf2 0.30+. FDA pembrolizumab MSI-H / dMMR pan-tumor approval is from 2017 (Le 2015 *NEJM*; KEYNOTE-016/164/158); approval extended to colorectal first-line in 2020. Before using code patterns, verify installed versions match. If versions differ: - Python: `pip show <package>` then `help(module.function)` to check signatures - CLI: `<tool> --version` If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying. MSIsensor-pro replaces MSIsensor for tumor-only assays; MSIsensor-ct is the bTMB-equivalent for ctDNA panels. # MSI Detection; The Companion ICI Biomarker to TMB **'Detect MSI status from this somatic sequencing data'** -> Profile microsatellite instability across canonical loci (Bethesda 5 panel + extended NGS-derived sites); classify MSI-H / MSS / MSI-L per Bethesda / FDA / KEYNOTE convention. - CLI (recommended tumor-only): `msisensor-pro msi -d microsatellites.list -t tumor.bam -o msi_out -b 16` - CLI (paired tumor-normal): `msisensor msi -d microsatellites.list -n normal.bam -t tumor.bam -o msi_out` - CLI (ctDNA / blood MSI): `msisensor-ct ...` - CLI (older WES standard): `mantis -t tumor.bam -n normal.bam -b targets.bed --threads 8` ## The Regulatory and Trial Landscape | Event | Year | Threshold | Notes | |-------|------|-----------|-------| | **Le 2015** *NEJM* | 2015 | MSI-H + ICI in CRC | The seminal paper: pembrolizumab in MSI-H CRC ORR 40% vs 0% MSS | | **FDA pembrolizumab MSI-H / dMMR pan-tumor** | 2017 | MSI-H | First tissue-agnostic FDA approval (KEYNOTE-016/164/158) | | **FDA pembrolizumab first-line MSI-H CRC** | 2020 | MSI-H + first-line CRC | KEYNOTE-177 | | **CheckMate 142** | 2017-2018 | MSI-H + nivolumab/ipilimumab | Pan-tumor MSI-H second-line | | **ESMO 2024** | 2024 | MSI-H | Maintained pan-tumor MSI-H biomarker | | **Universal Lynch screening** | -- | IHC + MSI on all CRC <= 70 yr | NCCN / ACG / EGAPP guidelines | ## MSI vs dMMR vs TMB-H: The Conceptual Hierarchy | Term | Definition | Method | Relationship | |------|-----------|--------|--------------| | **dMMR (deficient MMR)** | Loss of MMR protein function | IHC (MLH1, MSH2, MSH6, PMS2) | Causes MSI | | **MSI-H** | Microsatellite instability high | PCR-based Bethesda or NGS | Consequence of dMMR | | **Lynch syndrome** | Germline MMR mutation | Germline sequencing | Causes ~50% of MSI-H CRC; rest are sporadic (MLH1 hyper-methylation) | | **TMB-H** | >= 10 mut/Mb | NGS panel / WES | Statistical correlate of MSI-H | | **POLE-exo hypermutator** | POLE proofreading defect | Sequencing / signatures | Hypermutator WITHOUT MMR-D; MSI-stable typically | **MSI-H + TMB-H overlap** (Chalmers 2017 *Genome Med* 9:34): - ~83% of MSI-H tumors are TMB-H. - ~16% of TMB-H solid tumors are MSI-H. - Sha 2020 *Cancer Discov*: MSI-H is the more established dMMR biomarker for ICI decisions; TMB-H not additive. **POLE-exo vs MMR-D:** - POLE-exo (SBS10a/10b): hypermutator (100-300 mut/Mb pure); typically MSI-stable. - MMR-D (SBS6/15/26/44 + ID1/2): 30-50 mut/Mb typical; MSI-H. - POLE-exo + MMR-D (SBS14 + SBS20): ultra-hypermutator >=500 mut/Mb; MSI-H. ## Tool Taxonomy | Tool | Paired | Tumor-only | ctDNA | Algorithm | Fails when | |------|--------|-----------|-------|-----------|-----------| | **MSIsensor** (Niu 2014 *Bioinformatics*) | Yes | No | No | Bayesian + read-length distribution | Tumor-only data (no baseline); cohort baseline missing | | **MSIsensor-pro** (Jia 2020 *Genom Proteom Bioinform*) | Optional | **Yes** | No | Distribution comparison to baseline | Baseline cohort not provided; panel < 50 loci | | **MSIsensor-ct** (Han 2021 *Brief Bioinform*) | -- | -- | **Yes** | cfDNA-aware | Tumor fraction < 3%; low ctDNA shed | | **MANTIS** (Kautto 2017 *Oncotarget*) | Yes | No | No | Step-wise difference | Tumor-only; low coverage at microsatellites | | **mSINGS** (Salipante 2014 *Clin Chem*) | -- | Yes | No | Background panel (unstable-loci fraction) | Background panel poorly characterized for cohort | **Operational consensus 2024-2026:** - **Tumor + paired normal WES:** MSIsensor or MANTIS. - **Tumor-only assay** (commercial panels, often unpaired): MSIsensor-pro with reference baseline. - **ctDNA / liquid biopsy:** MSIsensor-ct. - **Lynch screening:** IHC FIRST (rules out 90%+); MSI-PCR / NGS confirmatory. ## Decision