SMAD4 Mutations Research Articles

Post-operative mortality and recurrence patterns in pancreatic cancer according to KRAS mutation and CDKN2A, p53, and SMAD4 expression.

Alterations in KRAS, CDKN2A (p16), TP53, and SMAD4 genes have been major drivers of pancreatic carcinogenesis. The clinical course of patients with pancreatic cancer in relation to these driver alterations has not been fully characterised in large populations. We hypothesised that pancreatic carcinomas with different combinations of KRAS mutation and aberrant expression of CDKN2A, p53, and SMAD4 might show distinctive recurrence patterns and post-operative survival outcomes. To test this hypothesis, we utilised a multi-institutional cohort of 1,146 resected pancreatic carcinomas and assessed KRAS mutations by droplet digital polymerase chain reaction and CDKN2A, p53, and SMAD4 expression by immunohistochemistry. Multivariable hazard ratios (HRs) and 95% confidence intervals (CIs) for disease-free survival (DFS) and overall survival (OS) were computed according to each molecular alteration and the number of altered genes using the Cox regression models. Multivariable competing risks regression analyses were conducted to assess the associations of the number of altered genes with specific patterns of recurrence. Loss of SMAD4 expression was associated with short DFS (multivariable HR, 1.24; 95% CI, 1.09-1.43) and OS times (multivariable HR, 1.27; 95% CI, 1.10-1.46). Compared to cases with 0-2 altered genes, cases with three and four altered genes had multivariable HRs for OS of 1.28 (95% CI, 1.09-1.51) and 1.47 (95% CI, 1.22-1.78), respectively (ptrend < 0.001). Patients with an increasing number of altered genes were more likely to have short DFS time (ptrend = 0.003) and to develop liver metastasis (ptrend = 0.006) rather than recurrence at local or other distant sites. In conclusion, loss of SMAD4 expression and an increasing number of altered genes were associated with unfavourable outcomes in pancreatic cancer patients. This study suggests that the accumulation of the four major driver alterations can confer a high metastatic potential to the liver, thereby impairing post-operative survival among patients with pancreatic cancer.

Not all treated KRAS-mutant pancreatic adenocarcinomas are equal: KRAS G12D and survival outcome.

4020 Background: KRAS is an oncogenic driver in pancreatic ductal adenocarcinoma (PDAC) with mutations identified in &gt; 90% of cases. G12D is the most frequent variant, followed by G12V and G12R. We recently reported on the prognostic impact of distinct KRAS mutations. The current study utilized a large clinical and genomic database, to further explore and characterize the prognostic and molecular differences between KRAS variants, focusing on KRAS G12D and G12R. Methods: PDAC samples were tested using whole transcriptome sequencing (WTS; Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ). Transcriptomic signatures including MPAS (MAPK activation score), T-cell inflamed score and tumor micro environment (TME) characterization were calculated on WTS data. Significance was determined by X2 and Fisher-Exact and p-value was adjusted for multiple comparisons (q). Real-world overall survival (rwOS) obtained from insurance claims data was calculated from tissue collection to last contact (comparison done by Kaplan-Meier test). Results: 5,555 PDAC patients harboring either KRAS G12D (n = 2,671), G12V (n=1,871) G12R (n = 904) or G12C (n = 109) variants were identified. Patients with KRAS G12R mutant tumors had significantly longer OS compared to G12D (396 vs 311 days, HR 0.81, CI 0.74-0.88, p&lt;0.0001). There was no difference among KRAS variants in the rate of TP53, CDKN2A and SMAD4 mutations. ARID1A and KMT2D were more frequently mutated in KRAS G12D vs. G12R. The MPAS gene signature reflecting MAPK pathway activation trended lower in G12R vs. G12D. Expression of multiple genes in this pathway was statistically lower in the G12R cohort. Immune profiling suggested that: PDL1 expression is significantly lower in G12R vs G12D (13% vs 19%), TMB-H and dMMR were comparable in G12D vs G12R and several glucose and glutamine metabolism genes were significantly lower in expression when comparing G12R vs G12D - table. OS was improved within the G12R cohort for those patients on metformin (n=273 patients) (416 vs 388 days, HR 0.84, CI 0.72-0.99, p =0.037) while no difference was seen in those with KRAS G12D based on metformin use. Conclusions: Patients with G12D mutations have significantly lower survival compared to G12R. Significant molecular differences were seen in MAPK pathway gene expression, markers of immune activation, and genes involved in glucose and glutamine metabolism. Intriguingly, metformin use appeared to impact survival in the KRAS G12R subgroup. We aim to further explore distinct vulnerabilities based on MAPK pathway activation and dysregulated metabolism. Based on this data, future studies should address the KRAS mutation status and explore distinct therapeutic vulnerabilities. [Table: see text]

