Wnt Pathway Alterations Research Articles

Abstract 998: Mechanism of action of RXC004, a Wnt pathway inhibitor, in genetically-defined models of cancer

Abstract Background: RXC004 is a porcupine inhibitor, and thus a potent and selective inhibitor of Wnt ligand-dependent signalling. RXC004 is currently being investigated in a safety and tolerability study in cancer patients with solid tumours (NCT03447470). Certain Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Fzd on the cell surface increasing Wnt-ligand dependent signalling. These alterations are implicated in colorectal and pancreatic cancer. We present pre-clinical data on the effects of RXC004 in genetically defined models of cancer. Methods: RXC004 in vitro effects on the metabolic activity of both sensitive (genetically selected RNF43 mutant or RSPO fusion models) and insensitive (beta-catenin mutant model) colorectal and pancreatic tumour cells lines were assessed. To test if these effects translated in vivo, RXC004 was also evaluated in an in vivo FDG-PET study in an RSPO fusion tumour cell line. In addition, as we had previously noted that RXC004 caused cell differentiation in an RSPO fusion model, we studied if pre-treatment with RXC004 would affect subsequent tumour growth post RXC004 induced cell differentiation in vivo. Results: In tumour cell lines in vitro, RXC004 significantly reduced glucose uptake only in genetically selected tumour cell lines. In vivo FDG-PET studies also showed a reduction in FDG-PET signals post 7 days treatment of 5mg/kg QD RXC004. Imaging demonstrated that MaxSUV (maximum standardised uptake value) decreased by 31% in RXC004 treated versus vehicle treated tumours, furthermore biodistribution of PET ligand was significantly inhibited in tumours post 7 days RXC004 treatment. Pre-treatment of RXC004 in an in vivo mouse xenograft model for 7 days resulted in a significant reduction in tumour growth when tumour fragments from these pre-treated mice were re-implanted into naïve mice (P<0.05). Conclusion: These data demonstrate that RXC004 monotherapy results in tumour cell differentiation accompanied by reduced metabolic activity in genetically selected tumours. RXC004-induced cell differentiation may therefore continue to provide patient benefit even post dosing. Moreover, FDG-PET imaging in clinical studies may allow early detection of the positive effects of RXC004 in a non-invasive manner, and prior to any changes in tumour volume by conventional RECIST measurements. Citation Format: Caroline Phillips, Simon Woodcock, Catherine Eagle, Andrew Saunders, Craig Tilston, Inder Bhamra, Richard Armer. Mechanism of action of RXC004, a Wnt pathway inhibitor, in genetically-defined models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 998.

Abstract 1674: WNT signal pathway activation correlates with innate resistant to immune checkpoint therapies in melanoma

Abstract Background: Immune checkpoint inhibitors targeting the programmed cell death-1 receptor (PD-1) improve survival in a subset of patients with melanoma. There are still lots of patients could not reach the clinical benefit, even though with the positive expression of the programmed cell death 1 ligand 1 (PD-L1). Here, we aimed to research the immune resistance mechanism in melanoma. Methods: The genomic data and clinical data of the discovery cohort was obtained from The Cancer Genome Atlas (TCGA). The clinical data of validation cohort in melanoma treated by immunotherapy was retrospective collected. And tissue from patients with melanoma were performed to whole exome sequencing in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab. Results: Activation of WNT signaling was inferred that somatic mutations or somatic copy number alterations in WNT signaling elements including APC, WNT1, WNT4, WNT6, WNT16, CTNNB1, BCL9L, SMAD3 and SMAD4. Two cohort have been enrolled in this study, the discovery cohort from TCGA, and the validation cohort from the Chinese patients with melanoma. The frequency of WNT pathway alterations from the TCGA cohort was 28.4%, and which was represented mutually exclusive molecular subsets. In TCGA cohort, activating alteration WNT signaling were associated with shorter median OS (22.5 vs 13.6 months, p=0.047, HR=0.61 (0.37-0.89)). Conclusions: Mutations predicted to activate the WNT pathway were associated with innate resistance to immune checkpoint blockade in melanoma. Citation Format: Fuyu Gong, Yuezong Bai, wenzhuan Xie, Chuanliang Cui. WNT signal pathway activation correlates with innate resistant to immune checkpoint therapies in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1674.

