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PURPOSE Appendiceal adenocarcinomas (ACs) are rare, histologically diverse malignancies treated as colorectal cancers despite having distinct biology and clinical behavior. To guide clinical decision making, we defined molecular subtypes of AC associated with patient survival, metastatic burden, and chemotherapy response. PATIENTS AND METHODS A comprehensive molecular analysis was performed in patients with AC to define molecular subtypes. Associations between molecular subtype and overall survival, intraoperative peritoneal cancer index, and first-line chemotherapy response were assessed adjusting for histopathologic and clinical variables using multivariable Cox proportional hazards, linear regression, and logistic regression models. RESULTS We defined distinct molecular lineages of mucinous appendiceal adenocarcinoma (MAAP) from co-occurring mutations in GNAS, RAS, and TP53. Of 164 MAAP tumors, 24 were RAS-mutant (mut) predominant ( RAS-mut/ GNAS-wild-type [wt]/ TP53-wt) with significantly decreased mutations and chromosomal alterations compared with tumors with GNAS mutations (GNAS-mut predominant) or TP53 mutations (TP53-mut predominant). No patient with RAS-mut predominant subtype metastatic MAAP died of cancer, and overall survival in this subgroup was significantly improved compared with patients with GNAS-mut ( P = .05) and TP53-mut ( P = .004) predominant subtypes. TP53-mut predominant subtypes were highly aneuploid; increased tumor aneuploidy was independently ( P = .001) associated with poor prognosis. The findings retained significance in patients with any metastatic AC. RAS-mut predominant metastases exhibited reduced peritoneal tumor bulk ( P = .04) and stromal invasion ( P < .001) compared with GNAS-mut or TP53-mut predominant tumors, respectively. Patients with RAS-mut predominant MAAP responded more to first-line chemotherapy (50%) compared with patients with GNAS-mut predominant tumors (6%, P = .03). CONCLUSION AC molecular patterns identify distinct molecular subtypes: a clinically indolent RAS-mut/GNAS-wt/TP53-wt subtype; a chemotherapy-resistant GNAS-mut predominant subtype; and an aggressive, highly aneuploid TP53-mut predominant subtype. Each subtype exhibits conserved clinical behavior irrespective of histopathology.

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance. Significance: Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.

BRAF-mutated advanced colorectal cancer is a relatively small but critical subset of this tumor type on the basis of prognostic and predictive implications. BRAF alterations in colorectal cancer are classified into three functional categories on the basis of signaling mechanisms, with the class I BRAF V600E mutation occurring most frequently in colorectal cancer. Functional categorization of BRAF mutations in colorectal cancer demonstrates distinct mitogen-activated protein kinase pathway signaling. On the basis of recent clinical trials, current standard-of-care therapies for patients with BRAF V600E -mutated metastatic colorectal cancer include first-line cytotoxic chemotherapy plus bevacizumab and subsequent therapy with the BRAF inhibitor encorafenib and antiepidermal growth factor receptor antibody cetuximab. Treatment regimens currently under exploration in BRAF V600E-mutant metastatic colorectal cancer include combinatorial options of various pathway-targeted therapies, cytotoxic chemotherapy, and/or immune checkpoint blockade, among others. Circumvention of adaptive and acquired resistance to BRAF-targeted therapies is a significant challenge to be overcome in BRAF-mutated advanced colorectal cancer.

PURPOSE Prospective data on the efficacy of a watch-and-wait strategy to achieve organ preservation in patients with locally advanced rectal cancer treated with total neoadjuvant therapy are limited. METHODS In this prospective, randomized phase II trial, we assessed the outcomes of 324 patients with stage II or III rectal adenocarcinoma treated with induction chemotherapy followed by chemoradiotherapy (INCT-CRT) or chemoradiotherapy followed by consolidation chemotherapy (CRT-CNCT) and either total mesorectal excision (TME) or watch-and-wait on the basis of tumor response. Patients in both groups received 4 months of infusional fluorouracil-leucovorin-oxaliplatin or capecitabine-oxaliplatin and 5,000 to 5,600 cGy of radiation combined with either continuous infusion fluorouracil or capecitabine during radiotherapy. The trial was designed as two stand-alone studies with disease-free survival (DFS) as the primary end point for both groups, with a comparison to a null hypothesis on the basis of historical data. The secondary end point was TME-free survival. RESULTS Median follow-up was 3 years. Three-year DFS was 76% (95% CI, 69 to 84) for the INCT-CRT group and 76% (95% CI, 69 to 83) for the CRT-CNCT group, in line with the 3-year DFS rate (75%) observed historically. Three-year TME-free survival was 41% (95% CI, 33 to 50) in the INCT-CRT group and 53% (95% CI, 45 to 62) in the CRT-CNCT group. No differences were found between groups in local recurrence-free survival, distant metastasis-free survival, or overall survival. Patients who underwent TME after restaging and patients who underwent TME after regrowth had similar DFS rates. CONCLUSION Organ preservation is achievable in half of the patients with rectal cancer treated with total neoadjuvant therapy, without an apparent detriment in survival, compared with historical controls treated with chemoradiotherapy, TME, and postoperative chemotherapy.