Correlation of survival in colorectal cancer with tumor mutational burden and mutations identified by next-generation sequencing.

e18861 Background: Next-generation sequencing (NGS) can identify specific mutations and molecular abnormalities such as high tumor mutational burden (TMB), defined as 10 or more mutations per megabase. Immune checkpoint inhibitors (ICI) have been approved for all solid tumors with high TMB, including colorectal cancer (CRC), though studies have been inconclusive regarding prognosis of high TMB as well as the efficacy of ICI’s in treating CRC specifically. We aimed to use data collected from next-generation sequencing (NGS) of CRC at our institution to correlate survival to TMB, correlate survival to presence of mutations of interest, and to investigate efficacy of ICI in this patient population. Methods: This was a retrospective cohort analysis of patients at a single institution, analyzing NGS of patients who were identified as TMB-high and microsatellite stable (MSS) (n = 161) from January 20218 to December 2020.Demographics, clinical-pathological factors, and NGS results were extracted along with clinical outcomes. We conducted a survival analysis looking at TMB status and selected mutations of interest. A descriptive subset analysis of the patients who underwent treatment with immunotherapy was performed. Results: Patients with high TMB MSS CRC trended to have worse survival with a HR of 1.45, though this was not significant (p = 0.22). Median survival for high TMB patients was 28.1 months and was 77.5 months for non-high TMB. Analysis of NGS revealed 112 unique mutations, with TP53 (82.0%), APC (80.1%), and KRAS (43.5%) occurring the most frequently. Patients were found to have statistically significant worse overall survival with KRAS (HR = 1.81, p = 0.023), CDKN2A (HR = 4.81, p = 0.0002), RB1 (HR = 2.83, p = 0.047), and SMAD4 (HR = 1.858, p = 0.039) mutations. Twelve patients underwent treatment with ICI with nine patients having between 0.7 to 4.1 months of progression-free survival (PFS) and three patients with long PFS of 13.2, 24.7, and 26.3 months. The two patients with the longest PFS were not high TMB. Conclusions: Presence of high TMB in MSS CRC did not show a statistically significant difference in outcome. Presence of KRAS, CDKN2A, RB1, and SMAD4 correlated with significantly worse prognosis. Treatment with ICI’s was effective in some CRC patients though survival did not correlate with high TMB. There is a need to identify a marker beyond TMB which better indicates which patients with MSS CRC will have good response to ICI therapy.

Clinical genomic implications of transcriptional subtypes in pancreatic cancer.

4145 Background: Transcriptional profiling of pancreatic cancers (PC) has defined classical and basal subtypes; basal tumors have worse outcomes. Mesenchymal (MES) and neural-like progenitor (NRP) subtypes are increasingly recognized and enriched post-therapy. Initial data suggests worse outcomes to FOLFIRINOX (FFX) compared with gemcitabine nab-Paclitaxel (GnP) in basal tumors. Several clinical trials are ongoing to investigate this. Here, we examined the clinical implications of transcriptional subtypes in a large, real-world dataset. Methods: Retrospective IRB exempt, deidentified data was examined from NextGen DNA and RNA sequencing performed on PCs at Caris Life Sciences (Phoenix, AZ). Classical and basal cell states were identified using RNA-seq and the PurIST algorithm in a genomic cohort or GATA6 and KRT5 expression levels in a clinical cohort. Tumor microenvironment immune cell composition on RNA seq was performed using QuantiSeq. Survival was obtained from insurance claims data and calculated from first treatment date to last known contact. Kaplan-Meier estimates were calculated for patient cohorts. P values were adjusted using Benjamini-Hochberg correction. Results: A total of 7,250 PCs were profiled in the genomic cohort. 3,063 tumors (42.2%) were strongly classical (SC), 2,015 tumors (27.8%) were strongly basal (SB) and the remaining had mixed phenotypes. MES and NRP marker genes were significantly co-expressed with each other, with basal genes, and anti-correlated with classical genes. When compared to SC, SB had significantly higher mutation rates in KRAS (93% vs. 88%), TP53 (83% vs. 72%) and ARID1A (12% vs. 8%), whereas SMAD4 (23% vs. 17%) mutations were more common in SC (all q &lt; 0.05). There were no differences in mutation rates in homologous recombination or mismatch repair genes. SB had a significantly higher fraction of M1 macrophages (fold change [FC]: 1.14) and neutrophils (FC 1.16), whereas SC tumors had higher M2 macrophages (FC 1.18), NK (FC 1.2), and dendritic cells. Overall proportions of CD4/8 T cells were low and not different. Interestingly, SB had higher levels of PD-L1 by IHC (4.8% vs. 35%) and higher expression of immune exhaustion genes including CTLA4 (FC 1.19), TIM3 (FC 1.22) and PD-1 (FC 1.43) (all q &lt; 0.05). The clinical cohort had 1,623 patients. Basal tumors had an inferior survival (median survival: 8.2 months (mo) vs 13.3 mo (Hazard Ratio (HR) 0.67, p &lt; 0.00001)) and showed a significant improvement in outcomes when treated with upfront FFX vs GnP (n = 80 vs 90, Median: 15.8 vs 7.4 mos., HR 0.68, p = 0.021). This difference between FFX vs GnP was less pronounced in classical tumors (n = 70 vs 89, Median: 17.3 vs 15.4 mos, HR 0.70, p = 0.049). Conclusions: Our work represents the largest known real world molecular comparison of transcriptional subtypes of PC. Differential outcomes for patients with basal tumors treated with FFX versus GnP warrants further investigation in prospective studies.

