Malignant mesothelioma remains a diagnostically and therapeutically challenging disease with a median overall survival of one to three years for pleural disease and approximately five years for peritoneal disease. Recent advances in immunotherapy, targeted agents, and molecular pathology have created an urgent need for a coherent, evidence-based biomarker framework that integrates diagnostic confirmation, prognostic stratification, predictive selection, and translational discovery into a single operational structure. This review synthesizes current guideline recommendations and key published evidence to provide actionable guidance for medical professionals and clinical trial designers.
1. Diagnostic Biomarkers: Confirming Malignancy and Excluding Mimics
Standard Immunohistochemical Panels
No single immunohistochemical (IHC) marker is sufficient for mesothelioma diagnosis. A structured dual-marker strategy is mandated: at least two positive mesothelial markers—calretinin, CK5/6, WT1, and D2-40/podoplanin—combined with at least two negative carcinoma markers, including claudin-4, TTF-1, Napsin A, polyclonal CEA, BerEP4/MOC31, and PAX8 2835. For peritoneal mesothelioma, important distinctions apply: TTF-1 and D2-40 are less informative for peritoneal disease, and PAX8 positivity can occur in peritoneal mesotheliomas, requiring careful interpretation to exclude ovarian serous carcinoma 35.
BAP1 Loss
Loss of BAP1 (BRCA1-associated protein 1) nuclear expression by IHC is one of the most diagnostically powerful molecular adjuncts. BAP1 inactivation is common in mesothelioma, particularly in epithelioid tumors, but reported frequencies vary substantially across studies and methodologies. Loss of BAP1 expression is rare in reactive mesothelial proliferations but may also occur in a subset of other malignancies, including some serous carcinomas 2836. BAP1 loss is defined as complete absence of nuclear expression or cytoplasmic-only staining; this distinction is clinically critical and must be applied with adequate positive internal controls (lymphocytes and stromal cells) 3621. In the specific context of well-differentiated papillary mesothelial tumor (WDPMT), all four cases that progressed to invasive mesothelioma in a retrospective series harbored BAP1 loss, supporting a proposal to restrict the WDPMT designation to BAP1-retained tumors and reclassify BAP1-deficient lesions as papillary mesothelioma in situ 15. Notably, BAP1 loss is more frequent in epithelioid histology (<20% in sarcomatoid cases) 35.
MTAP/CDKN2A Loss
MTAP immunohistochemistry serves as a practical and highly specific surrogate for CDKN2A homozygous deletion (confirmed by FISH as the gold standard). Loss of MTAP cytoplasmic staining is highly specific for malignant mesothelioma in the context of a mesothelial proliferation, although rare exceptions and technical pitfalls may occur, and the combination of BAP1 loss and/or MTAP loss achieves diagnostic separation from reactive lesions in up to 90% of mesothelioma cases 20. A multicenter study across five institutions in four countries confirmed excellent interobserver agreement (κ=0.85) and interlaboratory reproducibility (κ=0.77–0.89), with MTAP IHC demonstrating 78% sensitivity and 96% specificity for CDKN2A homozygous deletion 24. Critically, antibody selection is consequential: MTAP monoclonal antibody 1813 (NBP2-75730) demonstrated 96% sensitivity, 86% specificity, and zero equivocal interpretations, whereas the commonly used EPR6893 clone yielded equivocal results in 34% of cases 13. Concordance between MTAP IHC and NGS-based copy-number analysis is optimal at tumor purity >20%; in low-purity samples, MTAP IHC may actually outperform NGS, and FISH can confirm discordant cases 13. An important site-specific consideration is that CDKN2A/MTAP deletions are substantially less prevalent in peritoneal mesothelioma (8–35%) than in pleural disease (60–74%) 37.
For peritoneal disease, FISH analysis also reveals that minute and interstitial BAP1 deletions (detected by diminished signal rather than canonical signal loss) occur more frequently than in pleural disease, and that only canonical—not minute—CDKN2A deletions result in MTAP IHC loss, potentially explaining some IHC/FISH discordances 26.
