Non-mucinous Colorectal Cancer Research Articles

Abstract 1756: Characterizing the mutational spectrum of metastatic mucinous adenocarcinoma of the appendix

Abstract Background: Appendiceal cancer (AC) is an orphan malignancy with only 1-2 cases per 100,000 people in the US. The genomic composition of metastatic, mucinous AC is yet to be resolved at the whole exome level and few groups have assessed the clinical actionability of target genes. Here we performed whole-exome sequencing (WES) on AC specimens to 1) elucidate the mutational spectrum of AC, 2) identify recurrent mutations at frequencies unique to AC or in common with colorectal cancer (CRC), and 3) uncover recurrent AC mutations that are associated with patient survival outcomes. Methodology: Twenty-four FFPE samples, one from each of 24 AC patients treated at our institution with cytoreductive surgery (CRS) followed by heated intraperitoneal chemotherapy (HIPEC), were collected from our tumor bank with IRB approval. DNA from tumor and corresponding normal tissue (PBMCs) was extracted, assessed for integrity and quantity, then analyzed by WES using the Illumina NextSeq 6000 platform. The resulting data were processed using both GATK and DRAGEN pipelines. The frequencies of recurrent mutations in AC were compared with those of CRC from cBioPortal and statistically compared by Fisher’s Exact Test. Logrank test was performed to estimate survival associations based off mutation status. Mutational signatures derived from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium were cross-compared to our cohort to identify previously unknown mutational processes in AC. Results: The most frequent mutations were found in MUC6 (63%), KRAS (58%), HLA-B (54%), PABPC1 (50%), and MUC5AC (50%). Distinct from a 534-patient CRC cohort, AC had lower rates of alteration in APC (4% vs 73% CRC, p<0.001) and TP53 (21% vs. 62%, p<0.001), with higher rates of alteration in HLA-B (54% vs 3%, p<0.001), GNAS (38% vs 5%, p<0.001), and ABCA1 (29% vs 7%, p=0.001). Notably, ABCA1 mutation frequency was higher in mucinous CRC (n=56) when compared to non-mucinous CRC (n=478, 20% vs 7%; p<0.001), suggesting that ABCA1 may be uniquely altered in bowel cancers of mucinous phenotype. Alterations in Rho-A were a statistically significant predictor of poor survival in both AC (p<0.001) and CRC (p=0.05). PCAWG analysis showed that the predominant mutational signature in AC most resembles SBS5, suggesting that the mutational etiology of AC may be linked to age and/or smoking status. Discussion: Here we present the largest whole-exome sequencing analysis of AC analyzed to date and identify previously undescribed recurrently mutated genes unique to AC as compared to CRC. We demonstrate novel associations between altered genes and patient survival, which opens discussion regarding the clinical utility of molecular markers in AC for assisting treatment decision making. Citation Format: Daniel J. Gironda, Ming Leung, Guangxu Jin, Liang Liu, Konstantinos I. Votanopoulos, Edward A. Levine, Lance D. Miller. Characterizing the mutational spectrum of metastatic mucinous adenocarcinoma of the appendix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1756.

Utilizing multimodal mass spectrometry imaging for profiling immune cell composition and N-glycosylation across colorectal carcinoma disease progression.

Colorectal cancer (CRC) stands as a leading cause of death worldwide, often arising from specific genetic mutations, progressing from pre-cancerous adenomas to adenocarcinomas. Early detection through regular screening can result in a 90% 5-year survival rate for patients. However, unfortunately, only a fraction of CRC cases are identified at pre-invasive stages, allowing progression to occur silently over 10-15years. The intricate interplay between the immune system and tumor cells within the tumor microenvironment plays a pivotal role in the progression of CRC. Immune cell clusters can either inhibit or facilitate tumor initiation, growth, and metastasis. To gain a better understanding of this relationship, we conducted N-glycomic profiling using matrix-assisted laser desorption-ionization mass spectrometry imaging (MALDI-MSI). We detected nearly 100 N-glycan species across all samples, revealing a shift in N-glycome profiles from normal to cancerous tissues, marked by a decrease in high mannose N-glycans. Further analysis of precancerous to invasive carcinomas showed an increase in pauci-mannose biantennary, and tetraantennary N-glycans with disease progression. Moreover, a distinct stratification in the N-glycome profile was observed between non-mucinous and mucinous CRC tissues, driven by pauci-mannose, high mannose, and bisecting N-glycans. Notably, we identified immune clusters of CD20+ B cells and CD3/CD44+ T cells distinctive and predictive with signature profiles of bisecting and branched N-glycans. These spatial N-glycan profiles offer potential biomarkers and therapeutic targets throughout the progression of CRC.

