Shanequa Manuel

Association of SMAD4 mutation with patient demographics, tumor characteristics, and clinical outcomes in colorectal cancer.

SMAD4 is an essential mediator in the transforming growth factor-β pathway. Sporadic mutations of SMAD4 are present in 2.1–20.0% of colorectal cancers (CRCs) but data are limited. In this study, we aimed to evaluate clinicopathologic characteristics, prognosis, and clinical outcome associated with this mutation in CRC cases. Data for patients with metastatic or unresectable CRC who underwent genotyping for SMAD4 mutation and received treatment at The University of Texas MD Anderson Cancer Center from 2000 to 2014 were reviewed. Their tumors were sequenced using a hotspot panel predicted to cover 80% of the reported SMAD4 mutations, and further targeted resequencing that included full-length SMAD4 was performed on mutated tumors using a HiSeq sequencing system. Using The Cancer Genome Atlas data on CRC, the characteristics of SMAD4 and transforming growth factor-β pathway mutations were evaluated according to different consensus molecular subtypes of CRC. Among 734 patients with CRC, 90 (12%) had SMAD4 mutations according to hotspot testing. SMAD4 mutation was associated with colon cancer more so than with rectal cancer (odds ratio 2.85; p<0.001), female sex (odds ratio 1.71; p = 0.02), and shorter overall survival than in wild-type SMAD4 cases (median, 29 months versus 56 months; hazard ratio 2.08; p<0.001 [log-rank test]). SMAD4 mutation was not associated with age, stage at presentation, colonic location, distant metastasis, or tumor grade. A subset of patients with metastatic CRC (n = 44) wild-type for KRAS, NRAS, and BRAF who received anti-epidermal growth factor receptor therapy with mutated SMAD4 (n = 13) had shorter progression-free survival duration than did patients wild-type for SMAD4 (n = 31) (median, 111 days versus 180 days; p = 0.003 [log-rank test]). Full-length sequencing confirmed that missense mutations at R361 and P356 in the MH2 domain were the most common SMAD4 alterations. In The Cancer Genome Atlas data, SMAD4 mutation frequently occurred with KRAS, NRAS, and BRAF mutations and was more common in patients with the consensus molecular subtype 3 of CRC than in those with the other 3 subtypes. This is one of the largest retrospective studies to date characterizing SMAD4 mutations in CRC patients and demonstrates the prognostic role and lack of response of CRC to anti-epidermal growth factor receptor therapy. Further studies are required to validate these findings and the role of SMAD4 mutation in CRC.

Clinical utility of circulating cell-free DNA in advanced colorectal cancer

Background Circulating cell-free DNA (cfDNA) isolated from the plasma of cancer patients (pts) has been shown to reflect the genomic mutation profile of the tumor. However, physician and patient assessment of clinical utility of these assays in patients with metastatic colorectal cancer (mCRC) has not been previously described. Methods Patients were prospectively consented to a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC]), with collection of blood for cfDNA extraction and sequencing of a 54-gene panel in a CLIA-certified lab. Formalin-fixed, paraffin-embedded (FFPE) tissue from prior resections or biopsies underwent 50-gene sequencing. Results from both assays were returned to the treating physicians for patient care and clinical trial selection. Follow-up surveys of treating physicians and chart reviews assessed clinical utility. Results 128 mCRC pts were enrolled between 6/2014 and 1/2015. Results were returned in median of 13 and 26 days for cfDNA and FFPE sequencing, respectively. With cfDNA sequencing, 78% (100/128) of samples had a detectable somatic genomic alteration. 50% of cfDNA cases had potentially actionable alterations, and 60% of these could be genomically matched to at least one clinical trial in our institution. 50% (15/30) of these pts enrolled onto an identified matched trial. Physicians reported that the cfDNA testing improved the quality of care they could provide in 73% of the cases, and that 89% of pts reported greater satisfaction with the efforts to personalize experimental therapeutic agents. Conclusions cfDNA sequencing can provide timely information on potentially actionable mutations and amplifications, thereby facilitating clinical trial enrollment and improving the perceived quality of care.

