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Revised supplemental figures and tables

Post treatment and pre treatment DEG in RFS long vs short

Sensitivity analysis comparing mutation prevalence estimates by site of biopsy of tissue used for next generation sequencing panel in metastatic colorectal cancer.

Univariate logistic regression models for deleterious mutation among 133 sequenced subjects (n = 26 with deleterious mutation).

Supplementary Fig S3. Association of gene size and the differential expression of IFN-γ pathway in genes commonly mutated in MSS CRC

Purpose:Little is known about the genetic alterations characteristic of small bowel adenocarcinoma (SBA). Our purpose was to identify targetable alterations and develop experimental models of this disease.Experimental Design: Whole-exome sequencing (WES) was completed on 17 SBA patient samples and targeted-exome sequencing (TES) on 27 samples to confirm relevant driver mutations. Two SBA models with ERBB2 kinase activating mutations were tested for sensitivity to anti-ERBB2 agents in vivo and in vitro. Biochemical changes were measured by reverse-phase protein arrays.Results:WES identified somatic mutations in 4 canonical pathways (WNT, ERBB2, STAT3, and chromatin remodeling), which were validated in the TES cohort. Although APC mutations were present in only 23% of samples, additional WNT-related alterations were seen in 12%. ERBB2 mutations and amplifications were present in 23% of samples. Patients with alterations in the ERBB2 signaling cascade (64%) demonstrated worse clinical outcomes (median survival 70.3 months vs. 109 months; log-rank HR = 2.4, P = 0.03). Two ERBB2-mutated (V842I and Y803H) cell lines were generated from SBA patient samples. Both demonstrated high sensitivity to ERBB2 inhibitor dacomitinib (IC50 < 2.5 nmol/L). In xenografts derived from these samples, treatment with dacomitinib reduced tumor growth by 39% and 59%, respectively, whereas it had no effect in an SBA wild-type ERBB2 model.Conclusions:The in vitro and in vivo models of SBA developed here provide a valuable resource for understanding targetable mutations in this disease. Our findings support clinical efforts to target activating ERBB2 mutations in patients with SBA that harbor these alterations.

Comparison of (A) cell viability and (B) protein expression levels as assessed by reverse phase protein arrays in Ba/F3 transformation assay demonstrating (C) a lack of correlation between protein level and transformation, supporting that functional status rather than protein level are driving cellular transformation.

Classification accuracy on validation data set, the entire set (V2), out-of-sample prediction (V2o), Affymetrix subset (V2a), and TCGA RNAseq subset (TCGA) for various methods as function of number of genes.

Purpose:Despite the prognostic importance of immune infiltrate in colorectal cancer, immunotherapy has demonstrated limited clinical activity in refractory metastatic proficient mismatch-repair (pMMR) colorectal cancer. This study explores combining anti–CTLA-4 and an anti–PD-L1 therapy in the preoperative management of resectable colorectal cancer liver metastases with the intent to improve immune responses in this disease setting.Patients and Methods:Patients with resectable colorectal cancer liver-only metastases received one dose of tremelimumab and durvalumab preoperatively followed by single-agent durvalumab postoperatively. Primary objectives were to determine feasibility and safety.Results:A total of 24 patients were enrolled between November 2016 and November 2019. Twenty-three patients received treatment [21 pMMR and 2 deficient mismatch-repair (dMMR)] and subsequently 17 (74%; 95% CI: 53%–88%) underwent surgical resection. Grade 3/4 treatment-related immune toxicity and postoperative grade 3/4 toxicity were seen in 5/23 (22%; 95% CI: 10%–44%) and 2/17 (12%; 95% CI: 2%–38%) patients. The median relapse-free survival (RFS) was 9.7 (95% CI: 8.1–17.8) months, and overall survival was 24.5 (95% CI: 16.5–28.4) months. Four patients demonstrated complete pathologic response, two dMMR patients and two POLE mutation patients. Pre- and post-tumor tissue analysis by flow cytometry, immunofluorescence, and RNA sequencing revealed similar levels of T-cell infiltration, but did demonstrate evidence of CD8+ and CD4+ activation posttreatment. An increase in B-cell transcriptome signature and B-cell density was present in posttreatment samples from patients with prolonged RFS.Conclusions:This study demonstrates the safety of neoadjuvant combination tremelimumab and durvalumab prior to colorectal cancer liver resection. Evidence for T- and B-cell activation following this therapy was seen in pMMR metastatic colorectal cancer.

