Single-cell sequencing reveals the immune microenvironment landscape related to anti-PD-1 resistance in metastatic colorectal cancer with high microsatellite instability.
The objective response rate of microsatellite instability-high (MSI-H) metastatic colorectal cancer (mCRC) patients with first-line anti-programmed cell death protein-1 (PD-1) monotherapy is only 40-45%. Single-cell RNA sequencing (scRNA-seq) enables unbiased analysis of the full variety of cells comprising the tumor microenvironment. Thus, we used scRNA-seq to assess differences among microenvironment components between therapy-resistant and therapy-sensitive groups in MSI-H/mismatch repair-deficient (dMMR) mCRC. Resistance-related cell types and genes identified by this analysis were subsequently verified in clinical samples and mouse models to further reveal the molecular mechanism of anti-PD-1 resistance in MSI-H or dMMR mCRC.
The response of primary and metastatic lesions to first-line anti-PD-1 monotherapy was evaluated by radiology. Cells from primary lesions of patients with MSI-H/dMMR mCRC were analyzed using scRNA-seq. To identify the marker genes in each cluster, distinct cell clusters were identified and subjected to subcluster analysis. Then, a protein‒protein interaction network was constructed to identify key genes. Immunohistochemistry and immunofluorescence were applied to verify key genes and cell marker molecules in clinical samples. Immunohistochemistry, quantitative real-time PCR, and western blotting were performed to examine the expression of IL-1β and MMP9. Moreover, quantitative analysis and sorting of myeloid-derived suppressor cells (MDSCs) and CD8+ T cells were performed using flow cytometry.
Tumor responses in 23 patients with MSI-H/dMMR mCRC were evaluated by radiology. The objective response rate was 43.48%, and the disease control rate was 69.57%. ScRNA-seq analysis showed that, compared with the treatment-resistant group, the treatment-sensitive group accumulated more CD8+ T cells. Experiments with both clinical samples and mice indicated that infiltration of IL-1β-driven MDSCs and inactivation of CD8+ T cells contribute to anti-PD-1 resistance in MSI-H/dMMR CRC.
CD8+ T cells and IL-1β were identified as the cell type and gene, respectively, with the highest correlation with anti-PD-1 resistance. Infiltration of IL-1β-driven MDSCs was a significant factor in anti-PD-1 resistance in CRC. IL-1β antagonists are expected to be developed as a new treatment for anti-PD-1 inhibitor resistance.
Wu T
，Zhang X
，Liu X
，Cai X
，Shen T
，Pan D
，Liang R
，Ding R
，Hu R
，Dong J
，Li F
，Li J
，Xie L
，Wang C
，Geng S
，Yang Z
，Xing L
，Li Y
... -
《BMC Medicine》
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
... -
《-》
Real-World Treatment Patterns and Clinical Outcomes for Standard of Care Regimens in Patients with Deficient MMR or MSI-High Metastatic Colorectal and Non-Colorectal Cancer: A Retrospective Chart Review Study in France.
There is limited evidence on the effectiveness of standard of care (SOC) treatments in previously treated patients with deficient mismatch repair (dMMR) or microsatellite instability high (MSI-H) metastatic cancer. Immune checkpoints inhibitors (ICIs) have emerged as key treatments for dMMR/MSI-H tumors. However, clinical outcomes data with SOC regimens are still limited. Study objectives were to evaluate real-world treatment patterns and clinical outcomes in patients with dMMR/MSI-H metastatic colorectal cancer (mCRC) and non-CRC receiving SOC regimens. Given the resulting small cohort of patients with metastatic non-CRC, only summary results are provided.
Two French university hospitals participated in a retrospective chart review study in which adult patients with dMMR/MSI-H mCRC and non-CRC were enrolled. Treatment patterns, overall survival (OS) from the start of third-line (3L, for mCRC) or second-line (2L, for non-CRC) treatment, and the best overall response rate (BORR) were reported.
Thirty-six patients with dMMR/MSI-H mCRC were included. Almost all patients received combination treatments both in first-line (1L, 100%) and 2L (97%). For 3L and later, combination treatment was preferred over monotherapy but decreased in usage (75% at 3L and 57% at fourth-line, 4L). The BORR was 5.7% and median OS for patients with dMMR/MSI-H mCRC receiving 3L therapy was 9.0 months (95% confidence interval, CI 4.0-14.1); it decreased to 4.1 months (95% CI 4.0-9.0) when survival data of patients receiving ICIs at fourth or later lines were censored at progression date of prior treatment line.
Real-world clinical outcomes observed for patients with dMMR/MSI-H mCRC receiving 3L treatment(s) are suboptimal, suggesting a high unmet need that could be addressed with ICIs.
Roset M
，Amonkar M
，Patel R
，Lara N
，Kothari S
... -
《-》
ResearchGate
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