Increased Fusobacterium tumoural abundance affects immunogenicity in mucinous colorectal cancer and may be associated with improved clinical outcome

There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the association of Fusobacterium abundance with immune cell composition and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumour microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with an increased proportion of CD8 + lymphocytes (p = 0.018), regulatory T-cells (p = 0.001) and M2 macrophages (p = 0.001). In the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. High Fusobacterium abundance was associated with an increased proportion of CD4 + lymphocytes (p = 0.031) and M1 macrophages (p = 0.006), whilst M2 macrophages (p = 0.043) were under-represented in this cohort. Patients with increased Fusobacterium relative abundance in our mucinous CRC TCGA cohort tended to have better clinical outcomes (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance may be associated with improved outcomes in mucinous CRC, possibly due to a modulatory effect on the host immune response.Key messages• Increased Fusobacterium relative abundance was not found to be associated with microsatellite instability in mucinous CRC.• Increased Fusobacterium relative abundance was associated with an M2/M1 macrophage switch, which is especially significant in mucinous CRC, where M2 macrophages are overexpressed.• Increased Fusobacterium relative abundance was associated with a significant improvement in disease specific survival in mucinous CRC.• Our findings were validated at a protein level within our own in house mucinous and non-mucinous rectal cancer cohorts.

Correlation of CDX2 Protein Expression with Clinicopathologic Features and Survival Rate in Iraqi Patients with Colorectal Cancer

Predicting the prognosis of colorectal cancer (CRC) is challenging since these tumors exhibit a wide range of biological behaviors. It has been hypothesized that caudal-related homeobox gene 2 (CDX2), which is vital for intestinal growth and maintenance, has a tumor-suppressing effect and promising role in CRC prognosis but studies are still controversial. This study used the immunohistochemical (IHC) staining method to determine the expression of the CDX2 protein in mucinous and non-mucinous CRC adenocarcinoma, as well as in normal colorectal tissues as a control, and correlate this expression with clinicopathological features such as grade, tumor distant metastasis, tumor site, histological type, lymph node metastasis, tumor invasion, sex, age, and rate of 4 years Overall survival (OS) after diagnosis. A total of sixty three tissue samples were obtained from CRC patients (58.90±14.94) years and embedded in wax and thirty-seven normal non-tumoural colorectal tissue samples with (56.43±12.28) years as a control group. CDX2 protein expression decreased significantly (p<0.05) in CRC patients than control, advanced age, mucinous pattern of CRC, moderate and poorly differentiated grades, lymph node metastasis, advanced tumor invasion (T3, T4), and organs metastasis. Moreover, the (OS) for patients with low CDX2 expression was (17.943±1.7) months compared to (33.431±2.7) months for those with high CDX2 expression (p = 0.0001). This study concluded that protein expression of CDX2 is regarded as a prognostic and diagnostic marker for CRC patients.

Apoptotic and Necroptotic Mediators are Differentially Expressed in Mucinous and Non-Mucinous Colorectal Cancer.

BackgroundMucinous colorectal cancer (CRC) represents 10% of all CRC and is associated with chemotherapy resistance. This study aimed to determine expression of apoptosis and necroptosis mediators in mucinous CRC.MethodsRNA gene expression data were extracted from TCGA. Protein levels in 14 mucinous and 39 non-mucinous tumors were measured by multiplexed immunofluorescence. Levels of apoptosis and necroptosis signalling proteins were analysed in SW1463 (mucinous rectal), SW837 (non-mucinous rectal), LS174T (mucinous colon) and HCT116 (non-mucinous colon) cell lines by western blot. Cell death was investigated by flow cytometry measurement of propidium iodide stained cells.ResultsHigh cleaved-Caspase 3 expression was noted in resected mucinous tumors. Western blot identified alterations in apoptosis proteins in mucinous CRC, most prominently downregulation of Bcl-xL protein levels (p=0.029) which was also observed at the mRNA level in patients by analysis of TCGA gene expression data (p<0.001). Treatment with 5-FU did not significantly elevate cell death in mucinous cells, while non-mucinous cells showed robust cell death responses. However, 5-FU-induced phosphorylation of MLKL in mucinous cancer cells, suggestive of a switch to necroptotic cell death signaling.ConclusionApoptotic and necroptotic mediators are differentially expressed in mucinous and non-mucinous colorectal cancers and represent targets for investigation of cell death mechanisms in the mucinous subtype.