The differential response to immune checkpoint inhibitors (ICIs) according to mismatch repair alterations in gastrointestinal (GI) non-colorectal cancers (non-CRCs) and the impact of dual vs. monotherapy ICIs on survival in GI (CRC and non-CRC) cancers.

2620 Background: In patients with mismatch repair deficient (dMMR) colorectal cancers (CRCs), we previously reported that loss of expression of MSH2 and MSH6 (MutS co-loss) was associated with better response to ICIs and longer median overall survival (mOS) compared to loss of expression of MLH1 and PMS2 (MutL co-loss). Here, we expanded our analysis and included gastrointestinal (GI) non-CRCs and explored the impact of dual vs. monotherapy ICIs on mOS in GI (CRC and non-CRC) cancers. Methods: Specimens were profiled by next-generation sequencing (592, NextSeq; WES, WTS NovaSeq) (Caris Life Sciences, Phoenix, AZ). MMR/microsatellite instability (MSI) status was determined by immunohistochemistry (IHC) of MMR protein. Real world OS was extracted from insurance claims and calculated using Kaplan-Meier estimates for molecularly defined cohorts from first treatment with ICIs (Nivolumab, Nivo; Ipilumumab, ipi; or Pembrolizumab, pembro) to last contact. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q &lt; 0.05). Results: The GI non-CRC cohort (N= 19,767) included cancers of the esophagus, stomach, gastroesophageal junction, pancreas, bile duct and small bowel with 97 (0.49%) patients having MutS co-loss, and 494 (4.03%) patients having MutL co-loss. MutS co-loss was associated with increased KRAS (45.4% vs 25.2%, q&lt;0.01), CDKN2A (29.2% vs 9.0%), GNAS (27.8% vs 7.8%) and SMAD4 (17.5% vs 5.9%) mutations compared to MutL co-loss. Independent of treatment, MutS co-loss (N=74) had improved mOS compared to MutL co-loss (N=332) (40.4 m vs. 26.2 m, HR = 0.66; (95% CI: 0.46-0.95), P=0.024). In patients treated with ICIs, the mOS in MutS co-loss (N=21) was better compared to MutL co-loss (N=76) (not reached (NR) vs. 25.4 m, HR= 0.23 (95% CI: 0.07-0.75), p=0.008). At 3 years, more than 80% of the patients with MutS co-loss were alive. Of particular importance, when looking at all GI (CRC and non-CRC) patients, the mOS of MutL co-loss treated with ipi/nivo (N=18) trended for better mOS compared to MutL co-loss treated with pembro (N=215) (NR vs. 28.2m (HR=0.39; (95% CI:0.14-1.07), p=0.057), while the mOS of MutS co-loss treated with ipi/nivo (N=6) was not different compared to MutS co-loss treated with pembro (N=44) (NR vs. NR, HR=0.75 (95% CI: 0.094-5.92), p=0.78). Conclusions: In ICI-treated GI non-CRCs, the mOS was longer in MutS co-loss compared to MutL co-loss. In ICI-treated GI (CRC and non-CRC) patients with MutL co-loss, there was a trend for better survival with ipi/nivo compared to pembro. Our data suggest that the MutS vs. MutL status may guide the choice of ICIs regimen (Dual vs. Monotherapy) but more data are needed.

Therapeutic yield of extensive molecular profiling in cholangiocarcinoma: a retrospective single-center study.