FBXW7 missense mutation: a novel negative prognostic factor in metastatic colorectal adenocarcinoma

Background FBXW7 functions as a ubiquitin ligase tagging multiple dominant oncogenic proteins and commonly mutates in colorectal cancer. Data suggest missense mutations lead to greater loss of FBXW7 function than other gene aberrations do. However, the clinicopathologic factors and outcomes associated with FBXW7 missense mutations in metastatic colorectal cancer (mCRC) have not been described. Methods Data were obtained from mCRC patients whose tumors were evaluated by next-generation sequencing for hotspot mutations at The University of Texas MD Anderson Cancer Center. Alterations in FBXW7 were identified, and their associations with clinicopathologic features and overall survival (OS) were evaluated. Results Of 855 mCRC patients, 571 had data on FBXW7 status; 43 (7.5%) had FBXW7 mutations, including 37 with missense mutations. R465C mutations in exon 9 were the most common missense mutations (18.6%). PIK3CA mutations were associated with FBXW7 missense mutations (p=0.012). On univariate analysis, patients with FBXW7 missense mutations had significantly worse OS (median 28.7 mo) than those with wild-type FBXW7 (median 46.6 mo; p=0.003). On multivariate analysis including other known prognostic factors such as BRAF mutations, FBXW7 missense mutations were the strongest negative prognostic factor for OS (hazard ratio 2.0; p=0.003). Conclusions In the largest clinical dataset of mCRC to date, FBXW7 missense mutations showed a strong negative prognostic association.

Modeling of Patient Derived Xenografts in Colorectal Cancer

Developing realistic preclinical models using clinical samples that mirror complex tumor biology and behavior are vital to advancing cancer research. While cell line cultures have been helpful in generating preclinical data, the genetic divergence between these and corresponding primary tumors has limited clinical translation. Conversely, patient-derived xenografts (PDX) in colorectal cancer are highly representative of the genetic and phenotypic heterogeneity in the original tumor. Coupled with high-throughput analyses and bioinformatics, these PDXs represent robust preclinical tools for biomarkers, therapeutic target, and drug discovery. Successful PDX engraftment is hypothesized to be related to a series of anecdotal variables namely, tissue source, cancer stage, tumor grade, acquisition strategy, time to implantation, exposure to prior systemic therapy, and genomic heterogeneity of tumors. Although these factors at large can influence practices and patterns related to xenotransplantation, their relative significance in determining the success of establishing PDXs is uncertain. Accordingly, we systematically examined the predictive ability of these factors in establishing PDXs using 90 colorectal cancer patient specimens that were subcutaneously implanted into immunodeficient mice. Fifty (56%) PDXs were successfully established. Multivariate analyses showed tissue acquisition strategy [surgery 72.0% (95% confidence interval (CI): 58.2–82.6) vs. biopsy 35% (95% CI: 22.1%–50.6%)] to be the key determinant for successful PDX engraftment. These findings contrast with current empiricism in generating PDXs and can serve to simplify or liberalize PDX modeling protocols. Better understanding the relative impact of these factors on efficiency of PDX formation will allow for pervasive integration of these models in care of colorectal cancer patients. Mol Cancer Ther; 16(7); 1435–42. ©2017 AACR.

Abstract A36: Circulating cell-free DNA as a marker for response and resistance to BRAF and EGFR inhibition in BRAF-mutated metastatic colorectal cancer

This abstract has been withheld from publication due to its inclusion in the AACR-NCI-EORTC Molecular Targets Conference 2015 Official Press Program. It will be posted online at the time of its presentation in a press conference or in a session: 12:15 PM ET Friday, November 6. Citation Format: Van Morris, Filip Janku, Helen Huang, Siqing Fu, Michael Overman, Sarina Piha-Paul, Vivek Subbiah, Bryan Kee, Apostolia Tsimbierdou, David Fogelman, Imad Shurieqi, Shanequa Manuel, Antonio Scamardo, Richard Lanman, Nicolas Sommer, David Hong, Scott Kopetz. Circulating cell-free DNA as a marker for response and resistance to BRAF and EGFR inhibition in BRAF-mutated metastatic colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A36.

Abstract 4763: Correlation of CpG island methylation with clinical and pathologic characteristics in metastatic colorectal cancer patients

