Sinicrope, Frank A

Abstract Purpose: Immune checkpoint blockade (ICB) induces frequent and durable responses in metastatic deficient DNA mismatch repair (dMMR) colorectal cancer, yet substantial molecular heterogeneity and resistance remain. We sought to identify candidate tumor- and immune-related biomarkers associated with clinical outcomes following anti–PD-1 therapy. Experimental Design: Consecutive patients with metastatic dMMR colorectal cancer (N = 39) treated with anti–PD-1 therapy underwent tumor profiling using a validated immune-enhanced exome and transcriptome platform. Microsatellite instability (MSI) burden was quantified as the percentage of unstable microsatellite loci using MSIsensor-pro. Associations with objective response were evaluated, and progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models. Results: Higher MSI burden was associated with improved objective response (P = 0.018) and survival. The dichotomized MSI level (Q2–4 vs. Q1) was associated with longer PFS [hazard ratio (HR), 0.18; 95% confidence interval (CI), 0.06–0.56; P = 0.003] and OS (HR, 0.20; 95% CI, 0.07–0.58; P = 0.003), with similar results when modeled continuously. MSI burden correlated with neoantigen clonality but not burden (R = 0.53, P = 0.01). Responders exhibited significantly greater T-cell receptor repertoire diversity; both T- and B-cell receptor diversities were associated with survival. Although human leukocyte antigen (HLA) A, HLA-B, and HLA-C expression was not prognostic, the HLA-B*07:02 allele was associated with the best overall response. In contrast, overexpression of immune exhaustion–related genes and cytotoxic T-cell and natural killer cell exhaustion phenotypes were associated with ICB resistance and poorer prognosis. Conclusions: Integrated exome/transcriptome profiling with MSI quantification identified MSI burden and adaptive immune repertoire diversity as potential correlates of response and survival following ICB. These findings suggest a mechanistic link among genomic instability, antigen recognition diversity, and immunotherapy benefit.

PURPOSE Detection of molecular residual disease using circulating tumor DNA (ctDNA) may enable postoperative risk stratification and guide adjuvant therapy. We evaluated the prognostic value of a tissue-free, epigenomic ctDNA assay in patients with stage III colon cancer (CC) enrolled in a phase III adjuvant chemotherapy trial. METHODS Plasma samples were collected after surgery and before adjuvant infusional fluorouracil, leucovorin, and oxaliplatin alone or combined with cetuximab. ctDNA was analyzed using a tissue-free assay; in ctDNA-positive patients, tumor fraction (TF) was quantified and genotyping was performed with a 739-gene panel. Associations with disease-free survival (DFS), time to recurrence (TTR), and overall survival (OS) were assessed using multivariable Cox models adjusted for covariates. RESULTS Among 2,260 evaluable patients, 461 (20.4%) were ctDNA-positive with significantly higher detection in advanced T-/N-stage, high-grade, obstruction/perforation, and BRAF V600E tumors. At a median follow-up of 6.1 years, ctDNA positivity was independently associated with shorter TTR (hazard ratio [HR], 5.96 [95% CI, 5.11 to 6.96]), DFS (HR, 5.03 [95% CI, 4.36 to 5.81]), and OS (HR, 4.45 [95% CI, 3.76 to 5.27]; all P < .0001). The 5-year DFS was 27.7% (95% CI, 23.8 to 32.2) v 77.1% (95% CI, 75.1 to 79.1) in ctDNA-positive versus ctDNA-negative patients, and adverse prognostic impact was greater in lower T/N stage, low-risk, and dMMR subsets (interaction P = .0012-.041). Among ctDNA-positive patients, TF was nearly double in those who recurred or died ( P = .0002) and stratified patients for TTR, DFS, and OS (all adjusted P < .002). Genotyping identified mutations in FLT1 (OR, 8.99) and PREX2 (OR, 7.73) genes that were most strongly associated with recurrence ( P < .03). CONCLUSION Evaluation of ctDNA in resected stage III CC using a tissue-free assay provided robust and independent prognostic value. Higher ctDNA burden, dMMR, and specific mutations defined poor prognostic groups among ctDNA-positive patients.