Current available systemic therapies for advanced cholangiocarcinoma (CCA) are of limited effectiveness and prognosis is poor. Recently, introduction of next-generation sequencing (NGS) technologies led to a better understanding of the genetic pathophysiology and, consequently, identification of molecular alterations for targeted treatment. To determine the proportion of actionable alterations using extensive molecular profiling in a routine diagnostic setting and to study the effect of targeted treatment on disease control. Results of extensive molecular testing by either FoundationOne NGS or an in-house developed 96 cancer gene panel were retrospectively collected from patients with locally advanced or metastatic CCA diagnosed between 01/12/2018 and 01/08/2021 in a single center. Gene variants were classified according to ESCAT and correlated with efficacy endpoints. Of 125 patients included, 65 patients had an intrahepatic CCA (iCCA). FGFR2 fusions and IDH1/BAP1 mutations were more frequent in iCCA, while KRAS and SMAD4 mutations were predominant in extrahepatic CCA (eCCA). Targetable alterations (ESCAT tiers I-IV) were identified in 73,6% of patients. Overall survival was significantly better for higher tiers regardless of treatment. Thirteen patients (10.4%) received targeted treatment based on molecular profiling, with a median progression-free survival (PFS) of 7.3months. Extensive molecular characterization led to the identification of targetable and potentially targetable alterations in a significant proportion of patients with locally advanced or metastatic CCA. We confirmed the association between higher ESCAT tier and benefit of a targeted treatment. Molecular analysis should therefore be considered in all patients fit enough for systemic treatment.

Predictive ability of pancreatic cyst fluid biomarkers: A systematic review and meta-analysis

BackgroundMucinous pancreatic cysts harbor the potential to progress to highly lethal pancreatic ductal adenocarcinoma (PDAC). Since these precursor cysts require cancer surveillance or surgical resection, they need to be reliably distinguished from harmless pancreatic cysts. Current clinical and radiographic assessment is imperfect and the value of cyst fluid analysis for differential diagnosis is unclear. Therefore, we set out to investigate the value of cyst fluid biomarkers in distinguishing pancreatic cysts. MethodsWe performed a systematic review of the current literature to identify articles that evaluated the diagnostic performance of clinically relevant and promising candidate cyst fluid biomarkers, with a particular emphasis on DNA-based biomarkers. Meta-analysis was performed for biomarkers targeted at identifying cyst type and presence of high-grade dysplasia or PDAC. ResultsData from a total of 42 studies was analyzed. Mutations in KRAS and/or GNAS allowed identification of mucinous cysts with a sensitivity of 79% and specificity of 98%. This exceeded the performance of the traditional biomarker carcinoembryonic antigen (CEA; sensitivity 58%, specificity 87%). Mutations in VHL were specific for serous cystadenomas (SCAs; sensitivity 56%, specificity 99%) and help to exclude mucinous cysts. Mutations in CDKN2A, PIK3CA, SMAD4, and TP53 each had high specificities of 97%, 97%, 98%, and 95%, respectively, to identify high-grade dysplasia or PDAC in mucinous cysts. ConclusionsCyst fluid analysis can be a valuable tool in the characterization of pancreatic cysts, with relevant clinical implications. Our results support the use of DNA-based cyst fluid biomarkers in the multidisciplinary diagnostic work-up of pancreatic cysts.

TP53 Gain-of-Function and Non–Gain-of-Function Mutations Are Associated With Differential Prognosis in Advanced Pancreatic Ductal Adenocarcinoma

To examine the impact of TP53 gain-of-function (GOF) and non-GOF mutations on prognosis of advanced pancreatic ductal adenocarcinoma (PDAC) among patients with KRAS, CDKN2A, and SMAD4 comutations. This cohort included patients with locally advanced, recurrent, and de novo metastatic PDAC with next-generation sequencing performed from November 2017 to May 2022. We defined R175H, R248W, R248Q, R249S, R273H, R273L, and R282W as GOF and all other p53 mutations (mutp53) as non-GOF. We used Cox regression modeling to examine the association between GOF and non-GOF mutp53 and overall survival (OS), adjusting for demographics, performance status, Charlson comorbidity index, receipt of chemotherapy, and KRAS, CDKN2A, and SMAD4 comutations. Of 893 total eligible patients, 68.5% had tumors with mutp53, 90.1% had KRAS mutations (mutKRAS), 44.7% had CDKN2A mutations (mutCDKN2A), and 17.0% had SMAD4 mutations. Among patients with mutp53, 121 had GOF and 491 had non-GOF. GOF mutp53 was associated with worse OS than non-GOF mutp53 (hazard ratio [HR], 1.27; 95% CI, 1.02 to 1.59) and wild-type p53 (wtp53; HR, 1.24; 95% CI, 0.98 to 1.57), whereas non-GOF was not associated with worse OS than wtp53 (HR, 0.95; 95% CI, 0.80 to 1.13). In addition, mutKRAS was associated with worse OS than wild-type KRAS in patients with mutCDKN2A (HR, 1.57; 95% CI, 0.88 to 2.80) but not in patients with wild-type CDKN2A (HR, 1.03; 95% CI, 0.76 to 1.39). GOF and non-GOF mutp53 were associated with differential prognosis in advanced PDAC. The adverse effect of mutKRAS on OS appeared to be primarily driven by patients with mutCDKN2A. Our results provide new insight that could be helpful for prognostic stratification in clinical practice and for aiding future clinical trial designs.