Practical Diagnostic Algorithm
| Marker | Assay Platform | Preferred Antibody/Clone | Interpretation Rule | Pitfalls |
|---|---|---|---|---|
| BAP1 | IHC | C4 (Santa Cruz) | Complete nuclear loss = positive for malignancy | Sarcomatoid tumors often retain BAP1; require adequate internal controls |
| MTAP | IHC | mAb 1813 (Novus) preferred over EPR6893 | Cytoplasmic loss = CDKN2A deletion surrogate | Lower prevalence in peritoneal disease; equivocal results with EPR6893 |
| CDKN2A | FISH | Standard dual-color probe | Homozygous deletion = gold standard | Minute deletions may be missed; low tumor purity reduces sensitivity |
| Standard panel | IHC | Calretinin, WT1, D2-40, claudin-4, TTF-1, CEA | Dual-positive/dual-negative strategy | PAX8 positivity in peritoneal disease; D2-40 less informative for peritoneal disease |
| Mesothelin (surface) | IHC | VENTANA MSLN SP74 or equivalent | ≥30% 2+/3+ membrane intensity | Heterogeneous expression; bystander-killing mechanisms may complicate thresholds |
2. Prognostic Biomarkers: Baseline Risk Stratification
Histologic Subtype
Histologic subtype remains the most robust and reproducible prognostic variable. Epithelioid mesothelioma carries a median overall survival of approximately 39 months for peritoneal disease, compared to 14 months for biphasic disease; following CRS/HIPEC, these figures improve to 55 months and 13 months, respectively 39. Sarcomatoid and biphasic histologies are generally contraindications to surgery. Histologic subtype must be documented according to the 2021 WHO Classification, with explicit reporting of the sarcomatoid component at a threshold of at least 1% per consensus recommendations 214.
Nuclear Grade
In epithelioid mesothelioma, nuclear grading (grades I–III) is an independent and strong prognostic factor. A multivariate analysis of 117 patients confirmed that grade I nuclear grading was the most significant independent predictor of overall survival (p<0.0001) 22. The 2023 International Mesothelioma Interest Group consensus and Italian expert recommendations both require nuclear grade to be included in all pathology reports for epithelioid mesothelioma 421.
BAP1 Status as a Prognostic Variable
The prognostic role of BAP1 is context-dependent and not yet clinically actionable as a standalone factor. Patients with germline BAP1 mutations appear to have better prognosis than those with somatic mutations, and one study paradoxically found longer survival associated with BAP1 loss in epithelioid MPM 229. A single-center retrospective study in 52 patients found that BAP1 positivity (retained expression) and PD-L1 TPS ≥1% were numerically associated with shorter overall survival (11.3 vs. 20.0 months for BAP1 positivity; 15.3 vs. 20.0 months for PD-L1) 19. However, given contradictory results across studies and limited patient numbers, BAP1 is not yet suitable for clinical use as a standalone prognostic or predictive factor 9.
Serum Mesothelin (SMRP) and Inflammatory Markers
Serum soluble mesothelin-related peptide (SMRP) provides prognostic context in patients treated with immune checkpoint therapy (ICT). A retrospective analysis of 125 MPM patients confirmed SMRP as an independent prognostic factor by multivariable analysis, with lower levels (≤1.35 nmol/L) correlating with improved overall survival 7. Importantly, this association was validated in a prospective ICT cohort but not in a historical cohort treated without ICT, suggesting the prognostic value may be specific to the immunotherapy context 7. Serum CA-125 and SMRP are incorporated into NCCN-recommended initial evaluation for peritoneal mesothelioma 38.