Abstract 380: WNT signal pathway activation correlates with innate resistant to immune checkpoint therapies in NSCLC

Abstract Background: Immune checkpoint inhibitors targeting the programmed cell death-1 receptor (PD-1) improve survival in a subset of patients with NSCLC. There are still lots of patients could not reach the clinical benefit, even though with the positive expression of the programmed cell death 1 ligand 1 (PD-L1). Here, we aimed to research the immune resistance mechanism in NSCLC. Methods: The genomic data and clinical data of the discovery cohort was obtained from The Cancer Genome Atlas (TCGA). The clinical data of validation cohort in NSCLC treated by immunotherapy was retrospective collected. And tissue from patients with NSCLC were performed to whole exome sequencing in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab. Results: Activation of WNT signaling was inferred that somatic mutations or somatic copy number alterations in WNT signaling elements including APC, WNT16, AXIN2, WNT4, AXIN1, CTNNB1, BCL9L and SMAD4. Two cohort have been enrolled in this study, the discovery cohort from TCGA, and the validation cohort from the Chinese patients with NSCLC. The frequency of WNT pathway alterations from the TCGA cohort was 16%, and which was represented mutually exclusive molecular subsets. In TCGA cohort, activating alteration WNT signaling were associated with shorter median PFS (18.8 vs 3.9 months, p=0.036, HR=0.24 (0.09-0.68)). Conclusions: Mutations predicted to activate the WNT pathway were associated with innate resistance to immune checkpoint blockade in NSCLC Citation Format: Dian Ren, Bo Zhang, Renwang Liu, Fuyu Gong, Yuezong Bai, Wenzhuan Xie, Huandong Huo, Hao Zhang, Zuoqing Song. WNT signal pathway activation correlates with innate resistant to immune checkpoint therapies in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 380.

Wild-type APC Is Associated with Poor Survival in Metastatic Microsatellite Stable Colorectal Cancer.

The prognostic implication of wild-type APC (APC-WT) in microsatellite stable (MSS) metastatic colorectal cancer (mCRC) is not well defined. APC prognostic value was evaluated retrospectively in two independent cohorts of patient with MSS mCRC with a confirmatory analysis from a public data set from Memorial Sloan Kettering Cancer Center (MSKCC). In comparison with the APC-mutant (APC-MT) population (n = 255), APC-WT patients (n = 86) tended to be younger (59% of age < 40 vs. 26% of age > 50), right-sided (41.7% vs. 27%), BRAFV600E mutated (23.3% vs. 0.8%), and KRAS wild type (65.1% vs. 49.8%). Alternative WNT pathway alterations, RNF43 and CTNNB1, were over-represented in the APC-WT versus APC-MT population (7% vs. 0.4% and 4.7% vs. 0.4%, respectively). APC-WT patients had a worse overall survival (OS) than APC-MT patients (22.6 vs. 45.6 months, p < .0001). Using a multivariate model correcting for primary tumor location, RAS and BRAF status, APC-WT was predictive of poor survival (APC-MT vs. APC-WT, hazard ratio [HR], 0.62; 95% confidence interval [CI], 0.44-0.86, p = .0037). The prognostic implication of APC-WT on OS was confirmed further in a similar multivariate model of 934 stage IV patients from MSKCC public database (APC-MT vs. APC-WT, HR, 0.63, 95% CI, 0.49-0.81, p < .0001). APC-WT is associated with poor OS in MSS mCRC regardless of RAS and BRAF status. Compared with APC-MT mCRC tumors, APC-WT tumors were associated with other Wnt activating alterations, including RNF43 and CTNBB1. Our data suggest alternative therapy needs to be investigated in APC-WT patients. Patients with microsatellite stable metastatic colorectal cancer with wild-type APC had a worse overall survival than patients with mutated APC regardless of RAS/RAF status. APC status should be considered as a stratification factor in prospective trials, and novel therapeutic strategies need to be developed for this subgroup of patients.