Abstract 284: Pro-Inflammatory Adventitial Fibroblast Contribute To Aortic Aneurysms In Loeys Dietz Syndrome

Introduction: Vascular smooth muscle cell (SMC) phenotype modulation is implicated in thoracic aneurysm formation, however, the role of adventitial fibroblasts (AdvFib) in aneurysm formation remains poorly characterized. Loeys-Dietz Syndrome (LDS), caused by a spectrum of mutations in the TGF-β signaling cascade, leads to aggressive thoracic aneurysms. Given the ubiquitous nature of TGF-β signaling machinery, the exact cell type(s) responsible for initiation of LDS pathogenesis is poorly understood. Methods: We performed single cell transcriptomic characterization of aortic root and ascending aortic samples from LDS patients ((n=6 LDS (harboring TGFB2 , TGFBR1 , TGFBR2 , or SMAD3 mutation); n=2 Donor control)) as well as root/ascending aorta from a murine model of LDS ( Tgfbr2 G357W/+ ) to understand cell-state transitions and transcriptomic alterations in LDS. Results: Single cell RNA sequencing (scRNAseq) of human and mice LDS samples (in total &gt;40,000 cells) revealed a secondary pattern of cellular activation in LDS as compared to Marfan Syndrome. Instead of SMC phenotypic modulation as seen in MFS, LDS appears to preferentially modulate adventitial fibroblasts (AdvFib) phenotype in the Tgfbr2 G357W/+ mouse model. Human LDS patient’s AdvFib turn on high levels of macrophage-recruiting pro-inflammatory cytokines CCL2 and HLA-B, with a corresponding 1.8-fold increase in LDS aortic wall macrophages compared to Donor. A more extreme pattern was observed in the Tgfbr2 G357W/+ LDS mouse model. While Tgfbr2 G357W/+ SMC are transcriptomically similar to wild type counterparts, AdvFib were activated into a pro-inflammatory state associated with increased aortic macrophage recruitment ( Ccl2 , Il6 , Vcam1, and Cxcl2 ) and fibrotic response ( Col1a1 , Col1a2 , Col3a1, and Fn1 ). A 6-fold increase in the number of aortic wall macrophages was identified in Tgfbr2 G357W/+ compared to WT. Conclusions: LDS mouse and human AdvFib undergo phenotypic alterations into a pro-inflammatory state evidenced by increased expression of numerous cytokines, macrophage recruitment, and ECM deposition. AdvFib, in addition to SMCs, are potentially another important pathological cell population in LDS aneurysm formation.

ASO Visual Abstract: Predictors of Long-Term Survival in Pancreatic Ductal Adenocarcinoma After Pancreatectomy-TP53 and SMAD4 Mutation Scoring in Combination with CA19-9.

ASO Visual Abstract: Predictors of Long-Term Survival in Pancreatic Ductal Adenocarcinoma After Pancreatectomy-TP53 and SMAD4 Mutation Scoring in Combination with CA19-9.

Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (-/-) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (-/-) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.

Oncogenic signaling is coupled to colorectal cancer cell differentiation state.

Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC, KRAS, TP53, and SMAD4 enable oncogenic signaling and establish the hallmarks of cancer. Here, we use mass cytometry of isogenic human colon organoids and patient-derived cancer organoids to capture oncogenic signaling, cell phenotypes, and differentiation states in a high-dimensional single-cell map. We define a differentiation axis in all tumor progression states from normal to cancer. Our data show that colorectal cancer driver mutations shape the distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell promoting or restricting effects. Individual nodes of the cancer cell signaling network remain coupled to the differentiation state, regardless of the presence of driver mutations. We use single-cell RNA sequencing to link the (phospho-)protein signaling network to transcriptomic states with biological and clinical relevance. Our work highlights how oncogenes gradually shape signaling and transcriptomes during tumor progression.

Abstract 3121: Exercise and tumor genomic landscapes in 2,879 patients with cancer