Supplementary figures and supplemental methods

Operating characteristics. A summary of the probability of stopping the trial early for the indicated true response rates.

Supplemental legend

Purpose:Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory.Experimental Design:We performed an in silico experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials.Results:The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to “gold standard” CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1–3 and good prognosis for CMS2 in metastatic colorectal cancer (P < 0.001).Conclusions:We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.

Nanostring CMS Classifier Performance: Performance of Nanostring 100 gene CMS classifier applied to FFPE samples (6A and 6C) and FF samples (6B and 63D). The top two figures plot 4-class accuracy vs. classification confidence, with the dots marking individual samples either correctly (1.0) or incorrectly (0.0), with color indicat-ing correct CMS. The line contains a generalized additive model (GAM) fit to these data with 95% pointwise confidence bands, and demonstrates that samples classified with greater confidence were more likely to be cor-rectly classified. Figure 6C and 6D plot 4-class accuracy vs. RNA quality, defined as 0nt (FFPE) or RIN (FF). Note that there is little if any association of CMS accuracy with RNA quality, suggesting that the perfor-mance of classifier is robust to RNA quality in this study.

Supplemental material

Classification accuracy of different modelling strategies on training data set (V1) using 4-fold cross validation.

Supplementary Table S1

Comparison of Classifiers. Comparison of random forest (RF) and single sample predictors (SSP) in Guinney et al. (2015) paper with our wSVM classifier with 472 genes, either forcing a single CMS call for each sample (all calls) or only when classified with high confidence

Lack of correlation between Src activity in PBMCs and hepatic tumors. For the patients in the expansion cohort, the change in the expression of pSrc and total Src was not correlated with a change in the phosphorlyation of the Src substrate paxillin. Patients with no change or a decrease in the expression of pSrc or Src from baseline to C2D8 were included in the "no increase" category.

Supplementary Figure 2. ROC curves for CTC counts using CSV: ROC curves were determined to discriminate the responding/ stable patients from that of the non-responding/ progressive metastatic colon cancer patients. An AUC-ROC value of closer to 1 denotes a perfect method for discrimination.

Supplemental Figure Legends

RFS long and short microbiome-taxa abundance

Purpose: Research biopsies are frequently incorporated within clinical trials in oncology and are often a mandatory requirement for trial enrollment. However, limited information is available regarding the extent and completeness of research biopsy reporting.Experimental Design: We identified a cohort of therapeutic clinical trials where research biopsies were performed between January 2005 and October 2010 from an IR database at our institution. Clinical trial protocols were compared with the highest level of corresponding publication as a manuscript or registry report.Results: A total of 866 research biopsies were performed across 46 clinical trials, with a median of 8 patients biopsied/trial and 19 biopsies collected/trial. After a median follow-up time of 4.3 years from trial completion, 36 of 46 trials (78%) reported trial results: published manuscripts (n = 35), or registry report (n = 1). A total of 635 conducted biopsies were reported in 18 of the 46 trials (39%). Six (33%) of these 18 trials underreported the number of biopsies performed. Of 33 trials with mandatory research biopsies, 13 (39%) trials reported on these biopsies. Biopsy complications occurred in 8 trials [n = 39 patients, 6 grade 3/4 adverse events (AE)] but only 1 trial reported these. Factors associated with biopsy reporting included a larger number of biopsies (P ≤ 0.001) and serial biopsies (P < 0.001). Twelve of 16 (75%) trials with >12 biopsies performed reported on these biopsies compared with only 20% (6/30) that performed ≤12 biopsies.Conclusions: Despite ethical obligations to report research biopsies, the majority (61%) of trials do not report results from research biopsies. Complications are rarely reported in these studies. Improved reporting of results and AEs from research biopsies is needed. Clin Cancer Res; 23(21); 6450–7. ©2017 AACR.