3. Predictive Biomarkers for Immunotherapy: PD-L1, TMB, and Emerging Immune Signatures
PD-L1 Expression: Contextual, Not Predictive
The landmark CheckMate 743 trial established ipilimumab plus nivolumab as a first-line standard with a median OS of 18.1 versus 14.1 months (HR 0.74) 30. PD-L1 positivity (≥1%) was observed in 74–80% of enrolled patients. Non-epithelioid histology and PD-L1 ≥1% were associated with greater absolute OS benefit in subgroup analyses, but PD-L1 was not used as a stratification factor. Critically, both ASCO 2024 guidelines and NCCN explicitly state that PD-L1 should not be used to determine choice of chemotherapy or immunotherapy 240. PD-L1 testing is recommended for exploratory correlative purposes only, not for patient enrichment. Additional complexities reduce PD-L1's predictive utility: prevalence of high expressors (TPS >50%) is only approximately 5%; non-epithelioid histology itself drives higher PD-L1 expression, confounding independent predictive analysis; and PD-L1 expression across 11 IHC studies was confirmed as a negative prognostic—not predictive—factor (HR=1.50) when histology was accounted for 612.
Tumor Mutational Burden: Low and Uninformative
Mesothelioma is classified as a low-TMB malignancy (mean 4 Muts/MB in the EORTC-SPECTA Arcagen cohort), and MSI-high status was detected in only 2 of 56 pleural mesothelioma cases (4%) 5. Unlike non-small cell lung cancer, TMB does not predict ICI response in mesothelioma. The CONFIRM phase III trial of nivolumab in relapsed mesothelioma confirmed no consistent difference in non-synonymous mutation burden or neoantigen load between responders and non-responders 12. ASCO recommends that TMB and MSI should not be used for treatment selection 2.
Emerging Predictive Signals: TLS, Inflammatory Signatures, and Molecular Context
Translational analysis of the CONFIRM trial identified tertiary lymphoid structures (TLS) and their associated CD8+ T cells and CD19+ B cells as the strongest correlates of nivolumab response, with TLS density correlating with longer PFS and OS (r=0.47) 12. The IL24/EMT ratio emerged as a particularly discriminating predictive signal (AUROC ~0.89), with epithelial-to-mesenchymal transition (EMT) enrichment predicting resistance 12. At the molecular level, a study of 113 mesothelioma specimens found that MTAP or CDKN2A loss was associated with an immune desert phenotype and shorter survival on ipilimumab plus nivolumab, while BAP1 alterations correlated with improved survival on the same regimen 8. These findings collectively indicate that the functional composition of the tumor immune microenvironment, rather than PD-L1 or TMB alone, governs ICI efficacy in mesothelioma.
Tumor-Associated Macrophage Immunosuppression
Tumor-associated macrophages (TAMs) constitute 20–30% of the total immune infiltrate in pleural mesothelioma, with approximately 95% displaying PD-L1 positivity and a protumor phenotype characterized by IL-10 and TGF-β secretion 6. CTLs—5–15% of the infiltrate—exhibit widespread exhaustion with elevated PD-1, TIM-3, LAG-3, and TIGIT 6. This "altered" TIME phenotype, in which T cells infiltrate but are functionally suppressed, explains why conventional checkpoint blockade yields modest but real activity. CSF-1R inhibition to reprogram TAMs and TLS-targeted approaches represent rational combinatorial strategies 6.
4. Emerging Translational Targets
Mesothelin-Directed ADCs
Mesothelin is overexpressed in 85–87% of mesothelioma cases at 2+/3+ intensity on ≥30% of tumor cells by IHC 33. In the anetumab ravtansine phase I trial, all objective responses occurred in patients with high mesothelin expression, and complete and partial responses were concentrated in tumors with ≥60% expression (2+/3+ membrane intensity), suggesting a threshold effect 33. Critically, not all high-expressors responded, likely reflecting the bystander-killing mechanism of ADCs, in which released DM4 payload kills neighboring non-expressing dividing cells, making spatial heterogeneity a relevant concern 33. Anti-drug antibodies (ADAs) were detected in 8 of 22 ADA-negative baseline patients but had no influence on tumor response or toxicity, suggesting ADA monitoring is a safety correlate but not a predictive gating factor 33. A companion diagnostic using a validated mesothelin IHC assay (e.g., VENTANA MSLN SP74) with a ≥30% 2+/3+ cutoff is required for ADC trial enrollment, with exploratory stratification at ≥60% to refine the expression-response gradient.