An Analysis of Clinicopathological Outcomes and the Utility of Preoperative MRI for Patients Undergoing Resection of Mucinous and Non-Mucinous Colorectal Cancer Liver Metastases

Background and AimsMucinous colorectal cancer has traditionally been associated with high rates of recurrence and poor long-term survival. There is limited published data on outcomes for patients undergoing liver resection for metastatic mucinous colorectal cancer. The aim of this study was to compare the clinicopathological outcomes for patients with mucinous colorectal cancer liver metastases (CRCLM) undergoing liver resection to a matched group of patients with adenocarcinoma not otherwise specified (NOS) and to evaluate the accurary of preoperative magnetic resonance imaging (MRI) at detecting the presence of mucin in liver metastases.Materials and MethodsPatients with mucinous CRCLM undergoing liver resection were matched 1:3 to patients with adenocarcinoma NOS CRCLM. Clinicopathological data from the primary tumour and metastatic lesion were collected and compared between the groups. Hepatic recurrence-free, disease-free and overall survival were compared between the groups. The ability of preoperative MRI to detect mucin in CRCLM was also evaluated.ResultsA total of 25 patients with mucinous CRCLM underwent surgery over the 12-year period and were matched to 75 patients with adenocarcinoma NOS. Clinicopathological findings were similar between the groups. Resection of mucinous CRCLM was feasible and safe with similar levels of morbidity to adenocarcinoma NOS. There were no differences identified in hepatic recurrence-free (p=0.85), disease-free (p=0.25) and overall survival (p=0.98) between the groups. MRI had a sensitivity of 31.3% in detecting the presence of mucin in CRCLM.ConclusionPatients with mucinous CRCLM in this study had similar outcomes to patients with adenocarcinoma NOS. Based on our findings, histological subtype should not be taken into account when deciding on resectability of CRCLM.

Mucinous colorectal cancer: Disease characteristics, treatment outcomes and the impact of metastasectomy.

3586 Background: Mucinous colorectal cancer (CRC) differs from adenocarcinoma with regard to clinical and histological features and is reported to have inferior outcomes when compared to non-mucinous CRC. This study aims to evaluate the clinical features and outcomes of patients with mucinous CRC at our institution. Methods: Medical records of patients with CRC that were referred to medical oncology between September 1999 and September 2018 were retrospectively reviewed. Mucinous histology was defined as those containing &gt; 50% mucin identified on histology specimens. Statistical analysis was performed using Prism V9.0. Results: We identified 1,115 patients with CRC that were referred to medical oncology during this period. The tumours of 81 (7.3%) patients were classified as mucinous. Median age was 65 (28-94 years) and 45 (55.5%) were male. Forty-one patients (51%) had right sided tumours, 27 (33%) had left sided tumours and 13 (16%) had rectal tumours. Twenty-three (28.4%) had de novo metastatic disease. Eleven of 24 patients (46%) with stage II disease relapsed and 18 of 33 (55%) of those with stage III disease relapsed. Radiological surveillance identified 20/29 (69%) of relapsed disease, 5 (17%) were symptomatic and 4 (14%) had a rise in CEA. Median follow up for patients with stage II disease was 53 months and 3 year and 5-year disease free survival (DFS) was equal in both groups at 60.9%. For stage III disease 3- and 5-year DFS was 58.1% and 48.4% respectively with a median follow up of 43 months. In the metastatic setting, we observed no significant difference in overall survival (OS) between left and right sided tumours ( p = 0.550). Median OS for pts with stage IV mucinous CRC who received any treatment was 25 months. Metastasectomy was performed in 25/52 (48%) patients and was associated with a significant improvement in OS, 23 vs 51 months ( p &lt; 0.005, HR 0.4). Conclusions: Mucinous CRC has been associated with inferior responses to treatment and worse overall outcomes compared to non-mucinous histologies. Survival in advanced-stage disease in our cohort is higher than what has been reported in the literature. With an effective multi-disciplinary approach and the increasing use of metastasectomy as a treatment option, survival in the advanced disease setting may be comparable to non-mucinous CRC.

Mucinous Colorectal Cancer is Associated With Expression of the TIM-3 Immune Checkpoint Independently of Microsatellite Instability (MSI) Status.

Immune checkpoint inhibition has demonstrated success in overcoming tumor-mediated immune suppression in several types of cancer. However, its clinical use is limited to a small subset of colorectal cancer (CRC) patients, and response is highly variable between CRC subtypes. This study aimed to determine the profile of immune checkpoints and factors associated with immune checkpoint inhibitor response in mucinous CRC. Gene expression data from CRC was extracted from the TCGA PanCanAtlas data-freeze release. Gene expression data were reported as batch-corrected and normalized RNA expression derived from RNA-Seq quantification. Clinical, pathologic, and transcriptomic data were compared between mucinous and non-mucinous CRC cohorts. The 557 cases of CRC eligible for inclusion in this study comprised 486 cases of non-mucinous CRC (87.3 %) and 71 cases of mucinous CRC (12.7 %). High correlation was observed in the expression of the included immune checkpoints. Significantly higher expression of programmed cell death protein 1 ligand (PD-L1) and T cell immunoglobulin and mucin domain 3 (TIM-3) was observed in mucinous CRC than in non-mucinous CRC. In a multiple regression model, significant contributors to the prediction of TIM-3 gene expression were microsatellite instability (MSI) (unstandardized regression coefficient [B] = 1.223; p < 0.001), stage (American Joint Committee on Cancer [AJCC] 2; B = 0.423; p < 0.05), and mucinous status (B = 0.591; p < 0.01). Expression of TIM-3, an emerging immune checkpoint inhibition target, was significantly higher in mucinous CRC, and expression was predicted by mucinous status independently of MSI. These findings should prompt investigation of immune checkpoint signaling in the mucinous tumor microenvironment to clarify the potential for immune checkpoint inhibition in mucinous CRC.