Abstract Approximately two-thirds of cancer diagnoses globally are attributed to modifiable lifestyle factors such as smoking, diet and inactivity. Conversely, regular exercise is linked to decreased risk of multiple cancers. However, the underlying molecular mechanisms are not fully elucidated. Specifically, how exercise impacts tumor genomic landscapes has not been considered. To address this gap, we integrated clinical annotation of exercise exposure with tumor mutational profiling of 2,879 patients with cancer. Exercise exposure was evaluated by validated questionnaire and tumor genomic profiling of ≥ 505 commonly mutated cancer genes was performed using the Memorial Sloan Kettering Cancer Center Integrated Mutation Profiling of Actional Cancer Targets (MSK-IMPACT) assay. Among the most represented cancer types were breast (n = 623), lung (n = 524), endometrial (n = 260), colorectal (n = 217) and prostate (n = 176). We found that exercise influences the genomic landscapes of tumors in a cancer-type specific manner. For instance, breast, colorectal and lung cancers from exercising patients had significantly lower tumor mutation burden (TMB) compared with non-exercisers. TMB of melanoma was significantly higher in exercisers versus non-exercisers. A pan-cancer analysis showed that mutations in SMAD3 were more frequent among exercisers, while mutations in STK11, KEAP1 and RBM10 were more frequent among non-exercisers. Overall, this study shows that exercise regulates tumor genomic landscapes and does so in a cancer-site specific manner. Citation Format: Brandon L. Tsai, Lydia Y. Liu, Stefan E. Eng, Whitney P. Underwood, Catherine P. Lee, Joshua W. Bliss, Paul C. Boutros, Lee W. Jones. Exercise and tumor genomic landscapes in 2,879 patients with cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3121.

Abstract 1328: Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system

Abstract Despite the use of multi-agent conventional chemotherapy regimens, metastatic pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with a 5-year survival of 9%. PDAC is characterized by a complex tumor microenvironment with multilayered interplay between neoplastic and stromal cells and the natural extracellular matrix (ECM). To date, models that recapitulate the complexity of these features are only partially reproduced ex vivo and lack the capacity to recreate PDAC tumor microenvironment physiology, fluid flow and the ECM architecture. To address these issues, we developed Curiochips MPS platform for metastatic PDAC on-a-chip. To replicate an accurate PDAC complex tumor microenvironment, we investigated human pancreatic cancer cell lines with/without SMAD4 mutation as a test model. SMAD4 is among the most frequently altered genes in PDAC and its mutation accelerates pancreatic cancer cell migration. To tailor cell-ECM interactions and recreate natural biochemical environment of tumors, we examined various ECM conditions. ECM substitutes such as type I collagen and fibrin gel offer mechanical properties to closely mimic the tumor ECM. We accessed the effect of ECM condition on the pancreatic stellate cells (PSC) activation and pancreatic cancer cell migration using PANC-1 (SMAD4-WT) and BxP3 (SMAD4-null) cell lines. Notably, type I collagen induced more PSC activation than fibrin gel. Interestingly BxP3 spheroids migrated toward to the endothelial compartment compared to PANC-1. Angiogenesis of endothelial cells was more prominent in co-culture with BxP3 than PANC-1. Furthermore, we are building the metastatic model using PDAC patient derived organoids and co-culture with primary PSC in different types of ECM. Preliminary analysis showed functional responses such as 3D organoid migration and aggregation, PSC activation and migration controlled by specific properties of EMC. Our data highlight the importance of the ECM composition on PDAC migration, PSC activation and angiogenesis in the context of genetic heterogeneity and presence/absence oncogenic mutation. The 3D co-culture platform, Curiochips offers suitable and tailored solution to investigate tumor-stroma interaction and potentially can be used as a platform for drug discovery and precision oncology to target PDAC desmoplastic tumor microenvironment. Citation Format: Sumin Kim, Yeongmin Choi, Jihye Baek, Bharti Garg, Jan Pencik, Andrew M. Lowy, Herve Tiriac, Sanghee Yoo. Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1328.

Abstract 3641: Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma

Abstract Up to 50% of pancreatic ductal adenocarcinomas (PDA) harbor mutations in canonical TGFß pathway genes such as SMAD4 and TGFBR2. SMAD4 mutations have been shown to drive resistance to chemotherapy, metastasis, and margin positivity following surgical resection. To characterize the unique tumor microenvironment of SMAD4 mutant PDA, we developed isogenic, orthotopic mouse models using CRISPR mediated knockout of Smad4 in a cell line derived from KPfC (KrasLSL-G12D;Trp53fl/fl;Pdx1Cre) mice, a genetically engineered mouse model of pancreatic cancer. To characterize the alteration of the secretome caused by the loss of Smad4, we performed proteomic analysis with conditioned media collected from Smad4 wildtype and mutant cancer cell clones. Interestingly, the loss of Smad4 resulted in the production of neuroendocrine related factors. In addition, gene ontology analysis with the secretome suggested that these factors were associated with neuronal processes. These data indicate that loss of Smad4 in PDA cancer cells may lead to a neuroendocrine signature change. In particular, we found one of the most up-regulated secreted factors in the mutant cells was SPP1, which has been shown to promote tumor progression by affecting cancer cell stemness and macrophage polarization. To validate the proteomic results, we established orthotopic models with Smad4 wildtype and mutant clones. We found that loss of Smad4 resulted in larger tumor growth. Moreover, immunohistochemistry showed a significant up-regulation of SPP1 in the cancer cells of Smad4 mutant tumors. To further profile the alteration of immune landscape, we stained for markers of myeloid cells and lymphocytes. We found that Smad4 mutant tumors had an immunosuppressive microenvironment characterized by increased M2 macrophage, neutrophil and MDSC infiltration. Altogether, our study suggests that loss of SMAD4 in PDA may induce a neuroendocrine signature with increased SPP1 secretion in cancer cells. The production of this unique cancer cell secretome may result in a microenvironment marked by immunosuppressive myeloid cells. Citation Format: Gilbert Z. Murimwa, Francesca Rossi, Henry K. Fleming, Zeynep Yazgan, Dina Alzhanova, Huocong Huang, Rolf A. Brekken. Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3641.