Tree by Scenario | Scenario | Recommended path | Why | |----------|------------------|-----| | Tumor + paired normal WES | MSIsensor (standard) | Reference paired-normal comparison | | Tumor-only WES/panel | MSIsensor-pro with panel baseline | No matched normal needed | | ctDNA / liquid biopsy | MSIsensor-ct | cfDNA-aware | | Lynch syndrome screening | Universal IHC + MSI (NCCN) | IHC catches 90%+; MSI for IHC-equivocal | | FDA pembrolizumab eligibility | Validate per FoCR PCR + IHC + NGS concordance | Cross-platform required | | MSI-H + TMB-H concurrence | MSI-H is primary biomarker | Sha 2020; TMB-H not additive | | POLE+MMR ultra-hypermutator | Sigprofiler signatures (SBS14, SBS20) | Mechanism beyond MSI alone | | Sporadic MSI-H | Confirm MLH1 hypermethylation; rule out Lynch | Distinguishes sporadic vs germline | | MSI-stable + TMB-H | Investigate POLE-exo signature (SBS10a/10b) | POLE-exo causes hypermutator without MSI | | Pan-tumor screening | MSI + IHC + TMB combined | Multiple modalities for ICI eligibility | ## Bethesda Panel and Modern NGS-Derived Loci The original **NCI/Bethesda reference panel** (Boland 1998) used BAT-25 and BAT-26 plus three dinucleotide markers (D2S123, D5S346, D17S250); >= 2 of 5 loci unstable -> MSI-H. Modern PCR assays use the **mononucleotide pentaplex** (the current clinical standard), which replaced the dinucleotide markers for improved cross-population specificity: - **BAT-25** (chr4) - **BAT-26** (chr2) - **NR-21** (chr14) - **NR-24** (chr2) - **MONO-27** (chr2) NGS-based MSI panels use 50-1000+ microsatellite loci. MSI-H requires unstable status at >=40% of tested loci typically (varies by panel calibration). ## Standard Workflow: MSIsensor-pro Tumor-Only **Goal:** Compute MSI status from tumor-only WES/panel. **Approach:** Generate baseline from population reference; compare patient tumor. ```bash # Generate microsatellite list from reference genome (one-time) msisensor-pro scan -d /reference/GRCh38.fa -o microsatellites.list -p 1 -m 5 # Generate baseline from N normal control samples (one-time per panel) msisensor-pro baseline -d microsatellites.list -i normal_samples.list -o baseline.list -b 16 # Score tumor sample. The `-i sample_id` flag is uncommon: in typical msisensor-pro # usage the sample identifier is derived from the BAM file -- verify the flag set # against `msisensor-pro pro --help` for the installed release. msisensor-pro pro \ -d microsatellites.list \ -t tumor.bam \ -o msi_output \ -b 16 \ --baseline baseline.list # Output: msi_output_all (raw); msi_output_unstable (unstable loci); msi_output.txt (summary) # Critical column: %_unstable. Threshold MSI-H typically >= 20-30% depending on panel. ``` ## Paired Tumor-Normal MSIsensor ```bash msisensor msi \ -d microsatellites.list \ -n normal.bam \ -t tumor.bam \ -o msi_paired_out \ -b 16 # Output: %_unstable in paired comparison # MSI-H threshold: >= 20% by FoCR guidance; varies 10-30% across studies ``` ## MANTIS Step-wise Difference ```bash mantis.py \ -t tumor.bam \ -n normal.bam \ -b microsatellite_targets.bed \ --threads 8 \ -o mantis_output # Output: mantis_output.kmer_counts (raw), mantis_output (status) # Threshold MSI-H: stepwise difference > 0.4 (default) ``` ## MSI-H Classification Logic ```python import pandas as pd def classify_msi(unstable_percentage, panel_calibrated_cutoff=20.0): '''Classify MSI status from percentage of unstable loci. Bethesda PCR: >=2 of 5 unstable -> MSI-H (40% loci) NGS: panel-specific cutoffs typically 10-30% Concordance: MSI-PCR + IHC + NGS should agree (FoCR) ''' if unstable_percentage >= panel_calibrated_cutoff: return 'MSI-H' elif unstable_percentage >= panel_calibrated_cutoff / 2: return 'MSI-L (intermediate; treat as MSS clinically per FDA)' else: return 'MSS' def msi_lynch_workflow(msi_status, ihc_results, mlh1_methylation_status, germline_test): '''Standard Lynch syndrome workflow. Args: msi_status: 'MSI-H' / 'MSS' / 'MSI-L' ihc_results: dict {MLH1: 'retained' or 'loss', MSH2, MSH6, PMS2} mlh1_methylation_status: 'methylated' (sporadic) / 'unmethylated' (Lynch suspect) germline_test: 'positive' / 'negative' / 'not_performed' ''' if msi_status != 'MSI-H': return 'No further Lynch screening indicated' ihc_loss = [gene for gene, status