Abstract Purpose: Routine testing for mismatch repair deficiency (dMMR)/microsatellite instability (MSI) in colorectal cancers is recommended for Lynch syndrome screening, prognosis, and treatment guidance. In the metastatic setting, RAS/BRAF mutations guide treatment decisions. The impact of these mutations on the tumor immune microenvironment (TiME) in MSI/dMMR colorectal cancer is not known. Experimental Design: The study involved retrospective analysis of 448 patients with stage I to IV MSI/dMMR colorectal cancer profiled using next-generation sequencing (Tempus xT DNA sequencing of 595–648 genes at 500x coverage and Tempus xR whole-exome capture RNA sequencing). MSI status was determined by assessing 44 or 239 loci using next-generation sequencing. dMMR was determined using IHC. Tumor mutational burden, tumor neoantigen burden (NTB), PD-L1, immune infiltration, and canonical immune pathways (76 gene set signatures) were analyzed. Results: The median age at diagnosis was 67 years (range, 21–86); 59% of patients were female, and 70% were stage III to IV. Among the 448 patients, 100 (22%) harbored RAS mutations (RASmut), 119 (27%) a BRAFV600E mutation, and 229 (51%) were double wild type. RASmut exhibited lower NTB (median, 12 vs. 15 vs. 16; P = 0.003) and PD-L1 (3.6% vs. 13% vs. 24%; P &lt; 0.001) than BRAFV600E and wild-type tumors, respectively. The RASmut TiME had lower overall inflammation and fewer infiltrating CD8+ T cells than wild-type or BRAFV600E tumors. In contrast, BRAFV600E tumors exhibited hyperproliferative characteristics associated with broad metabolic reprogramming but a similarly inflamed TiME compared with wild-type tumors. Conclusions: Our data suggest that MSI/dMMR colorectal cancers harboring RASmut are less immunogenic, and the TiME contains a lower inflammatory profile than wild-type or BRAFV600E tumors. Further analysis and validation are required to confirm these findings.

PURPOSE The development of colorectal liver metastases (CRLMs) is associated with poor prognosis, and recent data suggest that metastasis to the liver is associated with resistance to immunotherapy. We characterized the microenvironment of primary colorectal carcinomas (CRCs) relative to their synchronous CRLMs using a validated segmentation algorithm (QuantCRC) that quantifies 15 distinct morphologic tumor features. MATERIALS AND METHODS Adult patients with CRC with synchronous CRLM (N = 57) at Mayo Clinic were identified from the electronic health record. Routine tumor hematoxylin and eosin sections were digitized and reviewed for quality control. QuantCRC (Aiforia, Inc) was applied to digitized images to extract 15 GI pathologist predefined morphological features. Tumor features were compared between paired primary tumors and their synchronous CRLMs using the Wilcoxon signed-rank test overall and within subgroups. Linear regression models were used to find predictors of the paired differences in the distinct tumor morphological features. RESULTS The study included 57 patients (median age 59 years [IQR, 50-73], 51% female) with CRC primary and synchronous CRLM, and among primaries, 37 (65%) were left-sided. QuantCRC identified six of 15 morphological features that differed significantly between primaries and their CRLMs. Compared with the primary, CRLM showed reduced stroma, more high-grade tumor and necrosis, higher tumor–stromal ratio (TSR; all P < .05), and a trend toward increased tumor-infiltrating lymphocyte (TIL) density ( P = .052). Synchronous CRLM from patients with left-sided primary tumors had significantly higher TSR, percent high-grade, percent necrosis, and TIL density compared with the primary (all P values ≤0.04). Among right-sided primaries, CRLM had a significantly reduced percent mature stroma ( P = .0296) and a higher percent necrosis ( P = .0027). CONCLUSION Compared with the primary, synchronous CRLMs showed a higher TSR and increased high-grade tumor and necrosis, which are features of tumor aggressiveness and a metastatic phenotype.

Abstract Purpose: KRAS G12C inhibitors can treat KRASG12C-mutant advanced colorectal cancers and pancreatic ductal adenocarcinomas (PDAC), but alterations in Kirsten rat sarcoma (KRAS), EGFR, BRAF, MAP2K1, and other genes bypass KRAS inhibition and reduce therapy efficacy. Our study evaluates the genetic landscape of candidate primary resistance alterations relevant to KRAS targeting in KRASG12C-mutant colorectal cancer and PDAC. Experimental Design: We analyzed two cohorts (national database and Mayo) of patients with advanced colorectal cancer or PDAC tested with next-generation sequencing of ctDNA via Guardant360. Cohorts were divided into three groups: KRASG12C alone (KRASG12C without a resistance gene), KRASG12C with resistance (KRASG12C and ≥1 candidate resistance gene), and KRAS not detected. Candidate resistance mutations were inferred from the reported literature. Results: Among the national (13,603 colorectal cancer and 5,016 PDAC cases) and Mayo (741 colorectal cancer and 422 PDAC cases) cohorts, resistance alterations were identified in a considerable number of KRASG12C cases (46.5% of national colorectal cancer, 16.4% of national PDAC, 53.8% of Mayo colorectal cancer, and 36.4% of Mayo PDAC). The presence of resistance alterations was associated with a trend toward worse overall survival in KRASG12C colorectal cancer (P = 0.05). Conclusions: Putative resistance alterations are prevalent in PDAC and colorectal cancer and may limit monotherapy efficacy. Identifying these alterations has potential implications in optimal patient selection for targeted therapies and the development of combination therapy strategies to overcome primary resistance.