The incidence of rectal cancer is increasing in patients younger than 50 years. Locally advanced rectal cancer is still treated with neoadjuvant radiation, chemotherapy and surgery, but recent evidence suggests that patients with a complete response can avoid surgery permanently. To define correlates of response to neoadjuvant therapy, we analyzed genomic and transcriptomic profiles of 738 untreated rectal cancers. APC mutations were less frequent in the lower than in the middle and upper rectum, which could explain the more aggressive behavior of distal tumors. No somatic alterations had significant associations with response to neoadjuvant therapy in a treatment-agnostic manner, but KRAS mutations were associated with faster relapse in patients treated with neoadjuvant chemoradiation followed by consolidative chemotherapy. Overexpression of IGF2 and L1CAM was associated with decreased response to neoadjuvant therapy. RNA-sequencing estimates of immune infiltration identified a subset of microsatellite-stable immune hot tumors with increased response and prolonged disease-free survival.

PURPOSE Adagrasib, a KRASG12C inhibitor, has demonstrated clinical activity in patients with KRASG12C-mutated non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). KRASG12C mutations occur rarely in other solid tumor types. We report evaluation of the clinical activity and safety of adagrasib in patients with other solid tumors harboring a KRASG12C mutation. METHODS In this phase II cohort of the KRYSTAL-1 study (NCT03785249; https://www.clinicaltrials.gov/ct2/show/NCT03785249 ; phase Ib cohort), we evaluated adagrasib (600 mg orally twice daily) in patients with KRASG12C-mutated advanced solid tumors (excluding NSCLC and CRC). The primary endpoint was objective response rate. Secondary endpoints included duration of response, progression-free survival, overall survival, and safety. RESULTS As of October 1, 2022, 64 patients with KRASG12C-mutated solid tumors were enrolled and 63 patients treated (median follow-up, 16.8 months). The median number of prior lines of systemic therapy was 2. Among 57 patients with measurable disease at baseline, objective responses were observed in 20 (35.1%) patients (all partial responses), including 7/21 (33.3%) responses in pancreatic and 5/12 (41.7%) in biliary tract cancers. The median duration of response was 5.3 months (95% confidence interval [CI], 2.8 to 7.3) and median progression-free survival was 7.4 months (95% CI, 5.3 to 8.6). Treatment-related adverse events (TRAEs) of any grade were observed in 96.8% of patients and grade 3 to 4 in 27.0%; there were no grade 5 TRAEs. TRAEs did not lead to treatment discontinuation in any patients. CONCLUSION Adagrasib demonstrates encouraging clinical activity and is well tolerated in this rare cohort of pretreated patients with KRASG12C-mutated solid tumors.

C106 resistant cells drug withdrawal and Patient #12 MSK-Impact data

Patients Progression Cell Free DNA data

Patients Characteristics

C106 cell lines single cell sequencing analysis

RW7213 cell lines sequencing analysis

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance.Significance:Clinical resistance to KRASG12C–EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches.This article is highlighted in the In This Issue feature, p. 1

Generation of acquired resistance PDX model

Characterization of acquired resistance in vitro models

Oncoprint of baseline alterations in CRC patients who developed resistance to KRAS G12C and EGFR inhibitors

Patients Longitudinal Cell Free DNA data

Table S1. Pharmacokinetic parameters of encorafenib and alpelisib in patients at steady state (cycle 2 day 1). Table S2. Criteria for defining dose-limited toxicities. Supplementary Figure 1. Radiological images of response for a patient treated with the dual-combination therapy of encorafenib and cetuximab. Supplementary Figure 2. Time on study by response for patients treated with the dual-combination therapy of encorafenib and cetuximab and patients treated with the triple-combination therapy of encorafenib, alpelisib, and cetuximab.

Supplementary Figure 2

Summary of organoid genotypes and WNT resistance status

Supplemental Tables

Primer sequences

Detected RTK alterations and MAP2K1 mutations in sequenced colorectal carcinomas.

Supplementary Figure 1

(a) Barplot of GISTIC2.0 chromosome 20q gene level copy number calls in TCGA COADREAD data and (b) chromosome 20q genes covered by MSK-IMPACT. (c) Interquartile range (IGR) of gene level RNASeq expression z-score by copy number group from 2a. (d) MSK-IMPACT and TCGA tumor purity estimates.

