# Example: GBM Molecular Subtype Cohort Analysis ## Clinical Context This example demonstrates a patient cohort analysis stratified by molecular biomarkers, similar to the GBM Mesenchymal-Immune-Active cluster analysis provided as reference. ## Cohort Overview **Disease**: Glioblastoma (GBM), IDH-wild-type **Study Population**: n=60 patients with newly diagnosed GBM treated with standard Stupp protocol (temozolomide + radiation → adjuvant temozolomide) **Molecular Classification**: Verhaak 2010 subtypes with immune signature refinement - **Group A**: Mesenchymal-Immune-Active subtype (n=18, 30%) - **Group B**: Other molecular subtypes (Proneural, Classical, Neural) (n=42, 70%) **Study Period**: January 2019 - December 2022 **Data Source**: Single academic medical center, retrospective cohort analysis ## Biomarker Classification ### Mesenchymal-Immune-Active Subtype Characteristics **Molecular Features**: - NF1 alterations (mutations or deletions): 72% (13/18) - High YKL-40 (CHI3L1) expression: 100% (18/18, median z-score +2.8) - Immune gene signature: Elevated (median ESTIMATE immune score +1250) - CD163+ macrophage infiltration: High density (median 195 cells/mm², range 120-340) - MES (mesenchymal) signature score: >0.5 (all patients) **Clinical Characteristics**: - Median age: 64 years (range 42-76) - Male: 61% (11/18) - Tumor location: Temporal lobe predominant (55%) - Multifocal disease: 33% (6/18) - higher than overall cohort ### Comparison Groups (Other Subtypes) **Molecular Features**: - Proneural: n=15 (25%) - PDGFRA amplification, younger age - Classical: n=18 (30%) - EGFR amplification, chromosome 7+/10- - Neural: n=9 (15%) - neuronal markers, may include normal tissue ## Treatment Outcomes ### Response Assessment (RANO Criteria) **Objective Response Rate** (after chemoradiation, ~3 months): - Mesenchymal-Immune-Active: 6/18 (33%) - CR 0, PR 6 - Other subtypes: 18/42 (43%) - CR 1, PR 17 - p = 0.48 (Fisher's exact) **Interpretation**: No significant difference in initial response rates ### Survival Outcomes **Progression-Free Survival (PFS)**: - Mesenchymal-Immune-Active: Median 7.2 months (95% CI 5.8-9.1) - Other subtypes: Median 9.5 months (95% CI 8.1-11.3) - Hazard Ratio: 1.58 (95% CI 0.89-2.81), p = 0.12 - 6-month PFS rate: 61% vs 74% **Overall Survival (OS)**: - Mesenchymal-Immune-Active: Median 12.8 months (95% CI 10.2-15.4) - Other subtypes: Median 16.3 months (95% CI 14.7-18.9) - Hazard Ratio: 1.72 (95% CI 0.95-3.11), p = 0.073 - 12-month OS rate: 55% vs 68% - 24-month OS rate: 17% vs 31% **Interpretation**: Trend toward worse survival in mesenchymal-immune-active subtype, not reaching statistical significance in this cohort size ### Response to Bevacizumab at Recurrence **Subset Analysis** (patients receiving bevacizumab at first recurrence, n=35): - Mesenchymal-Immune-Active: n=12 - ORR: 58% (7/12) - Median PFS2 (from bevacizumab start): 6.8 months - Other subtypes: n=23 - ORR: 35% (8/23) - Median PFS2: 4.2 months - p = 0.19 (Fisher's exact for ORR) - HR for PFS2: 0.62 (95% CI 0.29-1.32), p = 0.21 **Interpretation**: Exploratory finding suggesting enhanced benefit from bevacizumab in mesenchymal-immune-active subtype (not statistically significant with small sample) ## Safety Profile **Treatment-Related Adverse Events** (Temozolomide): No significant differences in toxicity between molecular subtypes: - Lymphopenia (any grade): 89% vs 86%, p = 0.77 - Thrombocytopenia (grade 3-4): 22% vs 19%, p = 0.79 - Fatigue (any grade): 94% vs 90%, p = 0.60 - Treatment discontinuation: 17% vs 14%, p = 0.77 ## Clinical Implications ### Treatment Recommendations **For Mesenchymal-Immune-Active GBM**: 1. **First-Line**: Standard Stupp protocol (no change based on subtype) - Evidence: No proven benefit for alternative first-line strategies - GRADE: 1A (strong recommendation, high-quality evidence) 2. **At Recurrence - Consider Bevacizumab Earlier**: - Rationale: Exploratory data suggesting enhanced anti-angiogenic response - Evidence: Mesenchymal GBM has high VEGF expression, angiogenic phenotype - GRADE: 2C (conditional recommendation, low-quality evidence from subset) 3. **Clinical Trial Enrollment - Immunotherapy Combinations**: - Rationale: High