Mucinous and non-mucinous colorectal cancers show differential expression of chemotherapy metabolism and resistance genes.

Previous research has identified differences in mutation frequency in genes implicated in chemotherapy resistance between mucinous and non-mucinous colorectal cancers (CRC). We hypothesized that outcomes in mucinous and non-mucinous CRC may be influenced by expression of genes responsible for chemotherapy resistance. Gene expression data from primary tumor samples were extracted from The Cancer Genome Atlas PanCancer Atlas. The distribution of clinical, pathological, and gene expression variables was compared between 74 mucinous and 521 non-mucinous CRCs. Predictors of overall survival (OS) were assessed in a multivariate analysis. Kaplan-Meier curves were constructed to compare survival according to gene expression using the log rank test. The median expression of 5-FU-related genes TYMS, TYMP, and DYPD was significantly higher in mucinous CRC compared to non-mucinous CRC (p < 0.001, p = 0.003, p < 0.001, respectively). The median expression of oxaliplatin-related genes ATP7B and SRPK1 was significantly reduced in mucinous versus non-mucinous CRC (p = 0.004, p = 0.007, respectively). At multivariate analysis, age (odds ratio (OR) = 0.96, p < 0.001), node positive disease (OR = 0.49, p = 0.005), and metastatic disease (OR = 0.32, p < 0.001) remained significant negative predictors of OS, while high SRPK1 remained a significant positive predictor of OS (OR = 1.59, p = 0.037). Subgroup analysis of rectal cancers demonstrated high SRPK1 expression was associated with significantly longer OS compared to low SRPK1 expression (p = 0.011). This study highlights that the molecular differences in mucinous CRC and non-mucinous CRC extend to chemotherapy resistance gene expression. SRPK1 gene expression was associated with OS, with a prognostic role identified in rectal cancers.

Mucinous adenocarcinoma is a pharmacogenomically distinct subtype of colorectal cancer.

Mucinous colorectal cancer is a unique histological subtype that is known to respond poorly to cytotoxic chemotherapy and radiotherapy. There are a number of genes known to be associated with resistance to 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. The aim of this study was to compare the somatic mutation frequency and copy number variation (CNV) in these genes between mucinous and non-mucinous colorectal cancer. A systematic search of PubMed was performed to identify papers investigating drug resistance in colorectal cancer. From this review, a list of 26 drug-resistance-associated genes was compiled. Using patient data from The Cancer Genome Atlas (TCGA), the somatic mutation rate and CNV was compared between patients with mucinous and non-mucinous colorectal cancer. Statistical analysis was carried out using GraphPad PRISM® version 5.00. Data were available on 531 patients (464 non-mucinous, 67 mucinous). A statistically significant difference in the somatic mutation rate between the two cohorts was identified in the TYMP (p = 0.0179), ATP7B (p = 0.0465), SRPK1 (p = 0.0135), ABCB1 (p = 0.0423), and ABCG2 (p = 0.0102) genes. A statistically significant difference in CNV was identified between the two cohorts in the GSTP1 (p = 0.0405), CCS (p = 0.0063), and TOP1 (p = 0.0048) genes. Differences in somatic mutation rate and CNV in genes associated with resistance to 5-FU, oxaliplatin, and irinotecan may partly account for the pattern of resistance observed in mucinous colorectal cancers. These genetic alterations may prove useful when deciding on a personalized approach to chemotherapy and may also represent potential therapeutic targets going forward.

Growth Factor-Independent 1 Is a Tumor Suppressor Gene in Colorectal Cancer.