Supplemental Data

(a) Plot of SCNA in a CRC with 20q amplification as detected by MSK-IMPACT and (b) high resolution SNP analysis on Oncoscan confirmed the results without showing focal amplifications

Mutations at multiple sites in MEK1 occur in cancer, suggesting that their mechanisms of activation might be different. We analyzed 17 tumor-associated MEK1 mutants and found that they drove ERK signaling autonomously or in a RAS/RAF-dependent manner. The latter are sensitive to feedback inhibition of RAF, which limits their functional output, and often cooccur with RAS or RAF mutations. They act as amplifiers of RAF signaling. In contrast, another class of mutants deletes a hitherto unrecognized negative regulatory segment of MEK1, is RAF- and phosphorylation-independent, is unaffected by feedback inhibition of upstream signaling, and drives high ERK output and transformation in the absence of RAF activity. Moreover, these RAF-independent mutants are insensitive to allosteric MEK inhibitors, which preferentially bind to the inactivated form of MEK1. All the mutants are sensitive to an ATP-competitive MEK inhibitor. Thus, our study comprises a novel therapeutic strategy for tumors driven by RAF-independent MEK1 mutants.Significance: Mutants with which MEK1 mutants coexist and their sensitivity to inhibitors are determined by allele-specific properties. This study shows the importance of functional characterization of mutant alleles in single oncogenes and identifies a new class of MEK1 mutants, insensitive to current MEK1 inhibitors but treatable with a new ATP-competitive inhibitor. Cancer Discov; 8(5); 648–61. ©2018 AACR.See related commentary by Maust et al., p. 534.This article is highlighted in the In This Issue feature, p. 517

The WNT pathway is a fundamental regulator of intestinal homeostasis, and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer. To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that although WNT suppression blocks tumor growth in most organoid and in vivo colorectal cancer models, the accumulation of colorectal cancer–associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of TP53 and SMAD4, transient TGFβ exposure drives YAP/TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work identifies genetic and microenvironmental factors that drive WNT inhibitor resistance, defines a new mechanism for WNT-independent colorectal cancer growth, and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs.Significance:Colorectal and intestinal cancers are driven by mutations in the WNT pathway, and drugs aimed at suppressing WNT signaling are in active clinical development. Our study identifies a mechanism of acquired resistance to WNT inhibition and highlights a potential strategy to target those drug-resistant cells.This article is highlighted in the In This Issue feature, p. 1426

Here, comprehensive analysis was performed on the molecular and clinical features of colorectal carcinoma harboring chromosome 20q amplification. Tumor and normal DNA from patients with advanced colorectal carcinoma underwent next-generation sequencing via MSK-IMPACT, and a subset of case samples was subjected to high-resolution microarray (Oncoscan). Relationships between genomic copy number and transcript expression were assessed with The Cancer Genome Atlas (TCGA) colorectal carcinoma data. Of the colorectal carcinoma patients sequenced (n = 401) with MSK-IMPACT, 148 (37%) had 20q gain, and 30 (7%) had 20q amplification. In both the MSK-IMPACT and TCGA datasets, BCL2L1 was the most frequently amplified 20q oncogene. However, SRC was the only recognized 20q oncogene with a significant inverse relationship between mRNA upregulation and RAS/RAF mutation (OR, −0.4 ± 0.2, P = 0.02). In comparison with 20q diploid colorectal carcinoma, 20q gain/amplification was associated with wild-type KRAS (P < 0.001) and BRAF (P = 0.01), microsatellite stability (P < 0.001), distal primary tumors (P < 0.001), and mutant TP53 (P < 0.001), but not stage. On multivariate analysis, longer overall survival from the date of metastasis was observed with chromosome 20q gain (P = 0.02) or amplification (P = 0.04) compared with diploid 20q.Implications: 20q amplification defines a subset of colorectal cancer patients with better overall survival from the date of metastasis, and further studies are warranted to assess whether the inhibition of 20q oncogenes, such as SRC, may benefit this subset of patients. Mol Cancer Res; 15(6); 708–13. ©2017 AACR.

Supplementary Material

Supplementary Figure 3

Supplemental Table

Supplementary Figures and Legends

Supplementary Table 3

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion–positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion–positive ICC.Significance:ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983

Supplemental Figure Legends

Supplementary Figure 4

Supplementary Table 2

Supplementary Table 1

Supplementary Figures 1-16

Most patients with KRASG12C–mutant non–small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRASG12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRASG12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRASG12C colorectal cancer.Significance:The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079

supplementary tables, figures, and methods