in ihc_results.items() if status == 'loss'] if not ihc_loss: return 'MSI-H with retained IHC; consider Lynch with germline testing' if 'MLH1' in ihc_loss: if mlh1_methylation_status == 'methylated': return 'Sporadic MSI-H (MLH1 hypermethylation); not Lynch' elif mlh1_methylation_status == 'unmethylated': return 'Lynch suspect (MLH1 loss without methylation); proceed with germline testing' else: return 'MLH1 loss; perform methylation test' return f'MSH2/6/PMS2 loss ({", ".join(ihc_loss)}); strong Lynch suspect; germline testing' def msi_tmb_ici_decision(msi_status, tmb_value, tumor_type=None, dmmr_ihc=None): '''Integrated ICI eligibility from MSI + TMB. Sha 2020: MSI-H is primary biomarker; TMB-H not additive. McGrail 2021: TMB-H NOT endorsed for breast/prostate/glioma alone. ''' msi_high = msi_status == 'MSI-H' dmmr_positive = dmmr_ihc == 'positive' tmb_h = tmb_value >= 10 if msi_high or dmmr_positive: return ('ICI eligible: MSI-H or dMMR (FDA pembrolizumab 2017 pan-tumor; KEYNOTE-016/164/158); ' 'TMB-H is not additive (Sha 2020).') if tmb_h and tumor_type and tumor_type.lower() in ('breast', 'prostate', 'glioma'): return ('TMB-H but tumor type excluded by ESMO 2024 / McGrail 2021. ' 'Consider tumor-type-specific cutoff.') if tmb_h: return 'TMB-H pan-tumor (FDA pembrolizumab 2020); ICI eligible.' return 'MSS + TMB-low. Standard chemo per tumor type.' ``` ## Per-Operation Failure Modes **1. Tumor-only with paired-normal tool** - Trigger: Run MSIsensor on tumor-only BAM. - Mechanism: MSIsensor requires paired normal for baseline comparison. - Symptom: Tool errors or produces unstable noisy result. - Fix: Use MSIsensor-pro for tumor-only; or use mSINGS background-panel approach. **2. Panel size too small** - Trigger: 30-locus panel called MSI-H based on 20% threshold (= 6 unstable loci). - Mechanism: Small panel + stochastic unstable rates produce high false-positive rates. - Symptom: False-positive MSI-H in WES-comparable panels with < 50 microsatellite loci. - Fix: Validate panel calibration with reference cohort; use panel-specific cutoff; minimum 50 informative loci. **3. IHC vs MSI discordance not investigated** - Trigger: IHC retains all four MMR proteins; MSI-H by sequencing. - Mechanism: IHC may miss subtle loss; MSI may include MSH6-only subtype (more variable); rare germline POLE+MMR ultra-hypermutators show MSI. - Symptom: Apparent discordance; classification ambiguous. - Fix: Cross-check with germline MMR sequencing; check for POLE-exo on Sigprofiler. **4. MSI-H + Lynch syndrome confusion** - Trigger: Report MSI-H tumor as "Lynch syndrome". - Mechanism: ~50% of MSI-H CRC is sporadic (MLH1 hypermethylation, not germline Lynch). - Symptom: Incorrect family counseling; wrong screening. - Fix: Apply IHC + MLH1 methylation + germline testing workflow. **5. POLE-exo hypermutator labeled MSI** - Trigger: Tumor with 200 mut/Mb POLE-exo signature labeled MSI-H. - Mechanism: Pure POLE-exo causes hypermutator WITHOUT MSI (different repair mechanism); apparent MSI-H call may be a false positive in high-mutation context. - Symptom: Misclassification; ICI eligibility still positive but for different mechanism. - Fix: Run Sigprofiler signatures (SBS10a/10b vs SBS6/15/26/44); confirm POLE-exo via SBS10 contribution. **6. ctDNA MSI without sufficient tumor fraction** - Trigger: Run MSIsensor-ct on cfDNA with <1% tumor fraction. - Mechanism: Low ctDNA fraction produces noise-dominated unstable locus counts. - Symptom: False-negative or unstable MSI call. - Fix: Estimate tumor fraction first (ichorCNA); require >= 3% for reliable cfDNA MSI. **7. Universal screening missed** - Trigger: CRC patient < 70 yr without IHC / MSI. - Mechanism: NCCN / ACG universal Lynch screening required; without it, Lynch syndrome undiagnosed. - Symptom: Family loses screening benefit. - Fix: Universal IHC + MSI on all CRC < 70; institute reflex testing. **8. MSI-L treated as actionable** - Trigger: Report MSI-L (intermediate) as ICI-eligible. - Mechanism: FDA approval specifies MSI-H; MSI-L = MSS clinically. - Symptom: ICI given on insufficient indication; reimbursement issues. - Fix: Apply MSI-H threshold strictly per FDA; MSI-L = MSS. ## Reconciliation: When Sources Disagree | Pattern | Likely cause | Action |