Abstract IA35: Novel biological insights into colorectal cancer from West Africa

Abstract The burden of colorectal cancer (CRC) is increasing in West Africa. Understanding the molecular and clinicopathologic profile of the region’s CRC patients is vital to optimizing early detection and therapeutic regimens. Clinicopathologic data from consecutive patients from a multicenter, prospectively maintained Nigerian CRC database were compared to a contemporary cohort from Memorial Sloan Kettering Cancer Center (MSKCC). Fresh-frozen tissue from Nigerian patients was analyzed with a multigene, next-generation sequencing assay, along with immunohistochemistry and methylation analyses; data were compared to molecular data from MSKCC and The Cancer Genome Atlas (TCGA) cohorts. Adult patients with histologically confirmed colorectal adenocarcinoma enrolled in a Nigerian CRC database from April 2013-November 2018 were included. All adult patients with colorectal adenocarcinoma diagnosed from January-June 2013 were included in the MSKCC cohort. Somatic and germline next-generation sequencing of cancer-associated genes, immunohistochemistry, and methylation analyses were performed. The primary outcomes were microsatellite instability status, somatic and germline mutation frequency, and overall survival. The median age at diagnosis was 55.8 years in the Nigerian cohort (n=380) and 60.0 years in the MSKCC cohort (n=458) (p&amp;lt;0.001). The rate of microsatellite instability was 28.1% in the Nigerian cohort (18/64), compared to 14.2% and 8.5% in the TCGA (65/459) and MSKCC (97/1145) cohorts, respectively (p&amp;lt;0.001). In microsatellite-stable cases, tumors from Nigerian patients were less likely to have APC mutations (39.1% vs. 76.0% p&amp;lt;0.001) and WNT pathway alterations (47.8% vs. 81.9%, p&amp;lt;0.001), and more likely to have RAS pathway alterations (76.1% vs. 59.6%, p=0.03) compared to MSKCC patients. Overall survival was significantly longer for MSKCC vs. Nigerian patients (not reached vs. 12 months, p&amp;lt;0.001). The biology of CRC in Nigeria appears to be distinct from that in high-income countries, emphasizing the need for regional data to guide diagnostic and treatment approaches for patients in West Africa. Citation Format: Olusegun I. Alatise, Gregory C. Knapp, Avinash Sharma, Walid K. Chatila, Olalekan Olasehinde, Adeleye D. Omisore, Aba Katung, Samuel A. Olatoke, T.Peter Kingham. Novel biological insights into colorectal cancer from West Africa [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr IA35.

742P Activation of the WNT signaling pathway correlates with innate resistance to immune checkpoint therapies in clear cell renal cell carcinoma

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) improve survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). However, there are still many patients who do not achieve clinical benefit with these agents, even with positive expression of the programmed death-ligand 1 (PD-L1). Here, we aimed to investigate the immune resistance mechanisms in ccRCC. The genomic data and clinical data of the discovery cohort was obtained from The Cancer Genome Atlas (TCGA). The clinical data for the validation cohort in ccRCC treated with immunotherapy was retrospectively collected from Chinese patients. Whole exome sequencing was performed on tissue from patients with ccRCC in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab. Activation of WNT signaling was inferred by somatic mutations or somatic copy number alterations in WNT signaling elements, including CTNNB1, APC, AXIN1, AXIN2, CSNK1A, WNT1, WNT2, WNT3, WNT4, WNT6, WNT7, WNT8, WNT9, WNT10, WNT16, GSK3, SMAD3, SMAD4 and BCL9L. Two cohorts were enrolled in this study: the discovery cohort from the TCGA, and the validation cohort from Chinese patients with ccRCC. The frequency of WNT pathway alterations in the TCGA cohort was 25.7%, and which represented mutually exclusive molecular subsets. In the TCGA cohort, activating WNT signaling alterations were associated with shorter median PFS (1.4 vs 3.5 months) and OS (10.2 vs 24.2 months). Mutations predicted to activate the WNT pathway were associated with innate resistance to immune checkpoint blockade in clear cell renal cell carcinoma

Abstract 5668: Genomic correlates of PD-L1 expression are associated with response to immunotherapy in non-small cell lung cancer