Characteristics and Reporting by Trial

Supplemental Table Legend

5A. Classification accuracy of CMS 3 samples on training data set V1 using 4-fold cross validation as function of number of genes. 5B.Classification accuracy of CMS 3 samples on validation data set (V2) as function of number of genes. 5C. Classification accuracy CMS3 samples with out of sample prediction set (V2o) as function of number of genes. 5D. Classification accuracy CMS 3 samples with TCGA dataset (RAN-seq subset) as function of number of genes. 5E. Classification accuracy CMS3 samples with Affymetrix dataset (V2a) as function of number of genes.

Supplementary material and method Supplementary Figure S1. Addition of a4-1BB does not lead CD8+ TIL into senescence. Supplementary Figure S2. Fold expansion of bulk and CD8+ PDAC TIL following the REP. Supplementary Figure S3. Level of MHC class I expression on PDAC tumor cell lines.

6A. Classification accuracy of CMS 4 samples on training data set V1 using 4-fold cross validation as function of number of genes. 6B. Classification accuracy of CMS 4 samples on validation data set (V2) as function of number of genes. 6C. Classification accuracy CMS 4 samples with out of sample prediction set (V2o) as function of number of genes. 6D. Classification accuracy CMS 3 samples with TCGA dataset (RAN-seq subset) as function of number of genes. 6E. Classification accuracy of CMS 3 samples with Affymetrix data set (V2a) as function of number of genes.

Purpose:Small-bowel adenocarcinoma (SBA) is rare, and no standard of care exists for metastatic disease beyond first-line FOLFOX/CAPOX. SBA has higher rates of microsatellite instability (MSI-H) and T-lymphocyte infiltration than other gastrointestinal cancers. We hypothesize that pembrolizumab, a PD-1 inhibitor, will induce antitumor response.Patients and Methods:Patients with previously treated advanced SBA received pembrolizumab 200 mg i.v. every 3 weeks until disease progression (PD), toxicity, or 35 doses maximum. Primary endpoint was confirmed overall response rate (ORR) with secondary progression-free survival (PFS), overall survival (OS), and toxicity assessment endpoints. Outcomes were stratified by tumor location, microsatellite stability (MSS) or instability (MSI-H), and PD-L1 level.Results:Forty patients were treated for a median duration of four cycles (range, 1–35). All patients are off study treatment due to PD (75%), death (10%), 35 cycles completed (8%), refusal (3%), and adverse effects (AEs, 5%). Three confirmed partial responses [PRs; 8%; 95% confidence interval (CI), 2–20] did not meet predefined success criteria of ORR 30%. Median OS (7.1 months; 95% CI, 5.1–17.1) and median PFS (2.8 months; 95% CI, 2.7–4.2) were similar across primary tumor sites. One confirmed PR (3%) was seen in patients with low MSS/MSI tumors and correlated with high tumor mutation burden (TMB). Fifty percent of patients with MSI-H tumors achieved PR and remain alive without progression. Twenty-five patients (63%) had grade ≥3 AEs and 11 patients (28%) had grade 4/5 AEs.Conclusions:In the largest study of SBA to date, pembrolizumab did not induce the hypothesized response rate; however, we did identify responses in key biomarker-selected cohorts.

Supplementary Figure 1. Cell surface vimentin expression is limited to cancer cells. Cell surface staining analysis for CSV in cancer cell line HLM-3 (liver cancer; top) and normal cell line NCM-356 (normal colon; bottom) using confocal microscopy. Permeabilized cells were stained for CSV (with 84-1 antibody; green), nuclei (with nuclear stain DRAQ5; blue), and the cell-surface marker WGA (red). 84-1 binding indicates cell surface vimentin that co-localizes with WGA. Scale bar, 10 µm.

Sample-wise and Gene-wise correlation of FF/FFPE paired Samples: Scatterplot of gene-specific Spearman correlation of FF/FFPE computed separately for CRCSC samples and non-CRCSC samples , with red and blue marking the overall top 100 and top 200 genes, respectively, in terms of FF/FFPE correlation computed across all samples. Note the high level of agreement across batches that genes with high FF/FFPE correlations for one batch tended to also have high FF/FFPE correlation in the other batch, suggesting high FF/FFPE correla-tion is a characteristic of the gene/probe set.