Abstract Purpose: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti–PD-1 therapy in metastatic colorectal cancer. Experimental Design: Primary colorectal cancers with deficient mismatch repair from patients treated with anti–PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini–Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. Results: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti–PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P &lt; 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti–PD-1; each marker was significantly associated with longer patient PFS (0.02 &lt; adjusted HR &lt; 0.17; P &lt; 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. Conclusions: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti–PD-1, highlighting their potential clinical significance.

Rates of adjuvant chemotherapy in patients with early-onset versus later-onset stage II colon cancer

Neoadjuvant immunotherapy effectively uses the in situ tumor as a reservoir of tumor antigens to promote systemic antitumor immunity. Studies indicate that intratumoral responses to immune checkpoint inhibitors (ICIs) are mediated by resident memory T cells cells that are sequestered in tumors and have specificity for a wide range of tumor antigens. ICI treatment produces de novo priming of CD8+ T cells in tumor and in tumor-draining lymph nodes, and can boost the antitumor immune response by blocking inhibitory checkpoint proteins that can turn off T cells within the tumor. Neoadjuvant ICI treatment can enhance both intratumoral and systemic antitumor immunity, including expansion of intratumoral T-cell clones which is strongly associated with pathological treatment response. Recent data have shown high rates of pathological response to neoadjuvant immunotherapy with prolongation of survival compared with adjuvant ICI therapy alone in patients with unresectable or advanced melanoma. These data suggest that removal of the reservoir of tumor-specific T cells in the tumor and draining nodes by surgical resection may remove a significant proportion of the patient’s antitumor immunity with the potential to compromise ICI outcomes.

Abstract Deep learning may detect biologically important signals embedded in tumor morphologic features that confer distinct prognoses. Tumor morphologic features were quantified to enhance patient risk stratification within DNA mismatch repair (MMR) groups using deep learning. Using a quantitative segmentation algorithm (QuantCRC) that identifies 15 distinct morphologic features, we analyzed 402 resected stage III colon carcinomas [191 deficient (d)-MMR; 189 proficient (p)-MMR] from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy. Results were validated in an independent cohort (176 d-MMR; 1,094 p-MMR). Association of morphologic features with clinicopathologic variables, MMR, KRAS, BRAFV600E, and time-to-recurrence (TTR) was determined. Multivariable Cox proportional hazards models were developed to predict TTR. Tumor morphologic features differed significantly by MMR status. Cancers with p-MMR had more immature desmoplastic stroma. Tumors with d-MMR had increased inflammatory stroma, epithelial tumor-infiltrating lymphocytes (TIL), high-grade histology, mucin, and signet ring cells. Stromal subtype did not differ by BRAFV600E or KRAS status. In p-MMR tumors, multivariable analysis identified tumor-stroma ratio (TSR) as the strongest feature associated with TTR [HRadj 2.02; 95% confidence interval (CI), 1.14–3.57; P = 0.018; 3-year recurrence: 40.2% vs. 20.4%; Q1 vs. Q2–4]. Among d-MMR tumors, extent of inflammatory stroma (continuous HRadj 0.98; 95% CI, 0.96–0.99; P = 0.028; 3-year recurrence: 13.3% vs. 33.4%, Q4 vs. Q1) and N stage were the most robust prognostically. Association of TSR with TTR was independently validated. In conclusion, QuantCRC can quantify morphologic differences within MMR groups in routine tumor sections to determine their relative contributions to patient prognosis, and may elucidate relevant pathophysiologic mechanisms driving prognosis. Significance: A deep learning algorithm can quantify tumor morphologic features that may reflect underlying mechanisms driving prognosis within MMR groups. TSR was the most robust morphologic feature associated with TTR in p-MMR colon cancers. Extent of inflammatory stroma and N stage were the strongest prognostic features in d-MMR tumors. TIL density was not independently prognostic in either MMR group.