Abstract PD-L1 expression is the primary clinical biomarker of immune checkpoint blockade (ICB) response in non-small cell lung cancer (NSCLC); patients with tumors that express higher levels of PD-L1 tend to have better responses. However, somatic genomic events correlating with PD-L1 expression are not fully defined. To investigate this, we utilized a DFCI cohort of 909 non-squamous NSCLC samples grouped into 3 categories of PD-L1 expression based on immunohistochemistry (IHC): &amp;lt;1% (negative, n=304), 1-49% (low, n=326), and ≥50% (high, n=279) expression. All samples were sequenced by hybrid capture for over 300 cancer-associated genes (OncoPanel), and we assessed somatic mutations and copy number (CN) variations for correlation with PD-L1 expression. Compared to PD-L1 high tumors, PD-L1 neg. cases were more likely to have mutations in STK11 (Permutation test, q=7e-4), EGFR (q=7e-4), CTNNB1 (q=0.04), APC (q=0.17), and SMARCA4 (q=0.2). CN losses that more significantly occurred in PD-L1 neg. tumors included deletions of PD-L1, PD-L2, and JAK2 (Fisher exact test, q&amp;lt;1e-3) as well as 9p chromosomal arm loss (q=0.04) and homozygous deletions in CDKN2A/B (q=0.05). We assessed the association of these somatic alterations with PD-L1 mRNA expression in an independent cohort of 529 TCGA primary lung tumors. STK11 mutation (Mann-Whitney test, q&amp;lt;1e-3), CN loss of PD-L1, PD-L2, and/or JAK2 (q&amp;lt;1e-3), and CN loss of chromosome arm 9p (q&amp;lt;1e-3) were all associated with decreased PD-L1 mRNA expression. Conversely, CN gain of PD-L1, PD-L2, and/or JAK2 and 9p were significantly (q&amp;lt;1e-3) associated with increased PD-L1 expression. Next, we examined if somatic alterations associated with PD-L1 expression were also correlated with progression free (PFS) or overall survival (OS) using a partially-overlapping DFCI cohort of 468 advanced stage NSCLCs. STK11 mutation (Log-rank test, q=0.1), EGFR mutation (q&amp;lt;1e-3), and CN loss of PD-L1, PD-L2, and/or JAK2 (q=0.02) were all significantly associated with worse PFS after ICB. EGFR mutation was significantly associated with worse OS after ICB (q=0.19). We observed the previously identified (Schabath, et al 2016) mutual exclusivity between EGFR and STK11 mutations. We further show that they may indicate distinct subtypes of PD-L1 neg. tumors with STK11 mutant tumors exhibiting strikingly worse prognosis after ICB (med. PFS 52 vs 64 days, p&amp;lt;1e-3; med. OS 212 vs 299 days, p=5e-3). In summary, we confirm the findings that STK11 and EGFR mutations are associated with reduced PD-L1 expression and worse prognosis after ICB (Rizvi, et al 2018) and identify CN loss of the PD-L1 locus as a potential novel biomarker of lower PD-L1 expression and worse ICB outcomes. In addition, we show a novel anticorrelation between Wnt pathway alterations and PD-L1 expression by IHC. Taken together, this work suggests a role of specific mutations and CNVs in affecting PD-L1 expression and response to ICB. Citation Format: Liam F. Spurr, Giuseppe Lamberti, Yvonne Y. Li, Biagio Ricciuti, Gonzalo Recondo, Renato Umeton, Lynette M. Sholl, Matthew L. Meyerson, Andrew D. Cherniack, Mark M. Awad. Genomic correlates of PD-L1 expression are associated with response to immunotherapy in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5668.

Specific Mutations in APC, but Not Alterations in DNA Damage Response, Associate With Outcomes of Patients With Metastatic Colorectal Cancer

In an integrated genomic and clinical analysis, we evaluate the effects of Wnt and DNA damage response pathway alterations on metastatic colorectal cancer.

Somatic Tissue Engineering in Mouse Models Reveals an Actionable Role for WNT Pathway Alterations in Prostate Cancer Metastasis.

To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with Trp53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer. SIGNIFICANCE: Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease.This article is highlighted in the In This Issue feature, p. 890.

Clinical and molecular correlates of PD-L1 expression in patients with lung adenocarcinomas

Programmed death-ligand 1 (PD-L1) expression is the only FDA-approved biomarker for immune checkpoint inhibitors (ICIs) in patients with lung adenocarcinoma, but sensitivity is modest. Understanding the impact of molecular phenotype, clinical characteristics, and tumor features on PD-L1 expression is largely unknown and may improve prediction of response to ICI. We evaluated patients with lung adenocarcinoma for whom PD-L1 testing and targeted next-generation sequencing (using MSK-IMPACT) was performed on the same tissue sample. Clinical and molecular features were compared across PD-L1 subgroups to examine how molecular phenotype associated with tumor PD-L1 expression. In patients treated with anti-PD-(L)1 blockade, we assessed how these interactions impacted efficacy. A total of 1586 patients with lung adenocarcinoma had paired PD-L1 testing and targeted next-generation sequencing. PD-L1 negativity was more common in primary compared to metastatic samples (P < 0.001). The distribution of PD-L1 expression (lymph nodes enriched for PD-L1 high; bones predominantly PD-L1 negative) and predictiveness of PD-L1 expression on ICI response varied by organ. Mutations in KRAS, TP53, and MET significantly associated with PD-L1 high expression (each P < 0.001, Q < 0.001) and EGFR and STK11 mutations associated with PD-L1 negativity (P < 0.001, Q= 0.01; P= 0.001, Q < 0.001, respectively). WNT pathway alterations also associated with PD-L1 negativity (P= 0.005). EGFR and STK11 mutants abrogated the predictive value of PD-L1 expression on ICI response. PD-L1 expression and association with ICI response vary across tissue sample sites. Specific molecular features are associated with differential expression of PD-L1 and may impact the predictive capacity of PD-L1 for response to ICIs.

Genomic characterization of rectal cancer and molecular determinants of response to neoadjuvant chemoradiotherapy.