immune cell infiltration may predict immunotherapy benefit - Targets: PD-1/PD-L1 blockade ± anti-CTLA-4 or anti-angiogenic agents - Evidence: Ongoing trials (CheckMate-498, CheckMate-548 showed negative results, but did not select for immune-active) - GRADE: R (research recommendation) **For Other GBM Subtypes**: - Standard treatment per NCCN guidelines - Consider tumor treating fields (Optune) after radiation completion - Clinical trials based on specific molecular features (EGFR amplification → EGFR inhibitor trials) ### Prognostic Information **Counseling Patients**: - Mesenchymal-immune-active subtype associated with trend toward shorter survival (12.8 vs 16.3 months) - Not definitive due to small sample size and confidence intervals overlapping - Prospective validation needed - Should not alter standard first-line treatment ## Study Limitations 1. **Small Sample Size**: n=18 in mesenchymal-immune-active group limits statistical power 2. **Retrospective Design**: Potential selection bias, unmeasured confounders 3. **Single Institution**: May not generalize to other populations 4. **Heterogeneous Recurrence Treatment**: Not all patients received bevacizumab; treatment selection bias 5. **Molecular Classification**: Based on bulk tumor RNA-seq; intratumoral heterogeneity not captured 6. **No Central Pathology Review**: Molecular classification performed locally ## Future Directions 1. **Prospective Validation**: Confirm survival differences in independent cohort (n>100 per group for adequate power) 2. **Biomarker Testing**: Develop clinically feasible assay for mesenchymal-immune subtype identification 3. **Clinical Trial Design**: Immunotherapy combinations targeting mesenchymal-immune-active GBM specifically 4. **Mechanistic Studies**: Investigate why mesenchymal-immune GBM may respond better to bevacizumab 5. **Longitudinal Analysis**: Track molecular subtype evolution over treatment course ## Data Presentation Example ### Baseline Characteristics Table ``` Characteristic Mesenchymal-IA (n=18) Other (n=42) p-value Age, years (median [IQR]) 64 [56-71] 61 [53-68] 0.42 Sex, n (%) Male 11 (61%) 24 (57%) 0.78 Female 7 (39%) 18 (43%) ECOG PS, n (%) 0-1 15 (83%) 37 (88%) 0.63 2 3 (17%) 5 (12%) Tumor location Frontal 4 (22%) 15 (36%) 0.35 Temporal 10 (56%) 16 (38%) Parietal/Occipital 4 (22%) 11 (26%) Extent of resection Gross total 8 (44%) 22 (52%) 0.58 Subtotal 10 (56%) 20 (48%) MGMT promoter methylated 5 (28%) 18 (43%) 0.27 ``` ### Survival Outcomes Summary ``` Endpoint Mesenchymal-IA Other HR (95% CI) p-value Median PFS, months (95% CI) 7.2 (5.8-9.1) 9.5 (8.1-11.3) 1.58 (0.89-2.81) 0.12 6-month PFS rate 61% 74% Median OS, months (95% CI) 12.8 (10.2-15.4) 16.3 (14.7-18.9) 1.72 (0.95-3.11) 0.073 12-month OS rate 55% 68% 24-month OS rate 17% 31% ``` ## Key Takeaways 1. **Molecular heterogeneity exists** in GBM with distinct subtypes 2. **Mesenchymal-immune-active subtype** characterized by NF1 alterations, immune infiltration 3. **Trend toward worse prognosis** but not statistically significant (power limitations) 4. **Potential bevacizumab benefit** hypothesis-generating, requires prospective validation 5. **Immunotherapy target**: High immune infiltration rational for checkpoint inhibitor trials 6. **Clinical implementation pending**: Need prospective validation before routine subtyping ## References 1. Verhaak RG, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010;17(1):98-110. 2. Wang Q, et al. Tumor Evolution of Glioma-Intrinsic Gene Expression Subtypes Associates with Immunological Changes in the Microenvironment. Cancer Cell. 2017;32(1):42-56. 3. Stupp R, et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. NEJM. 2005;352(10):987-996. 4. Gilbert MR, et al. Bevacizumab for Newly Diagnosed Glioblastoma. NEJM. 2014;370(8):699-708. 5. NCCN Clinical Practice Guidelines in Oncology: Central Nervous System Cancers. Version 1.2024. --- **This example demonstrates**: - Biomarker-based stratification methodology - Outcome reporting with appropriate statistics - Clinical contextualization of findings - Evidence-based recommendations with grading - Transparent limitation discussion - Structure suitable for pharmaceutical/clinical research documentation