MTAP Synthetic Lethality
MTAP deletion creates a dependency on PRMT5 and MAT2A, providing a synthetic lethality therapeutic strategy 2328. PRMT5 inhibitors (e.g., AMG 193) have demonstrated favorable safety profiles and early antitumor activity across MTAP-deleted solid tumors, though mesothelioma-specific clinical data are limited in the retrieved materials 28. Given that MTAP deletions are far more prevalent in pleural (60–74%) than peritoneal mesothelioma (8–35%), enrichment strategies for these trials will be more feasible in pleural disease 37. MTAP IHC (preferred antibody: mAb 1813) is the recommended screening assay, with FISH or NGS confirmation 13.
ALK Rearrangements in Young Patients
A small but clinically important subset of young peritoneal mesothelioma patients—particularly those without asbestos exposure—harbor ALK rearrangements, with documented responses to ceritinib and crizotinib 41. ALK FISH or NGS should be performed in patients under 40 years of age with mesothelioma and absent typical asbestos history.
YAP/Hippo Pathway
Aberrant Hippo pathway signaling and YAP overexpression have been identified as potential prognostic biomarkers and therapeutic targets, though clinical-stage data are early 25.
5. Comprehensive Genomic Profiling: Feasibility and Clinical Utility
The EORTC-SPECTA Arcagen study demonstrated that comprehensive genomic profiling (>300 genes, TMB, MSI) is operationally feasible in mesothelioma, with a median turnaround time of 8 days from sample receipt 5. Tissue-based FFPE profiling identified molecular alterations in 90% of 42 pleural mesothelioma samples versus only 43% of 14 ctDNA cases, confirming tissue superiority over liquid biopsy for molecular characterization 5. The molecular tumor board advised treatment options in 30% of patients, with 75% directed to clinical trials, 22% to off-label drugs, and 3% to early access programs 5. Germline BAP1 testing was triggered in five patients based on suspected pathogenic variants, highlighting that comprehensive profiling may identify germline risk. Triggers for germline BAP1 testing should include: age <50 years, family history of BAP1-associated cancers (uveal melanoma, renal cell carcinoma, pancreatic cancer), mesothelioma without asbestos exposure, or multiple primary cancers 42.
6. Practical Biomarker Sampling Schedule and Trial-Design Recommendations
Tiered Sampling Schedule
| Timepoint | Specimen | Biomarker | Purpose | Classification |
|---|---|---|---|---|
| Baseline (pretreatment) | FFPE tumor tissue | BAP1 IHC (C4), MTAP IHC (mAb 1813), mesothelial/carcinoma panel, nuclear grade, histologic subtype | Diagnosis, risk stratification | Required |
| Baseline | FFPE or fresh core | Mesothelin IHC (≥30% 2+/3+ cutoff), CDKN2A FISH | ADC trial enrichment; synthetic lethality enrichment | Required for targeted trials |
| Baseline | Plasma | SMRP, ctDNA (NGS-based) | Prognostic context; translational monitoring | Required (SMRP); optional (ctDNA) |
| Baseline | FFPE tumor tissue | PD-L1 IHC (22C3 clone), RNA-seq, WES, mIF (TLS, CD8, CD19, CD4, PD-1, TIM-3, TIGIT) | Exploratory immune profiling | Exploratory/correlative |
| On-treatment (C2D1, C3D1) | Plasma | ctDNA kinetics, SMRP dynamics | Response monitoring hypothesis | Translational |
| Progression | Plasma or tissue | ctDNA, re-biopsy if feasible | Resistance mechanisms | Translational |
| ADC trials (predose and D8 C1; D1 every even cycle; EOT) | Serum | Anti-drug antibodies (ADA), PK | Safety/PK correlate (not predictive gating) | Required for ADC trials |
Biomarker Role Classification for Trial Design
| Biomarker | Role | Rationale |
|---|---|---|
| Histologic subtype (epithelioid vs. non-epithelioid) | Mandatory stratification variable | Strongest independent prognostic factor; affects surgery eligibility and ICI benefit |
| BAP1 IHC | Required diagnostic; exploratory stratification for ICI | Diagnostic sensitivity; emerging predictive signal in peritoneal disease |
| MTAP IHC (mAb 1813) | Required diagnostic; enrichment for PRMT5/MAT2A inhibitor trials | 100% specific for malignancy; dominant alteration in pleural disease |