192 Background: Rectal cancers are clinically different from colon cancers and have not yet been molecularly characterized due to the scarcity of pretreatment specimens. Discovery of molecular determinants of response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II and III) are needed to select those who can avoid surgery and benefit from watch-and-wait strategies. Methods: We profiled 371 pre-treatment specimens using targeted-exome sequencing of 468 cancer genes (n = 325), whole-exome sequencing (n = 100), and RNA-sequencing (n = 113). The targeted-sequencing cohort included patients with stage I (n = 44), II (n = 41), III (n = 176), and IV (n = 64) disease. Primary tumors were divided into lower (LR: 0-4 cm to anal verge, n = 62), middle (MR: 4-8 cm, n = 115), and upper rectum (UR: 8-12 cm, n = 107). We examined molecular determinants of complete response (CR) and relapse free survival (RFS) in LARC patients treated with chemoradiotherapy only (CRT: n = 39), induction chemotherapy + CRT (INCT: n = 87) and consolidation chemotherapy after CRT (CCNT: n = 63). Results: Among MSS cases, oncogenic gene and signaling pathway alterations did not vary by clinical stage. WNT pathway alterations, driven by APC mutations, were more frequent in the UR (89% UR v 86% MR v 60% LR, p &lt; 0.001) while RTK/RAS alterations were more frequent in the LR (54% UR v 69% MR v 72% LR, p &lt; 0.03). A set of genes enriched in mTOR signaling, G2M checkpoint, EMT transition, and DNA repair were overexpressed in the UR (FDR &lt; 0.1). The 5-yr RFS rate for LARC was 75% (CI: 68%-82%) and 24% of the cases had a CR (n = 45). MSI cases had a higher rate of CR compared to MSS cases (50% v 23%, p = 0.07) and none relapsed (n = 8). KRAS-altered MSS tumors exhibited worse RFS in cases treated with CNCT (5-yr: 74% v 97%, p = 0.01), but not in cases treated with INCT (5-yr: 67% v 72%, p = 0.7). Conclusions: WNT alterations are more frequent in the UR while RTK/RAS alterations are more frequent in the LR, suggesting differences in tumor biology between proximal and distal rectal cancer. Further, we report correlations between distinct molecular profiles and response to treatment paradigms that could guide the design of future clinical trials.

Abstract 3874: Efficacy of the Wnt/Beta-Catenin pathway inhibitor RXC004 in genetically-defined models of cancer

Abstract Background: RXC004, a potent and selective inhibitor of the Wnt/β-Catenin pathway regulator porcupine, is being investigated in a safety and tolerability study in cancer patients with solid tumours (NCT03447470). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Fzd on the cell surface, increasing Wnt-ligand dependent signalling. These alterations are implicated in colorectal, gastric, pancreatic and biliary cancer. We present pre-clinical data on the direct tumour-targeting effects of RXC004 in genetically-defined models of cancer. Methods: RXC004 in vitro effects on sensitive (RNF43 mutant or RSPO fusion) or insensitive (APC/β-Catenin mutant) colorectal and pancreatic tumour cells lines were assessed against a panel of potential Wnt target genes measured using qPCR. To test if these effects translated in vivo, RXC004 was evaluated in several RNF43 mutant or RSPO fusion tumour line efficacy, PK and PD studies. A range of oral doses and schedules of RXC004 were tested for either 7 or 26 days, for PK/PD or efficacy measurements respectively. Efficacy was measured by tumour volume/weight, RXC004 PK was measured in plasma and tumour, and PD effects were measured by tumour qPCR and histological methods. Results: In sensitive cell lines, RXC004 inhibited expression of the Wnt pathway negative feedback genes Axin2 and RNF43. Decreases in c-Myc correlated with the anti-proliferative effects of RXC004. Additionally, MMP7 and CD44 were downregulated, whilst Mucin gene expression increased. In contrast, RXC004 had no anti-proliferative effects on APC mutant colorectal cancer cells in vitro, and no modulation of these Wnt target genes. In vivo, RXC004 demonstrated significant efficacy and PD effects in multiple RNF43 mutant and RSPO fusion xenograft models. Focusing on an RSPO fusion model, RXC004 reduced tumour volume in a dose-dependent manner at 1.5 and 5 mg/kg QD, and 1.5mg/kg BID. In addition, 1.5mg/kg BID of RXC004 scheduled 5 days on, 2 days off also gave significant efficacy. PK analysis was consistent with the predicted dose response, showing excellent absorption and tumour distribution. PD analysis confirmed the RXC004-induced Wnt target gene changes identified in vitro translated in vivo, whilst histology showed a clear reduction in the proliferation marker Ki67 and concomitant increase in the staining of Mucins, indicative of mucinous differentiation of the tumour cells. Conclusion: Taken together, these data demonstrate that RXC004 monotherapy has the potential to benefit patients with tumours bearing RNF43 mutations or RSPO fusions, supporting a genetically-defined patient selection strategy for ongoing RXC004 clinical studies. Citation Format: Simon Woodcock, Inder Bhamra, Cliff Jones, Alicia Edmenson Cook, Catherine Eagle, Caroline Phillips. Efficacy of the Wnt/Beta-Catenin pathway inhibitor RXC004 in genetically-defined models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3874.