Colorectal cancer is the third most common cancer and the third leading cause of cancer death in the United States. Growth factor-independent 1 (GFI1) is a zinc finger transcriptional repressor responsible for controlling secretory cell differentiation in the small intestine and colon. GFI1 plays a significant role in the development of human malignancies, including leukemia, lung cancer, and prostate cancer. However, the role of GFI1 in colorectal cancer progression is largely unknown. Our results demonstrate that RNA and protein expression of GFI1 are reduced in advanced-stage nonmucinous colorectal cancer. Subcutaneous tumor xenograft models demonstrated that the reexpression of GFI1 in 4 different human colorectal cancer cell lines inhibits tumor growth. To further investigate the role of Gfi1 in de novo colorectal tumorigenesis, we developed transgenic mice harboring a deletion of Gfi1 in the colon driven by CDX2-cre (Gfi1F/F; CDX2-cre) and crossed them with ApcMin/+ mice (ApcMin/+; Gfi1F/F; CDX2-cre). Loss of Gfi1 significantly increased the total number of colorectal adenomas compared with littermate controls with an APC mutation alone. Furthermore, we found that compound (ApcMin/+; Gfi1F/F; CDX2-cre) mice develop larger adenomas, invasive carcinoma, as well as hyperplastic lesions expressing the neuroendocrine marker chromogranin A, a feature that has not been previously described in APC-mutant tumors in mice. Collectively, these results demonstrate that GFI1 acts as a tumor suppressor gene in colorectal cancer, where deficiency of Gfi1 promotes malignancy in the colon. IMPLICATIONS: These findings reveal that GFI1 functions as a tumor suppressor gene in colorectal tumorigenesis.

Survival difference between mucinous vs. non-mucinous colorectal cancer following cytoreductive surgery and intraperitoneal chemotherapy

Background: It is believed that the oncologic behavior of mucinous colorectal adenocarcinoma (MC) is different from non-mucinous adenocarcinoma (NMC). The aim of the study is to compare long-term survivals between patients with MC and those with NMC following cytoreductive surgery (CRS) and intraperitoneal chemotherapy (IPC).Methods: This was a retrospective study of prospectively collected data of patients with peritoneal metastases of colorectal origin following CRS and IPC. Group I included patients with MC which was defined as being composed of >50% extracellular mucin. Group II included those with NMC. Subgroup analysis was performed according to the location of primary tumor.Results: A total of 213 patients were included in this study. The two groups had similar hospital mortality, high dependency unit stay. MC group had a significantly longer mean intensive care unit (ICU) stay (p = .037) and total hospital stay (p = .037). There was no significant difference in overall survival (OS) and disease-free survival (DFS) between two groups (p = .657 and p = .938, respectively). Multivariate analysis showed that the presence of mucin was not an independent negative prognostic factor for OS (p = .190).Conclusion: In summary, patients with MC had a similar long-term survival outcome with those with NMC following CRS and IPC.

Advanced Mucinous Colorectal Cancer: Epidemiology, Prognosis and Efficacy of Chemotherapeutic Treatment

Background: The clinicopathological significance of the mucinous subtype of colorectal cancer (CRC) remains controversial. As of today, none of the current guidelines differentiate treatment with respect to mucinous or nonmucinous cancer. Due to the lack of substantiated data, best treatment remains unclear and the mucinous subtype of CRC is usually treated along the lines of recommendations for adenocarcinoma of the colon. Methods: We investigated an East-Bavarian cohort of 8,758 patients with CRC. These included 613 (7.0%) patients with a mucinous subtype, who were analyzed for assessing their characteristics in clinical course and for evaluating the efficacy of common chemotherapy protocols. Results and Conclusion: Mucinous CRC was predominantly located in the right hemicolon; it was diagnosed at more advanced stages and occurred with preponderance in women. A higher rate of G3/4 grading was observed at diagnosis (all p < 0.001). An association of mucinous CRC with younger age at initial diagnosis, previously reported by other groups, could not be confirmed. Patients with mucinous stage IV colon cancer demonstrated poorer survival (p = 0.006). In contrast, no differences in survival were observed for specific stages I–III colon cancer. Stage-dependent analysis of rectal cancer stages I–IV also showed no differences in survival. However, univariable overall analysis resulted in significant poorer survival of mucinous compared to nonmucinous rectal cancer (p = 0.029). Also, combined analysis of all patients with mucinous CRC revealed poorer overall survival (OS) of these patients compared to nonmucinous CRC patients (median 48.4 vs. 60.2 months, p = 0.049) but not in multivariable analysis (p = 0.089). Chemotherapeutic treatment showed comparable efficacy regarding OS for mucinous and nonmucinous cancers in both an adjuvant and palliative setting for colon cancer patients (p values comparing mucinous and nonmucinous cancers < 0.001–0.005).