Pathogenic genomic alterations in Chinese pancreatic cancer patients and their therapeutical implications.

Approximately 90% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by the untargetable non-G12C KRAS mutations, and only a small subset of patients are eligible for FDA-approved precision therapies. The practice of precision therapy in pancreatic cancer was limited by the paucity of targetable genetic alterations, especially in the Asian population. To explore therapeutic targets in 499 Chinese PDAC patients, a deep sequencing panel (OncoPanscan™, Genetron health) was used to characterize somatic alterations including point mutations, indels, copy number alterations, gene fusions as well as pathogenic germline variants. We performed genomic profiling in 499 Chinese PDAC patients, which revealed somatic driver mutations in KRAS, TP53, CDKN2A, SMAD4, ARID1A, RNF43, and pathogenic germline variants (PGVs) in cancer predisposition genes including BRCA2, PALB2, and ATM. Overall, 20.4% of patients had targetable genomic alterations. About 8.4% of patients carried inactivating germline and somatic variants in BRCA1/2 and PALB2, which were susceptible to platinum and PARP inhibitors therapy. Patients with KRAS wild-type disease and early-onset pancreatic cancer (EOPC) harbored actionable mutations including BRAF, EGFR, ERBB2, and MAP2K1/2. Compared to PGV-negative patients, PGV-positive patients were younger and more likely to have a family history of cancer. Furthermore, PGVs in PALB2, BRCA2, and ATM were associated with high PDAC risk in the Chinese population. Our results demonstrated that a genetic screen of actionable genomic variants could facilitate precision therapy and cancer risk reduction in pancreatic cancer patients of Asian ethnicity.

Oncomicrobial Community Profiling Identifies Clinicomolecular and Prognostic Subtypes of Colorectal Cancer

Dysbiosis of gut microbiota is linked to the development of colorectal cancer (CRC). However, microbiota-based stratification of CRC tissue and how this relates to clinicomolecular characteristics and prognosis remains to be clarified. Tumor and normal mucosa from 423 patients with stage I to IV CRC were profiled by bacterial 16S rRNA gene sequencing. Tumors were characterized for microsatellite instability (MSI), CpG island methylator phenotype (CIMP), APC, BRAF, KRAS, PIK3CA, FBXW7, SMAD4, and TP53 mutations, subsets for chromosome instability (CIN), mutation signatures, and consensus molecular subtypes (CMS). Microbial clusters were validated in an independent cohort of 293 stage II/III tumors. Tumors reproducibly stratified into 3 oncomicrobial community subtypes (OCSs) with distinguishing features: OCS1 (Fusobacterium/oral pathogens, proteolytic, 21%), right-sided, high-grade, MSI-high, CIMP-positive, CMS1, BRAF V600E, and FBXW7 mutated; OCS2 (Firmicutes/Bacteroidetes, saccharolytic, 44%), and OCS3 (Escherichia/Pseudescherichia/Shigella, fatty acid β-oxidation, 35%) both left-sided and exhibiting CIN. OCS1 was associated with MSI-related mutation signatures (SBS15, SBS20, ID2, and ID7) and OCS2 and OCS3 with SBS18 related to damage by reactive oxygen species. Among stage II/III patients, OCS1 and OCS3 both had poorer overall survival compared with OCS2 for microsatellite stable tumors (multivariate hazard ratio [HR], 1.85; 95% confidence interval [CI], 1.15-2.99; P= .012; and HR, 1.52; 95% CI 1.01-2.29; P= .044, respectively) and left-sided tumors (multivariate HR, 2.66; 95% CI, 1.45-4.86; P= .002; and HR, 1.76; 95% CI, 1.03-3.02; P= .039, respectively). OCS classification stratified CRCs into 3 distinct subgroups with different clinicomolecular features and outcomes. Our findings provide a framework for a microbiota-based stratification of CRC to refine prognostication and to inform the development of microbiota-targeted interventions.

Loss of function SMAD4 nonstop mutations in human cancer.