Clinical and molecular factors predicting resistance to first-line (1L) FOLFOX in patients (pts) with advanced esophagogastric cancer (EGA) and patterns of subsequent therapy.

4047 Background: In the US, FOLFOX is the most widely used 1L treatment for advanced EGA. We evaluated if FOLFOX resistance (FR) impacted subsequent therapy and characterized the clinical and molecular factors that predict resistance to 1L FOLFOX. Methods: We reviewed pts with advanced Her2-negative EGA treated with 1L FOLFOX from Jan 2013 to Aug 2017. Response or stable disease (SD) at time of first restaging scan defined pts as FOLFOX sensitive (FS); progression defined FR. Pt characteristics were compared using Fisher’s exact and Wilcoxon Rank-Sum tests. Outcomes were correlated with clinical variables and MSK-IMPACT data. Microsatellite instable (MSI) pts were excluded from gene and pathway analysis. Overall survival (OS) was calculated from start of FOLFOX using Kaplan-Meier methods. Landmark analysis (2 months [mo] after starting FOLFOX) was used to compare OS between groups. Results: We identified 311 pts, median age 62, 73% male, 82% ECOG 0/1. 246 pts (79%) were FS and 65 (21%) were FR. FR pts had a higher number of metastatic sites, p = 0.001. Median OS was 13.4 mo in FS pts vs 4.3 mo in FR pts (p &lt; 0.001). At time of analysis, 213 pts (172 FS pts and 41 FR pts) and 110 pts (90 FS pts and 20 FR pts) had received 2nd-line (2L) and 3rd-line (3L) chemo respectively. In pts who received 2L chemo (ctx), there was no difference in ctx duration between FS and FR pts (2.1 vs 1.4 mo, p = 0.67). However, in pts who received 3L ctx, the duration was longer in FS vs FR pts (1.6 vs 0.5 months, p = 0.002). In IMPACT tested pts (n = 130), univariate analysis identified EGFR (33.3% vs 10.7%, p = 0.032) and WNT pathway (26.7% vs 4.9%, p = 0.015) alterations more frequently in FR pts vs FS pts. 12 pts were MSI, all were FS (3 SD, 8 PR, 1 CR). Of 63 pts who had IMPACT germline testing, 7 (11%) had germline mutations identified, including CDH1 (n = 2) and ATM (n = 1), all were FS. No pts were enrolled on genotype-matched trials as a result of IMPACT testing. Conclusions: From time of first restaging scan, FR pts have inferior OS to FS pts. The duration of 3L ctx was longer in FS vs FR pts. Higher number of metastatic sites and EGFR and WNT pathway alterations were associated with FR; MSI was not. IMPACT did not facilitate enrolment on genotype-matched studies.

Influence of WNT and DNA damage response pathway alterations on outcomes in patients with unresectable metastatic colorectal cancer.

3585 Background: We assembled a large series of consecutive patients with unresectable metastatic colorectal cancer (mCRC) to identify genomic biomarkers of response and survival. Methods: Patients with unresectable mCRC treated at Memorial Sloan Kettering with genomic tumor profiling between 2014 and 2017 were included. Patients who underwent upfront metastasectomy or received neoadjuvant/conversion chemotherapy were excluded. Clinical information was retrieved from electronic medical records, and we evaluated associations between genomic profiles with progression free survival (PFS) on first-line chemotherapy and overall survival (OS). Categorical data were analyzed by Fisher exact test and time-to-event data were analyzed by Cox proportional hazards models. Results: Of 1453 mCRCs profiled in this period, 471 patients met the study criteria. Median age was 59 years (range, 18 to 95), and 73% of patients were stage IV at diagnosis. Most tumors (91%) were microsatellite stable (MSS). The most frequent first-line regimen was FOLFOX +/- bevacizumab (66%). Among MSS patients treated with oxaliplatin-containing regimens (n = 305), 7% harbored alterations in genes associated with DNA damage response (DDR) (BRCA1, BRCA2, ATM, PALB2). DDR gene alterations were not associated with PFS (P = 0.94) nor were different quartiles of large-state transitions (P = 0.54). Genomic alterations that significantly varied by duration of response included BRAF (16%, 10%, and 5% for PFS &lt; 6 months, 6-12 months, and &gt; 12 months, respectively) and APC (62%, 74%, and 80% for PFS &lt; 6 months, 6-12 months, and &gt; 12 months, respectively). APC mutation, single or dual, was associated with significantly longer PFS (HR 0.67) and OS (HR 0.59) in multivariate analysis versus no WNT pathway alteration or alterations in other WNT pathway genes (RNF43, AXIN2, CTNNB1). Conclusions: In unresectable mCRC patients, mutations in APC were associated with better outcomes; absence of an APC alteration or the occurrence of other WNT pathway alterations was associated with shorter survival. Somatic alterations in DDR genes were not associated with outcomes in mCRC patients receiving oxaliplatin-containing regimen.