| Mesothelin IHC | Required for ADC trial enrollment (≥30% 2+/3+) | All ADC responders showed high expression in phase I data |
| PD-L1 IHC (22C3) | Collect at baseline; exploratory subgroup analysis only | Not predictive for ICI selection; retain for correlative science |
| TMB/MSI | Exploratory only | Low TMB universally; MSI-high rare (4%); no predictive utility |
| TLS density + IL24/EMT ratio | Exploratory predictive composite | Strongest predictive signals from CONFIRM trial; not yet validated for enrichment |
| Nuclear grade | Stratification variable in epithelioid trials | Independent prognostic factor; reproducible in routine practice |
| SMRP | Baseline prognostic context; longitudinal monitoring | Independent prognostic factor in ICT setting |
| ctDNA | Translational; prespecified correlative endpoint | Feasibility data limited; incorporate with hypothesis-driven analyses |
| ALK FISH/NGS | Required in young patients (<40 years) without asbestos exposure | Rare actionable target with documented therapeutic responses |
| Germline BAP1 | Triggered genetic counseling for at-risk patients | Identifies BAP1 tumor predisposition syndrome; cascade family testing |
Enrichment, Stratification, and Endpoint Strategy by Trial Phase
Early-phase trials (Phase I/II): Enrich ADC trials with mesothelin IHC ≥30% 2+/3+; enrich PRMT5/MAT2A inhibitor trials with MTAP IHC loss (confirmed by FISH or NGS). Collect baseline FFPE for WES, RNA-seq, and mIF; collect plasma for ctDNA and SMRP. Exploratory endpoints should pre-specify associations between mesothelin expression level (stratified at 30–60% vs. ≥60%), TLS density, IL24/EMT ratio, and response 1233. Do not use PD-L1 or TMB for ICI trial enrichment 2.
Late-phase trials (Phase III): Mandatory stratification by histologic subtype. Consider BAP1 status and PD-L1 as exploratory stratification factors if Phase II signals support pre-specification. Primary endpoints remain overall survival and progression-free survival; biomarker-response correlations and ctDNA kinetics should constitute pre-specified correlative endpoints 44. Adaptive designs with biomarker-stratified expansion cohorts—for example, BAP1-altered peritoneal mesothelioma for ICI trials—should be considered to address the underrepresentation of peritoneal disease in immunotherapy trials 2940.
Peritoneal-specific considerations: Given lower MTAP/CDKN2A deletion rates and distinct immunophenotypic features, peritoneal mesothelioma cohorts should be analyzed separately in stratification and subgroup analyses. For CRS/HIPEC surgical eligibility, biomarker-informed high-risk features triggering perioperative systemic therapy include Ki-67 >9%, nodal metastases, peritoneal cancer index >17, incomplete cytoreduction (CC>1), biphasic histology, and bicavitary disease 1.
Conclusions
A coherent biomarker framework for mesothelioma requires a tiered approach that distinguishes diagnostic markers—BAP1 loss and MTAP IHC (mAb 1813 preferred), confirmed by CDKN2A FISH where needed—from prognostic markers (nuclear grade, histologic subtype, SMRP), predictive markers (PD-L1 and TMB, which are exploratory only, in contrast to emerging TLS-based and molecular composite scores), and translational targets (mesothelin for ADCs, MTAP loss for synthetic lethality, ALK in young patients). Mandatory baseline tissue collection enabling multi-omics profiling within an 8-day turnaround is feasible and can identify actionable options in approximately 30% of patients 5. Plasma sampling for SMRP and ctDNA provides longitudinal translational opportunities, though dynamic changes require prospective validation as surrogate endpoints. This framework—grounded in reproducible histopathologic practice, guided by emerging molecular biology, and operationalized through practical sampling and pre-specified statistical analyses—represents the current evidence-based foundation for advancing precision oncology in both pleural and peritoneal mesothelioma.