Factors Correlating with Research Biopsy Reporting from Published Manuscripts

Summary of the relative prognostic and mutational impact of tumor location in metastatic colorectal cancer. Sub-caption: Relative prevalence of mutations at each site was compared to the median prevalence across all sites and used to calculate relative font size for each gene name. Hazard ratios displayed in the figure utilize rectal location as the reference category.

Sensitivity analysis demonstrating optimal left vs right cut-point to maximize prognostic differences based on side in metastatic colorectal cancer. Sub-caption: Numbers in figure represent the hazard ratio followed by the 95% confidence interval within square brackets. The hazard ratio estimate occurs at the location used to divide left from right in each sensitivity analysis. For example, if the division between right and left occurs between the hepatic flexure and transverse colon, the hazard ratio estimate will be placed on the graph half way between the hepatic flexure and transverse colon (ie. 1.66).

Purpose: Colorectal cancers are classified as right/left-sided based on whether they occur before/after the splenic flexure, with established differences in molecular subtypes and outcomes. However, it is unclear if this division is optimal and whether precise tumor location provides further information.Experimental Design: In 1,876 patients with colorectal cancer, we compared mutation prevalence and overall survival (OS) according to side and location. Consensus molecular subtype (CMS) was compared in a separate cohort of 608 patients.Results: Mutation prevalence differed by side and location for TP53, KRAS, BRAFV600, PIK3CA, SMAD4, CTNNB1, GNAS, and PTEN. Within left- and right-sided tumors, there remained substantial variations in mutation rates. For example, within right-sided tumors, RAS mutations decreased from 70% for cecal, to 43% for hepatic flexure location (P = 0.0001), while BRAFV600 mutations increased from 10% to 22% between the same locations (P < 0.0001). Within left-sided tumors, the sigmoid and rectal region had more TP53 mutations (P = 0.027), less PIK3CA (P = 0.0009), BRAF (P = 0.0033), or CTNNB1 mutations (P < 0.0001), and less MSI (P < 0.0001) than other left-sided locations. Despite this, a left/right division preceding the transverse colon maximized prognostic differences by side and transverse colon tumors had K-modes mutation clustering that appeared more left than right sided. CMS profiles showed a decline in CMS1 and CMS3 and rise in CMS2 prevalence moving distally.Conclusions: Current right/left classifications may not fully recapitulate regional variations in tumor biology. Specifically, the sigmoid-rectal region appears unique and the transverse colon is distinct from other right-sided locations. Clin Cancer Res; 24(5); 1062–72. ©2017 AACR.See related commentary by Dienstmann, p. 989

4A. Classification accuracy of CMS2 samples on training data set V1 using 4-fold cross validation as function of number of genes.4B. Classification accuracy of CMS2 samples on validation data set (V2) as function of number of genes.4C. Classification accuracy CMS2 samples with out of sample(V2o) prediction set as function of number of genes. 4D. Classification accuracy CMS2 samples with TCGA dataset (RNA-seq subset) as function of number of genes.4E. Classification accuracy CMS2 samples with Affymentrix dataset (V2a) as function of number of genes.

Prespecified dose modifications for adverse events related to study drug in Phase IB and Phase II.

Purpose: Survival for pancreatic ductal adenocarcinoma (PDAC) patients is extremely poor and improved therapies are urgently needed. Tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) has shown great promise in other tumor types, such as metastatic melanoma where overall response rates of 50% have been seen. Given this success and the evidence showing that T-cell presence positively correlates with overall survival in PDAC, we sought to enrich for CD8+ TILs capable of autologous tumor recognition. In addition, we explored the phenotype and T-cell receptor repertoire of the CD8+ TILs in the tumor microenvironment.Experimental Design: We used an agonistic 4-1BB mAb during the initial tumor fragment culture to provide 4-1BB costimulation and assessed changes in TIL growth, phenotype, repertoire, and antitumor function.Results: Increased CD8+ TIL growth from PDAC tumors was achieved with the aid of an agonistic 4-1BB mAb. Expanded TILs were characterized by an activated but not terminally differentiated phenotype. Moreover, 4-1BB stimulation expanded a more clonal and distinct CD8+ TIL repertoire than IL2 alone. TILs from both culture conditions displayed MHC class I-restricted recognition of autologous tumor targets.Conclusions: Costimulation with an anti-4-1BB mAb increases the feasibility of TIL therapy by producing greater numbers of these tumor-reactive T cells. These results suggest that TIL ACT for PDAC is a potential treatment avenue worth further investigation for a patient population in dire need of improved therapy. Clin Cancer Res; 23(23); 7263–75. ©2017 AACR.