An immunohistochemical approach to detect oncogenic CTNNB1 mutations in primary neoplastic tissues

The Wnt/β-catenin signaling pathway is dysregulated in different types of neoplasms including colorectal cancer (CRC). Aberrant activation of this signaling pathway is a key early event in the development of colorectal neoplasms, and is mainly caused by loss of function mutations in Adenomatous Polyposis Coli (APC), and less frequently by β-catenin stabilization mutations via missense or interstitial genomic deletions in CTNNB1. In this study, we have defined an immunohistochemical algorithm to dissect Wnt pathway alterations in formalin-fixed and paraffin-embedded neoplastic tissues. Basically, consecutive sections of tumor specimens were stained by immunohistochemistry with two different monoclonal antibodies against β-catenin: one (anti-active β-catenin antibody) recognizes hypo-phosphorylated β-catenin and the other recognizes the total pool of β-catenin. We validated the strategy in the HCT116 CRC cell line which has an in-frame deletion of β-catenin serine 45, and then studied human tumor microarrays containing colon adenomas, CRCs, solid pseudopapillary neoplasms of the pancreas as well as the whole tissue sections of CRCs, desmoid fibromatosis, and pilomatrixoma of the skin. In some tumors, we found strong β-catenin cytoplasmic and/or nuclear staining with the total β-catenin antibody but no staining with the anti-active β-catenin antibody. This was inferred to be an altered/mutant β-catenin staining pattern. All six colon adenomas of the 126 total adenomas studied for the altered/mutant β-catenin staining pattern had presumptively pathogenic point mutations or deletions in CTNNB1. Four of 10 CRCs with the alterated/mutant β-catenin staining pattern studied in depth, from 181 total CRCs from tissue microarray, had pathogenic CTNNB1 mutations. The frequencies of CTNNB1 alterations in non-colonic tumors with altered/mutant β-catenin staining ranged between 46 and 100%. Our results demonstrate that the immunohistochemical approach described here can detect oncogenic forms of β-catenin in primary tissue samples and can also highlight other tumors with presumptive novel defects activating the Wnt/β-catenin pathway.Wnt/β-catenin signaling pathway activation is frequent event in the development of neoplasms. In this study, the authors defined an immunohistochemical approach to dissect CTNNB1 (β-catenin) mutations in formalin-fixed and paraffin-embedded primary neoplasms. They validated an immunohistochemical mutation detection algorithm in colon adenomas, carcinomas and various non-colonic Wnt pathway activated tumors.

Recurrent WNT pathway alterations are frequent in relapsed small cell lung cancer

Nearly all patients with small cell lung cancer (SCLC) eventually relapse with chemoresistant disease. The molecular mechanisms driving chemoresistance in SCLC remain un-characterized. Here, we describe whole-exome sequencing of paired SCLC tumor samples procured at diagnosis and relapse from 12 patients, and unpaired relapse samples from 18 additional patients. Multiple somatic copy number alterations, including gains in ABCC1 and deletions in MYCL, MSH2, and MSH6, are identifiable in relapsed samples. Relapse samples also exhibit recurrent mutations and loss of heterozygosity in regulators of WNT signaling, including CHD8 and APC. Analysis of RNA-sequencing data shows enrichment for an ASCL1-low expression subtype and WNT activation in relapse samples. Activation of WNT signaling in chemosensitive human SCLC cell lines through APC knockdown induces chemoresistance. Additionally, in vitro-derived chemoresistant cell lines demonstrate increased WNT activity. Overall, our results suggest WNT signaling activation as a mechanism of chemoresistance in relapsed SCLC.