SMAD4 is a tumour suppressor gene that is mutated in a variety of cancers. SMAD4 nonstop mutations, which convert stop codons to sense codons that extend transcription, have been identified in genomic databases but have not been characterised in human pathology samples. The frequency of SMAD4 nonstop mutations and the consequences of nonstop mutations on SMAD4 protein expression are unknown. We retrospectively analysed our cancer sequencing database of 38,002 tumour specimens and evaluated the spectrum of SMAD4 mutations. SMAD4 protein expression was evaluated by immunohistochemistry in tumours with SMAD4 nonstop mutations. In total, 1956 SMAD4 mutations were identified in 1822 tumours. SMAD4 mutations were most common in tumours of the gastrointestinal tract and included nonsense variants (n=344), frameshift indels (n=258), splice site variants (n=104), and missense variants at codon R361 (n=245). In a subset of cases with immunohistochemistry, SMAD4 expression was lost in 23 of 25 tumours (92%) with protein truncating variants and in 7 of 27 tumours (26%) with missense variants. Four cases harboured SMAD4 nonstop mutations. SMAD4 nonstop mutations were identified in two pancreatic adenocarcinomas, one colonic adenocarcinoma, and one non-small cell lung carcinoma. Immunohistochemistry demonstrated loss of SMAD4 protein expression in each of the four tumours with SMAD4 nonstop mutations. SMAD4 nonstop mutations are associated with loss of SMAD4 protein expression in multiple tumour types. SMAD4 nonstop mutations should be clinically interpreted as pathogenic loss of function alterations when identified in cancer sequencing panels.

The targeted next-generation sequence revealed SMAD4, AKT1, and TP53 mutations from circulating cell-free DNA of breast cancer and its effect on protein structure – A computational approach

Breast cancer biomarkers that detect marginally advanced stages are still challenging. The detection of specific abnormalities, targeted therapy selection, prognosis, and monitoring of treatment effectiveness over time are all made possible by circulating free DNA (cfDNA) analysis. The proposed study will detect specific genetic abnormalities from the plasma cfDNA of a female breast cancer patient by sequencing a cancer-related gene panel (MGM455 – Oncotrack Ultima), including 56 theranostic genes (SNVs and small INDELs). Initially, we determined the pathogenicity of the observed mutations using PredictSNP, iStable, Align-GVGD, and ConSurf servers. As a next step, molecular dynamics (MD) was implemented to determine the functional significance of SMAD4 mutation (V465M). Lastly, the mutant gene relationships were examined using the Cytoscape plug-in GeneMANIA. Using ClueGO, we determined the gene’s functional enrichment and integrative analysis. The structural characteristics of SMAD4 V465M protein by MD simulation analysis further demonstrated that the mutation was deleterious. The simulation showed that the native structure was more significantly altered by the SMAD4 (V465M) mutation. Our findings suggest that SMAD4 V465M mutation might be significantly associated with breast cancer, and other patient-found mutations (AKT1-E17K and TP53-R175H) are synergistically involved in the process of SMAD4 translocate to nuclease, which affects the target gene translation. Therefore, this combination of gene mutations could alter the TGF-β signaling pathway in BC. We further proposed that the SMAD4 protein loss may contribute to an aggressive phenotype by inhibiting the TGF-β signaling pathway. Thus, breast cancer’s SMAD4 (V465M) mutation might increase their invasive and metastatic capabilities. Communicated by Ramaswamy H. Sarma

Analysis of the relative risks of first aortic events linked to hereditary thoracic aortic diseases

Objective: Heritable Thoracic Aortic (HTA) disease is characterized by pathogenic variants in eleven genes, even though there is insufficient data to categorise the First Aortic Events (FAE) risk related to such genes. Methods: The chance of FAE was estimated for a retrospective cohort of probands and families with rare mutations in seven genes for HTA disease (n=1,028) using the gene variables, pathogenic variation class, gender, proband status, and region of recruitment. Results: Both the Smooth Muscle Contraction (SMC) genes (MYLK, ACTA2, and PRKG1; P =0.002) and the Loeys-Dietz syndrome genes (TGFBR1, TGFBR2, TGFB2, and SMAD3; P&lt; 0.0001), which encodes a protein in the Transforming Growth Factor (TGF)-b pathway, showed significant variations in aortic event risk. In contrast to those who received elective aneurysm treatment, participants with variations in TGFBR2 had a reduced cumulative incidence of type-A aortic dissection. Participants with mutations in MYLK, ACTA2, PRKG1, and SMAD3 had consistently higher incidences of type-A aortic dissection. Compared to other genes, PRKG1, ACTA2, and TGFBR2 exhibited a higher Cumulative Incidence (CI) of type B aortic dissection. Even if proband status, gender, and recruiting location were taken into consideration, there was a noticeably higher risk of an FAE with childhood onset associated with specific polymorphisms in the gene ACTA2 and TGFBR2. Conclusion: Data on FAE in people with HTA disease that are gene and variant-specific assist personalized aortic monitoring and clinical care.