Purpose: This study aimed to detect cell-surface vimentin (CSV) on the surface of epithelial–mesenchymal transitioned (EMT) circulating tumor cells (CTC) from blood of patients with epithelial cancers.Experimental Design: In this study, 101 patients undergoing postsurgery adjuvant chemotherapy for metastatic colon cancer were recruited. EMT CTCs were detected from blood of patients using the 84-1 monoclonal antibody against CSV as a marker. EMT CTCs isolated were characterized further using EMT-specific markers, fluorescent in situ hybridization, and single-cell mutation analysis.Results: Using the 84-1 antibody, we detected CSV exclusively on EMT CTCs from a variety of tumor types but not in the surrounding normal cells in the blood. The antibody exhibited very high specificity and sensitivity toward different epithelial cancer cells. With this antibody, we detected and enumerated EMT CTCs from patients. From our observations, we defined a cutoff of Conclusion: Taken together, the number of EMT CTCs detected correlated with the therapeutic outcome of the disease. These results establish CSV as a universal marker for EMT CTCs from a wide variety of tumor types and thus provide the foundation for emerging CTC detection technologies and for studying the molecular regulation of these EMT CTCs. Clin Cancer Res; 21(4); 899–906. ©2014 AACR.

Algorithm showing strategy to de-velop the Nanostring based FFPE CMS classifier using Affymetrix gene expression data. FF: flash frozen, FFPE: Formalin fixed par-affin embedded, NFFPE: Nanos-tring FFPE, AFF: Flash frozen Affymetrix, GES: Gene expres-sion score, CRCSC: Colorectal cancer subtyping consortium

Treatment related adverse events and list of antibodies used.

Supplementary Table 1. Clinical Parameters in patients

Legend Supplemental Table S1 : Point mutations tested by plasma analysis in the KPLEX R study, (A). Comparison of point mutations tested by tumor tissue and plasma analysis, (B). Legend Supplemental Table S2: Concordance between tumor-tissue analysis and cfDNA analysis before treatments. Legend Supplemental Table S3: Mutation load at baseline in mutant patients with the equivalent type of mutation as determined by tumor tissue and plasma analysis are heterogneous from 31.83% to 0.009% Legend Supplemental Table S4: Mutation load values of emerging mutant subclones during treatments for both cohorts are reported in (A). Most emerging mutant subclones appears with a mutation load below 0.5% including down to 0.1%, (B). Sensitive methods is needed to track emergence of mutant subclones during targeted therapies. ND, non detected; NQ, scored positive but non quantified. Legend Supplemental table S5 : Compilation of cfDNA data, CEA level and imaging for both cohorts before and during treatments. Legend Supplemental Table S6: Concordance between tumor-tissue analysis and cfDNA analysis before treatments when considering if patients having primary tumor in place or not in place when entering in the study. Legend Supplemental Table S7: Mutant patients and point mutations determined at baseline with different mA% thresholds. Legend supplemental table S8: Description of all point mutations found from plasma analysis at baseline according to increasing allelic frequencies and the mutational status as determined with tumor tissue analysis. Legend Supplementary Table S9: Evolution of increasing mA% from baseline to end of treatment Supplemental Figure S1: Patient's Flow chart Legend Supplemental Figure S2 : Time lag between tumor tissue collection and blood drawing. Legend Supplemental Figure S3A: Total concentration of cfDNA (RefA) at baseline as determined by targeting BRAF and KRAS wild type sequence. Legend Supplemental Figure S3B: KRAS/BRAF ratio before treatment as determined by targeting BRAF and KRAS wild type sequence. KRAS/BRAF ratio before initiation of treatments (n=41). Supplemental Figure S4: RefA values do not differ at baseline between cohorts 1 and 2 Supplemental Figure S5: RefA values do not differ at baseline between patients stopped treatment before C4 and others Supplemental Figure S6: Concentrations of mutant cfDNA do not differ at baseline between cohorts 1 and 2 Supplemental Figure S7: Concentration of mutant cfDNA do not differ at baseline between patients stopped treatment before C4 and others Supplemental Figure S8: Mutation load do not differ at baseline between cohorts 1 and 2 Legend supplementary Figure S9: Validation of point mutation detection/quantification under the Poisson law. Supplemental Figure S10: Illustration of point mutation detection at very low frequency with IntPlex ASB QPCR method in mCRC patients in the study.