Abstract 138: FOXO proteins mediate adaptive resistance to PI3K inhibition in mucinous colorectal cancers

Abstract Purpose: Mucinous colon adenocarcinomas (MCAs) comprise a substantial fraction (10-15%) of sporadic colorectal cancers (CRCs). MCAs show a distinct range of genetic modifications compared to nonmucinous CRCs and are more prone to peritoneal dissemination and distant metastasis. These aggressive cancers show a poorer response to chemotherapy and are often difficult to treat. A higher mutation rate in effectors of the KRAS-RAF-MEK (80% vs 42%) and PI3K-AKT-mTOR (60% vs 22%) pathways in MCAs over nonmucinous CRCs suggested potential targets for therapy. Our previous results showed that, MCAs, though initially sensitive to PI3K inhibition (PI3Ki), later develop adaptive resistance. The purpose of this study was to test whether FOXO transcription factors mediate this adaptive resistance to PI3Ki single agent treatment. Methods: PI3K was inactivated in MCA cell lines, LS174T (KRAS G12D, PI3K H1047R) and RW7213 (KRAS G12C, PI3K WT), by treatment with GDC0941 (Pictilisib), a small molecule inhibitor of class I PI3K. Alterations in phosphorylation of effectors in the PI3K-AKT or MEK-ERK pathways were determined using western blots. FOXO nuclear localization was assessed by immunocytochemistry, confocal microscopy and nuclear fractionation studies. Expression of FOXO proteins (FOXO1, FOXO3 and FOXO4) was reduced by short inhibitory RNAs (siRNAs). FOXO1 was also inhibited in MCA cells using AS1842856, a small molecule inhibitor. Results: Immunohistochemical analysis and nuclear fractionation studies showed significant nuclear enrichment of FOXO1 and FOXO3 in MCA cell lines in response to PI3K inhibitor treatment. Upon siRNA-mediated knockdown of FOXOs, rebound ERK phosphorylation seen in response to PI3Ki treatment was substantially reduced. Concomitant with this decrease was a reduction in expression of receptor tyrosine kinases (RTKs) IR, IGFR, HER2 and HER3. Combinatorial treatment of PI3K and FOXO1 inhibitors resulted in a more pronounced loss of cell viability compared to single agent treatment. Conclusions: Our results suggest that resistance to PI3K single agent inhibition seen in MCA cells is mediated, at least in part, by nuclear translocation of FOXO proteins, particularly FOXO1. Our results support a model where PI3K inhibition results in increased FOXO nuclear localization which in turn, leads to transcriptional upregulation and activation of RTKs which then lead to rebound activation of the MEK-ERK pathway and increased survival and resistance. A role for FOXO proteins in mediating PI3Ki resistance in KRAS-mutant MCA cells has not been described before. Our results suggest that PI3Ki single agent therapy is unlikely to be successful in KRAS-mutant MCAs due to FOXO-mediated resistance thus providing a rationale for combination treatment strategies. Citation Format: Murali R. Kuracha, Peter Thomas, Brian W. Loggie, Venkatesh Govindarajan. FOXO proteins mediate adaptive resistance to PI3K inhibition in mucinous colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 138. doi:10.1158/1538-7445.AM2017-138

Bilateral blockade of MEK- and PI3K-mediated pathways downstream of mutant KRAS as a treatment approach for peritoneal mucinous malignancies.

Mucinous colorectal adenocarcinomas (MCAs) are clinically and morphologically distinct from nonmucinous colorectal cancers (CRCs), show a distinct spectrum of genetic alterations (higher KRAS mutations, lower p53, high MUC2), exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement) and are associated with poorer response to chemotherapy with limited treatment options. Here, we report the effectiveness of combinatorial targeting of two KRAS-mediated parallel pathways in reducing MUC2 production and mucinous tumor growth in vitro and in vivo. By knockdown of mutant KRAS we show that, mutant KRAS (a) is necessary for MUC2 production in vitro and (b) synergistically engages PI3K/AKT and MEK/ERK pathways to maintain MUC2 expression in MCA cells. These results define a novel and a previously undescribed role for oncogenic KRAS in mucinous cancers. MCA cells were sensitive to MEK inhibition suggesting cellular dependence (‘addiction’) of KRAS-mutant MCA cells on hyperactivation of the MEK-driven pathway. Interestingly, MCA cells, though initially sensitive, were later resistant to PI3K single agent inhibition. Our studies suggest that this resistance involves dynamic rewiring of signaling circuits mediated through relief of RTK inhibition and MEK-ERK rebound activation. This resistance however, could be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEK- and PI3K-targeted combination therapy for not only KRAS mutant MCA but also for other related mucinous neoplasms that overproduce MUC2 and have a high rate of KRAS mutations such as pseudomyxoma peritonei.