2 Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

IntroductionClinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.Material and methodsExome analysis was performed on a panel of CRC cells that became resistant to commonly used targeted agents to uncover genetic alterations acquired under drug selective pressure. Tumour evolution of resistant cell population was then inferred through EXPANDs software. β-catenin activity, cell growth and death were measured upon functional and pharmacological modulation of WNT pathway in resistant cells and preclinical models established from tissues biopsy obtained from CRC patients who relapsed during treatment with targeted therapies.Results and discussionsExome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, each associated with distinct genomic landscapes. Functional and pharmacological inhibition of WNT signalling induces cell death in CRC preclinical models, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT pathway resulted in translocation of β-catenin to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones and prevent onset of secondary resistance.ConclusionIn summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumours face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments.

Abstract 205: Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

Abstract Clinical effectiveness of targeted therapies is often transitory and virtually all patients develop secondary resistance. The heterogeneous mechanisms of resistance and the sub-clonal evolution pattern of cell populations that emerge upon pharmacological selection limit disease control with subsequent lines of therapy. We used colorectal cancer (CRC) to test the hypothesis that ancestral oncogenic event (such as WNT pathway alterations) shared by every cell sub-clone might be better suited as therapeutic targets than heterogeneous events in the branches, as the former remain present in each drug-resistant cell independently from its genetic drift.To test our assumption, we first generated a panel of CRC cells resistant to commonly used targeted agents, including EGFR and BRAF inhibitors. Treatment with targeted therapies, although initially effective, fuels clonal evolution and further amplifies molecular diversity. Exome sequencing and phylogenetic tracking unveiled a complex sub-clonal architecture in CRC resistance cell populations, indicating parallel evolution of multiple independent cellular lineages, associated with distinct genomic landscapes.Functional and pharmacological modulation of WNT signaling induces cell death in CRC preclinical models from patients that relapsed during treatment, regardless of drug type or resistance mechanisms. Pharmacological blockade of WNT ligands secretion resulted in translocation of β-catenin from the cytoplasm and nucleus to the plasma membrane, decreased of β-catenin dependent Tcf/LEF transcriptional activity, and cell growth impairment despite massive molecular drift of resistant derivatives. Concomitant blockade of WNT and MAPK signalling restrained evolution of drug-resistant clones.In summary, reliance of CRC on the WNT pathway is preserved in later phases of colorectal carcinogenesis when tumors face genomic bottlenecks and sub-clonal evolution driven by administration of target therapies; thus offering the possibility of a common therapeutic strategy to overcome secondary resistance to treatments. Citation Format: Mariangela Russo, Simona Lamba, Annalisa Lorenzato, Alberto Sogari, Giorgio Corti, Giuseppe Rospo, Monica Montone, Luca Lazzari, Sabrina Arena, Daniele Oddo, Michael Linnebacher, Andrea Sartore-Bianchi, Filippo Pietrantonio, Salvatore Siena, Federica Di Nicolantonio, Alberto Bardelli. Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 205.

Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies

Attempts at eradicating metastatic cancers with targeted therapies are limited by the emergence of resistant subclones bearing heterogeneous (epi)genetic changes. We used colorectal cancer (CRC) to test the hypothesis that interfering with an ancestral oncogenic event shared by all the malignant cells (such as WNT pathway alterations) could override heterogeneous mechanisms of acquired drug resistance. Here, we report that in CRC-resistant cell populations, phylogenetic analysis uncovers a complex subclonal architecture, indicating parallel evolution of multiple independent cellular lineages. Functional and pharmacological modulation of WNT signalling induces cell death in CRC preclinical models from patients that relapsed during the treatment, regardless of the drug type or resistance mechanisms. Concomitant blockade of WNT and MAPK signalling restrains the emergence of drug-resistant clones. Reliance upon the WNT–APC pathway is preserved throughout the branched genomic drift associated with emergence of treatment relapse, thus offering the possibility of a common therapeutic strategy to overcome secondary drug resistance.

Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer

Metastatic colorectal cancers (mCRCs) are clinically heterogeneous, but the genomic basis of this variability remains poorly understood. We performed prospective targeted sequencing of 1,134 CRCs. We identified splice alterations in intronic regions of APC and large in-frame deletions in CTNNB1, increasing oncogenic WNT pathway alterations to 96% of CRCs. Right-sided primary site in microsatellite stable mCRC was associated with shorter survival, older age at diagnosis, increased mutations, and enrichment of oncogenic alterations in KRAS, BRAF, PIK3CA, AKT1, RNF43, and SMAD4 compared with left-sided primaries. Left-sided tumors frequently had no identifiable genetic alteration in mitogenic signaling, but exhibited higher mitogenic ligand expression. Our results suggest different pathways to tumorigenesis in right- and left-sided microsatellite stable CRC that may underlie clinical differences.