Classification Confidence of Affymetrix Validation Samples vs. Classification Accuracy of 472-gene wSVM clas-sifier: Distribution of classification confidence (�i) across 713 validation samples (top), and classification accura-cy (1=correct, 0=incorrect) as a function of classification confidence (�i), with fitted loess curve and 95% confidence interval.

Classification accuracy for training data V1 (based on 4-fold cross val-idation, 2A) and the full validation data V2 (2B) for various methods as function of number

Symptom-specific dose adjustment tables.

Supplementary Table 2. Diagnostic cutoff

PURPOSE Although electronic patient-reported outcomes (ePROs) are efficacious in symptom management, much is unknown about the utility of vital signs surveillance. We examined the feasibility of a remote patient monitoring platform that integrates ePROs and biometrics into the ambulatory management of symptom burden. METHODS Using a decentralized workflow, patients with gastrointestinal or thoracic cancer were approached for a 1-month study. Patients reported symptom burden via ePROs and biometrics (blood pressure, oxygen saturation, pulse, weight, and temperature) using bluetooth-enabled devices daily. Alerts on the basis of prespecified thresholds were managed via nurse-led triage. Adherence was defined as the completion of > 70% of daily symptom and biometric reporting requirements. Pilot acceptability, appropriateness, and feasibility were measured using validated instruments. Net promoter score, system usability scale, and emergency department (ED) admission rates were collected. RESULTS Over 8 months, 36 patients were enrolled and 25 (60% gastrointestinal) completed the study. Participants had a mean age of 58.0 years, mean Eastern Cooperative Oncology Group score of 0.88, were 52% female, and predominantly had stage IV or recurrent disease (72%). Program adherence was 73% and associated with high acceptability (4.63), feasibility (4.56), and appropriateness (4.46). System usability scale and net promoter score scores were 88 and 55, respectively. Seventy percent of alerts were generated by biometrics, 28% for symptoms, and 2% were patient-initiated communication. Finally, the ED visitation rate over the pilot period was 8%. CONCLUSION Our remote patient monitoring pilot program was highly acceptable, feasible, and appropriate. It had high rates of patient adherence and satisfaction and was associated with low ED visitation rates.

PURPOSE The College of American Pathologists (CAP) has developed a guideline on testing for mismatch repair (MMR) and microsatellite instability (MSI) for patients considered for immune checkpoint inhibitor therapy. ASCO has a policy and set of procedures for endorsing clinical practice guidelines that have been developed by other professional organizations. METHODS The CAP guideline was reviewed for developmental rigor by methodologists. An ASCO Endorsement Panel subsequently reviewed the content and the recommendations. RESULTS The ASCO Endorsement Panel determined that the recommendations from the CAP guideline, published on August 3, 2022, are clear, thorough, and based on the most relevant scientific evidence. ASCO endorses Mismatch Repair and Microsatellite Instability Testing for Immune Checkpoint Inhibitor Therapy: Guideline From the College of American Pathologists in Collaboration With the Association for Molecular Pathology and Fight Colorectal Cancer. RECOMMENDATIONS Within the guideline, MMR immunohistochemistry (IHC), MSI polymerase chain reaction, and MSI next-generation sequencing are all recommended testing options for colorectal cancer, MMR-IHC and MSI-polymerase chain reaction for gastroesophageal and small bowel cancer, and only MMR-IHC for endometrial cancer. No recommendation in favor of any testing method over another could be made for any other cancer. Tumor mutational burden was not recommended as a surrogate for DNA MMR deficiency. If MMR deficiency consistent with Lynch syndrome is detected, it should be communicated to the treating physician. Additional information is available at www.asco.org/molecular-testing-and-biomarkers-guidelines .