Clinical and Pathological Characteristics of Mucinous Colorectal Adenocarcinoma: A Comparative Study

Background Mucinous adenocarcinoma accounts for approximately 5% - 15% of all colorectal cancers. Objectives The aim of this study was to investigate the clinicopathological characteristics of patients with mucinous colorectal adenocarcinoma. Patients and Methods This retrospective study was carried out by reviewing the medical records of 70 mucinous colorectal cancer (MCC) patients who were diagnosed and treated at a tertiary academic hospital between 2005 and 2010. For the comparative analysis, 491 patients with non-mucinous colorectal cancer (NMCC) were included. Results Of 561 patients with colorectal adenocarcinoma, 70 patients (12.5%) had the mucinous type. There were 42 (60%) men and 28 (40%) women, with a median age of 55 years old (range 24 - 81 years) included in the study. We did not find any differences regarding the patients’ mean age (P = 0.408) and male/female ratio (P = 0.700) between the MCC and NMCC; however, there was a predilection for the right colon and sigmoid colon in the MCC, when compared to the NMCC (P = 0.012). In addition, the MCC tended to have a larger tumor size (P = 0.004), higher histological grade (P < 0.001), higher node stage (P < 0.001), higher number of dissected nodes (P = 0.013), higher number of positive nodes (P < 0.001), and a higher rate of perineural invasion (P = 0.013) compared to the NMCC. Conclusions This study indicates that most clinicopathological characteristics of MCC are different from those of NMCC. In addition, there was an association between the mucinous subtype and adverse pathological features in the patients with colorectal cancer.

Abstract 2661: Bilateral blockade of MEK- and PI3K-driven pathways is effective in the treatment of KAS mutant mucinous colorectal cancer cells

Abstract Purpose. A significant proportion of sporadic colorectal cancers (CRCs) (10-15%) are of the mucinous subtype (&amp;gt;50% of the tumor). Mucinous colorectal adenocarcinomas (MCAs) are clinically, morphologically and molecularly distinct from nonmucinous CRCs as they show a different spectrum of genetic alterations (higher KRAS mutations, MSI-H, lower p53, high MUC2) and exhibit more aggressive behavior (more prone to peritoneal dissemination and lymph node involvement). MCAs are associated with poorer response to chemotherapy and have limited treatment options. The purpose of this study was to test the effectiveness of combinatorial targeting of two KRAS-associated parallel pathways in an attempt to counter adaptive resistance associated with single pathway inhibition. Methods. Two KRAS mutant MCA cell lines LS174T and RW7213 were treated with two small molecule inhibitors, GDC0973 and GDC0941, to block mitogen-activated extracellular regulated kinase kinase (MEK) and phosphoinositide 3-kinase (PI3K) respectively. Adaptive resistance of these two cell lines to the PI3K inhibitor (PI3Ki) was examined by protein phospho-receptor tyrosine kinase (RTK) arrays, western blots and immunocytochemistry. Results. Bioinformatic analysis of the colon cancer datasets in the cancer genome atlas (TCGA) showed increased mutation rates in mucinous over nonmucinous CRCs for effectors of RAS-RAF-MEK-ERK (80% versus 41.9%) and PI3K-AKT-mTOR (60% versus 21.8%) pathways. Both MCA cell lines were sensitive to MEK inhibitor (MEKi) treatment. In contrast, these cell lines, though initially sensitive to PI3Ki, later became resistant. Concomitant with the development of resistance were a) increase in phosphorylated RTKs including IR, IGF-1R, EGFR, ErbB2 b) increase in phosphorylated ERK and AKT, c) nuclear translocation of the transcription factor FOXO3A and d) a decrease in proapoptotic protein BIM. Combinatorial treatment of PI3Ki and MEKi synergistically reduced viability of MCA tumor cells with attendant decrease in phosphorylated ERK and AKT and increase in BIM. Conclusions. Our data suggest cellular dependence (‘addiction’) of KRAS-mutant mucinous CRC cells on hyperactivation of MEK and PI3K pathways. Interestingly, PI3K single agent inhibition initially triggers pathway downregulation and reduced tumor cell viability but later leads to development of adaptive resistance. Our studies suggest this resistance involves dynamic rewiring of signaling circuits mediated through FOXO3A nuclear localization, relief of RTK inhibition, MEK-ERK activation and BIM reduction. Adaptive resistance however, can be overcome by co-targeting of PI3K and MEK. Our studies thus provide a rational basis for MEKi and PI3Ki combination therapy for KRAS mutant CRCs. Citation Format: Murali R. Kuracha, Peter Thomas, Brian W. Loggie, Venkatesh Govindarajan. Bilateral blockade of MEK- and PI3K-driven pathways is effective in the treatment of KAS mutant mucinous colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2015-2661

Contemporary Management of Non-Appendicular Mucinous and Signet Cell Colorectal Cancer in Community Setting