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Colorectal cancer morbidity and mortality rates vary by race and ethnicity. The combined contribution of genetics and environment to response to chemotherapy, progression-free and overall survival in colorectal cancer patients is unclear, but CpG island hypermethylation (CIMP), a discrete molecular subtype of colorectal cancer, is associated with inflammation and environmental exposures. Hence our main objective is to assess correlation between various modifiable risk factors and CIMP status among metastatic colorectal cancer patients. Methods: We characterized CIMP methylation (MINT1, MINT2, MINT31, p14, p16, and MLH1) in 229 metastatic colorectal cancer patients using PCR amplification of bisulfite treated DNA followed by pyrosequencing. The number of methylated probes were averaged to obtain the% methylation with CIMP-High (CIMP-H) being defined as ≥40% of probes methylated. Associations of demographic and clinical characteristics, including BMI, diabetes, obesity, physical activity, smoking and drinking status, as well as presence or absence of other molecular alterations (BRAF, KRAS, NRAS, PIK3CA and PTEN loss) with overall survival were assessed in multivariable-adjusted Cox proportional hazards models. Results: When treating methylation as a continuous variable, patients with BRAF mutation had higher methylation as compared to participants with BRAF wildtype (40.6% vs 20.5%, p = 0.001). White, non-Hispanic (WNH) patients had greater degree of tumor methylation (24.8%) as compared to other racial categories (p = 0.02). When classified as a categorical variable, moderate to vigorous physical activity was associated with higher rate of having any methylated probes, as compared to sedentary patients (p = 0.02). CIMP status was not associated with OS in these patients in multivariable-adjusted Cox models. However when stratified by CIMP status. When stratified by CIMP status; among patients with 0-40% methylation, KRAS mutation was associated with poor OS(HR = 3.19, p = 0.006) and diabetes was protective (HR = .14, p = 0.025). Among patients with high methylation(41-100%) methylation, obesity was associated with poor OS (HR = 5.20, p = 0.038) and former smoking was associated with poor OS(HR = 2.92, p = 0.05). When stratified by KRAS mutation status, among patients with KRAS wildtype, obesity was associated with poor OS(HR = 2.86, p = 0.019) and among patients with KRAS mutation, diabetes was protective (HR = 0.18, p = 0.033) Conclusion and Impact: CpG island methylator phenotype was associated with unique clinicopathologic characteristics. Methylation, as assessed by the 6-gene CIMP panel, was not associated with worse outcomes after correcting for the KRAS mutations, a well-established genetic marker of poor prognosis. Hypermethylation did appear to modulate outcomes in obese patients with hypermethylated tumors. Citation Format: Shailesh M. Advani, Michael Sangmin Lee, Michael James Overman, David Fogelman, Bryan K. Kee, Shanequa D. Manuel, Jennifer Davis, Van Karlyle Morris, Callisia Nathelee Clarke, Carrie R. Daniel, David G. Menter, Stanley R. Hamilton, Dipen Maheshbhai Maru, Scott Kopetz. Correlation of CpG island methylation with clinical and pathologic characteristics in metastatic colorectal cancer patients. [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 4763. doi:10.1158/1538-7445.AM2015-4763

Abstract 5236: Prospective clinical application of circulating cell-free DNA sequencing in metastatic colorectal cancer

Background:Recent finding have suggested that discordance between primary and metastatic colorectal tumors is influenced by time, intervening therapy, and sites of metastatic disease. CfDNA allows a non-invasive assessment of the present molecular features of the tumor, but the degree of information provided and the clinical utility of the testing remains to be defined.Methods: Patients were prospectively consented for collection of cfDNA from plasma samples for sequencing on a 54-gene platform optimized for very low allele frequencies (Guardant360), a CLIA certified lab, and concurrent sequencing of clinically available and FFPE tissue following institutional standard of care (Ion-Torrent panel). Results were returned to patients and physicians within 12-19days, and used for evaluating clinical trial options. Surveys of treating physicians were provided to assess clinical utility of the additional ctDNA testing.Results: 105 patients were enrolled after a median of two prior lines of therapy for metastatic disease. Plasma testing was successful in all patients, and tissue sequencing was only able to be performed in 90% of patients, due to limited tissue availability. In the plasma testing, 85% of samples had a detectable genomic alterations (including 21% with detectable amplifications in EGFR, MET, or ERBB2), with an average of 4.6 mutations detected per patient. In the cases with tissue sequencing results, 38% of the cases had mutations detected in the plasma that were not present in the historic tissue. Only 8% of these were the previously described ‘acquired’ mutations in KRAS, NRAS, and EGFR with anti-EGFR monoclonal inhibition. Mutation in TP53 and SMAD4 genes were the most common newly detected in plasma. In many cases, it appeared that these represented minor clones not represented in the sequenced FFPE tumor. In support of this, the median allele quantification for these newly detected mutations was only 4% (IQR = 1.7% to 31%) of the dominant clone, in contrast to concordant mutations that were present at 87% (IQR = 54% to 100%) of the allele burden of the dominant clone (P Citation Format: Maria Pia Morelli, Michael Overman, Eduardo Vilar, Van Morris, David Fogelman, Imad Shureiqi, Chris Garret, Raghav Kanwal, Cathy Eng, Brian Kee, Shanequa Manuel, Robert Wolff, Dragon Sebisanovic, LaiMun Sew, Aubey Zapanta, Ben Shiller, Gangwu Mei, Helmy Eltoukhy, AmirAli Talasaz, Scott Kopetz. Prospective clinical application of circulating cell-free DNA sequencing in metastatic colorectal cancer. [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 5236. doi:10.1158/1538-7445.AM2015-5236