Performance of Next-Generation Sequencing for the Detection of Microsatellite Instability in Colorectal Cancer With Deficient DNA Mismatch Repair.
Next-generation sequencing (NGS) was recently approved by the United States Food and Drug Administration to detect microsatellite instability (MSI) arising from defective mismatch repair (dMMR) in patients with metastatic colorectal cancer (mCRC) before treatment with immune checkpoint inhibitors (ICI). In this study, we aimed to evaluate and improve the performance of NGS to identify MSI in CRC, especially dMMR mCRC treated with ICI.
CRC samples used in this post hoc study were reassessed centrally for MSI and dMMR status using the reference methods of pentaplex polymerase chain reaction and immunohistochemistry. Whole-exome sequencing (WES) was used to evaluate MSISensor, the Food and Drug Administration-approved and NGS-based method for assessment of MSI. This was performed in (1) a prospective, multicenter cohort of 102 patients with mCRC (C1; 25 dMMR/MSI, 24 treated with ICI) from clinical trials NCT02840604 and NCT033501260, (2) an independent retrospective, multicenter cohort of 113 patients (C2; 25 mCRC, 88 non-mCRC, all dMMR/MSI untreated with ICI), and (3) a publicly available series of 118 patients with CRC from The Cancer Genome Atlas (C3; 51 dMMR/MSI). A new NGS-based algorithm, namely MSICare, was developed. Its performance for assessment of MSI was compared with MSISensor in C1, C2, and C3 at the exome level or after downsampling sequencing data to the MSK-IMPACT gene panel. MSICare was validated in an additional retrospective, multicenter cohort (C4) of 152 patients with new CRC (137 dMMR/MSI) enriched in tumors deficient in MSH6 (n = 35) and PMS2 (n = 9) after targeted sequencing of samples with an optimized set of microsatellite markers (MSIDIAG).
At the exome level, MSISensor was highly specific but failed to diagnose MSI in 16% of MSI/dMMR mCRC from C1 (4 of 25; sensitivity, 84%; 95% confidence interval [CI], 63.9%-95.5%), 32% of mCRC (8 of 25; sensitivity, 68%; 95% CI, 46.5%-85.1%), and 9.1% of non-mCRC from C2 (8 of 88; sensitivity, 90.9%; 95% CI, 82.9%-96%), and 9.8% of CRC from C3 (5 of 51; sensitivity, 90.2%; 95% CI, 78.6%-96.7%). Misdiagnosis included 4 mCRCs treated with ICI, of which 3 showed an overall response rate without progression at this date. At the exome level, reevaluation of the MSI genomic signal using MSICare detected 100% of cases with true MSI status among C1 and C2. Further validation of MSICare was obtained in CRC tumors from C3, with 96.1% concordance for MSI status. Whereas misdiagnosis with MSISensor even increased when analyzing downsampled WES data from C1 and C2 with microsatellite markers restricted to the MSK-IMPACT gene panel (sensitivity, 72.5%; 95% CI, 64.2%-79.7%), particularly in the MSH6-deficient setting, MSICare sensitivity and specificity remained optimal (100%). Similar results were obtained with MSICare after targeted NGS of tumors from C4 with the optimized microsatellite panel MSIDIAG (sensitivity, 99.3%; 95% CI, 96%-100%; specificity, 100%).
In contrast to MSISensor, the new MSICare test we propose performs at least as efficiently as the reference method, MSI polymerase chain reaction, to detect MSI in CRC regardless of the defective MMR protein under both WES and targeted NGS conditions. We suggest MSICare may rapidly become a reference method for NGS-based testing of MSI in CRC, especially in mCRC, where accurate MSI status is required before the prescription of ICI.
Ratovomanana T
，Cohen R
，Svrcek M
，Renaud F
，Cervera P
，Siret A
，Letourneur Q
，Buhard O
，Bourgoin P
，Guillerm E
，Dorard C
，Nicolle R
，Ayadi M
，Touat M
，Bielle F
，Sanson M
，Le Rouzic P
，Buisine MP
，Piessen G
，Collura A
，Fléjou JF
，de Reyniès A
，Coulet F
，Ghiringhelli F
，André T
，Jonchère V
，Duval A
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Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency.
Patients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs.
MSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation.
Among 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (37%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (P < 0.0001), metastatic resection (P = 0.001) and LS-like mCRC (P = 0.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR) = 3.19, P = 0.01) and metastatic resection (HR = 4.2, P = 0.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HR = 0.28, P = 0.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; P = 0.44).
LS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations.
Cohen R
，Buhard O
，Cervera P
，Hain E
，Dumont S
，Bardier A
，Bachet JB
，Gornet JM
，Lopez-Trabada D
，Dumont S
，Kaci R
，Bertheau P
，Renaud F
，Bibeau F
，Parc Y
，Vernerey D
，Duval A
，Svrcek M
，André T
... -
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BRAF + EGFR +/- MEK inhibitors after immune checkpoint inhibitors in BRAF V600E mutated and deficient mismatch repair or microsatellite instability high metastatic colorectal cancer.
Immune checkpoint inhibitors (ICIs) are the guideline endorsed first choice for patients with deficient mismatch repair or microsatellite instability high (dMMR/MSI-H) mCRC, however a significant proportion experience primary or secondary resistance. BRAF V600E mutated (BRAFm) and dMMR/MSI-H mCRC can be treated with BRAF + EGFR inhibitors but specific data on the efficacy after progression to ICIs are missing.
We collected consecutive patients with BRAFm dMMR/MSI-H mCRC treated from 2017 to 2024 with a combination of BRAFi+EGFRi+/-MEKi, after disease progression on ICIs. A control cohort of BRAFm pMMR/MSS mCRC patients treated with encorafenib+cetuximab+/-binimetinib from 2nd line was used.
dMMR/MSI-H (n = 50) BRAFm mCRC patients were more often > 70-year-old, with right-sided primary tumors, without liver but more lymphnode metastases than pMMR/MSS (n = 170). They were treated more frequently beyond 2nd line and 45 % were primary progressors to ICIs. Lower ORR (18 % versus 32 %, p = 0.09) and DCR (60 % versus 73 %, p = 0.11) was seen without reaching significance in dMMR/MSI-H as compared to pMMR/MSS patients. After a median follow-up of 14.04 months, no differences in PFS (median 5.13 versus 4.50 months, HR 0.83, 95 %CI: 0.57-1.20, p = 0.31) and OS (median 10.75 versus 9.11 months, HR 0.89, 95 %CI: 0.59-1.32, p = 0.55) were observed.
Our results show that BRAFm dMMR/MSI-H mCRC patients benefit from BRAFi+EGFRi+/-MEKi after progression under ICIs. Despite lower ORR and DCR, the outcome is not different from that observed in pMMR/MSS BRAFm CRC and is in line with the results of the BEACON registration trial.
Ambrosini M
，Tougeron D
，Modest D
，Guimbaud R
，Kopetz S
，Decraecker M
，Kim S
，Coutzac C
，Perkins G
，Alouani E
，Marmorino F
，Pernot S
，Sinicrope FA
，Elez E
，Parent P
，Cremolini C
，Pietrantonio F
，Lonardi S
，Gallois C
，Taieb J
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