Introduction: Colon adenocarcinoma, the most common gastrointestinal cancer, has three distinct histological types namely, non-mucinous, mucinous and signet cell type. To date, the clinical implication of mucinous histology is not completely elucidated. Most studies discussing the impact of mucinous pathology in colon cancer are retrospectives analyses from large tertiary centers and report non-concordant results. The aim of our study is to analyze the results of management of non appendicular mucinous and signet colorectal cancer in a community setting. Methods: Under IRB approval, a prospectively maintained database of patients treated for colorectal cancer (1996-2013) at a university-affiliated community cancer center was accessed. Data was collected on excel documents and reported demographic data (age at initial diagnosis, sex), pathological data (site, stage, histological type of cancer) and management data (treatment modalities and survival). Two groups were created: Non mucinous, and mucinous - signet cell colorectal cancer. Differences between groups were analyzed using chi-square, fisher test and Kaplan Meier curves for survival analysis. Results: 1595 patients were included. Of these, 91.7% were non-MSCA (non-mucinous signet cell adenocarcinoma), remaining 7.2% were MCA and 1.1% was SRCA (signet ring cell carcinoma). Mean age was 67 in the three groups and there was no difference in age distribution. Right colon cancer represented 38% of non-mucinous colorectal cancer (n=556), and 58% (n=77) of the mucinous signet cell type. 5 year survival was 92.8%, 89.9%, 72.3% and 19.8% for stage I-IV; respectively for non-mucinous cancer. 5 year survival for mucinous-signet cell was 67.1%, 79%, 55.4% and 0%; respectively for stage I-IV; p<0.05. Conclusion: Mucinous and signet cell histology is a clinicopathological distinct type of colorectal cancer that carries a negative impact on patient survival. Despite multimodality management and advance in chemotherapy, stage IV mucinous-signet cell

REG4 independently predicts better prognosis in non-mucinous colorectal cancer.

IntroductionColorectal cancer (CRC) is one of the world’s three most common cancers and its incidence is rising. To identify patients who benefit from adjuvant therapy requires novel biomarkers. The regenerating islet-derived gene (REG) 4 belongs to a group of small secretory proteins involved in cell proliferation and regeneration. Its up-regulated expression occurs in inflammatory bowel diseases also in gastrointestinal cancers. Reports on the association of REG4 expression with CRC prognosis have been mixed. Our aim was to investigate tumor REG4 expression in CRC patients and its coexpression with other intestinal markers.MethodsTumor expression of REG4 was evaluated by immunohistochemistry in 840 consecutive surgically treated CRC patients at Helsinki University Central Hospital. Expression of MUC1, MUC2, MUC5AC, synapthophysin, and chromogranin was evaluated in a subgroup of 220 consecutively operated CRC patients. REG4 expression with clinicopathological parameters, other intestinal markers, and the impact of REG4 expression on survival were assessed.ResultsREG4 expression associated with favorable clinicopathological parameters and with higher overall survival from non-mucinous CRC (p = 0.019). For such patients under 65, its expression was an independent marker of lower risk of death within 5 years that cancer; univariable hazard ratio (HR) = 0.57; 95% confidence interval (CI) (0.34–0.94); multivariable HR = 0.55; 95% CI (0.33–0.92). In non-mucinous CRC, REG4 associated with positive MUC2, MUC4, and MUC5AC expression.ConclusionWe show, to our knowledge for the first time, that REG4 IHC expression to be an independent marker of favorable prognosis in non-mucinous CRC. Our results contradict those from studies based on quantification of REG4 mRNA levels, a discrepancy warranting further studies.

Aberrant SOX2 expression in colorectal cancers does not correlate with mucinous differentiation and gastric mucin MUC5AC expression.

Colorectal cancer (CRC) can be divided into non-mucinous and mucinous subtypes, of which the latter portends to have a worse clinical prognosis. A previous study suggested a putative link between SOX2 expression observed selectively in mucinous CRC and the induction of the gastric mucin MUC5AC. In this study, we re-evaluated the expression behavior of SOX2, MUC5AC, and CDX2 in both types of CRC. We performed immunohistochemical analysis on 90 cases of non-mucinous CRCs, 57 cases of mucinous CRCs, and 15 case-matched normal intestinal mucosa. In contrast to the previously suggested link between SOX2 and mucinous CRC, we observe aberrant expression of SOX2 at equal levels in both subtypes. Fluorescence in situ hybridization (FISH) analysis shows that expression is not attributed to genomic amplification. While SOX2 and CDX2 are normally expressed in a reciprocal manner, SOX2-positive tumor cells co-express CDX2. Furthermore, we show that MUC5AC is expressed independently of SOX2. In conclusion, we show that aberrant SOX2 expression is specifically linked neither to mucinous CRCs nor to the induction of MUC5AC, in contrast to previous suggestions.