Locally Advanced Rectal Adenocarcinoma Research Articles

Improved Pathologic response to chemoradiation in MGMT methylated locally advanced rectal cancer.

With the growing interest in total neoadjuvant treatment for locally advanced rectal adenocarcinoma (LARC) there is an urgent unmet need to identify predictive markers of response to long-course neoadjuvant concurrent chemoradiotherapy (LCRT). O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) gene methylation has been associated in some malignancies with response to concurrent chemoradiotherapy. We attempted to find if pathologic response to LCRT was associated with MGMT promoter hypermethylation (MGMTh). Patients were identified with LARC, available pre-treatment biopsy specimens, and at least 1year of follow-up who received LCRT followed by surgical resection within 6months. Biopsies were tested for MGMTh using a Qiagen pyrosequencing kit (Catalog number 970061). The primary outcome of LCRT responsiveness was based on tumor regression grade (TRG), with grades of 0-1 considered to have excellent response and grades of 2-3 considered to be non-responders. Secondary outcomes included overall survival (OS) and recurrence free survival (RFS). Of 96 patients who met inclusion criteria, 76 had samples which produced reliable assay results. MGMTh corresponded with higher grade and age of the biopsy specimen. The percentage of responders to LCRT was higher amongst the MGMTh patients than the MGMTn patients (60.0% vs 27.5%, p value=0.0061). MGMTh was not significantly associated with improved OS (2-year OS of 96.0% vs 98.0%, p=0.8102) but there was a trend for improved RFS (2-year RFS of 87.6% vs 74.2%, p=0.0903). Significantly greater tumor regression following LCRT was seen in MGMTh LARC. Methylation status may help identify good candidates for close observation without surgery following LCRT.

The role of MRI after neochemoradiotherapy in predicting pathological tumor regression grade and clinical outcome in patients with locally advanced rectal adenocarcinoma.

To evaluate the predictive value of tumor regression grade assessed by MRI (mr-TRG) after neoadjuvant chemoradiotherapy (neo-CRT) for postoperative pathological TRG (pTRG) and prognosis in patients with locally advanced rectal adenocarcinoma (LARC). This was a retrospective study from a single center experience. The patients who were diagnosed with LARC and received neo-CRT in our department between January 2016 and July 2021 were enrolled. The agreement between mrTRG and pTRG was assessed with the weighted κ test. Overall survival (OS), progress-free survival (PFS), local recurrence-free survival (LRFS), and distant metastasis-free survival (DMFS) were calculated by Kaplan-Meier analysis and log-rank test. From January 2016 to July 2021, 121 LARC patients received neo-CRT in our department. Among them, 54 patients had complete clinical data, including MRI of pre- and post-neo-CRT, postoperative tumor samples, and follow-up. The median follow-up time was 34.6 months (range: 4.4-70.6 months). The estimated 3-year OS, PFS, LRFS and DMFS were 78.5%, 70.7%, 89.0%, and 75.2%, respectively. The median time from the completion of neo-CRT to preoperative MRI and surgery was 7.1 weeks and 9.7 weeks, respectively. Out of 54 patients, 5 patients achieved mrTRG1 (9.3%), 37 achieved mrTRG2 (68.5%), 8 achieved mrTRG3 (14.8%), 4 achieved mrTRG4 (7.4%), and no patient achieved mrTRG5 after neo-CRT. Regarding pTRG, 12 patients achieved pTRG0 (22.2%), 10 achieved pTRG1 (18.5%), 26 achieved pTRG2 (48.1%), and 6 achieved pTRG3 (11.1%). The agreement between three-tier mrTRG (mrTRG1 vs. mrTRG2-3 vs. mrTRG4-5) and pTRG (pTRG0 vs. pTRG1-2 vs. pTRG3) was fair (weighted kappa=0.287). In a dichotomous classification, the agreement between mrTRG(mrTRG1 vs. mrTRG2-5)and pTRG(pTRG0 vs. pTRG1-3) also resulted in fair agreement (weighted kappa=0.391). The sensitivity, specificity, positive, and negative predictive values of favorable mrTRG (mrTRG 1-2) for pathological complete response (PCR) were 75.0%, 21.4%, 21.4%, and 75.0%, respectively. In univariate analysis, favorable mrTRG (mrTRG1-2) and downstaging N were significantly associated with better OS, while favorable mrTRG (mrTRG1-2), downstaging T, and downstaging N were significantly associated with superior PFS (p<0.05). In multivariate analysis, downstaging N was an independent prognostic factor for OS. Meanwhile, downstaging T and downstaging N remained independent prognostic factors for PFS. Although the consistency between mrTRG and pTRG is only fair, favorable mrTRG after neo-CRT may be used as a potential prognostic factor for LARC patients.

Rectal tumor fragmentation as a response pattern following chemoradiation

Tumor response to neoadjuvant therapy is heterogenous and prognostically important for locally advanced rectal adenocarcinoma (LARC) patients. Commonly applied response classification approaches including tumor regression grading (TRG) and TN downstaging can be discordant. The aim of this study is to compare the prognostic value of discordant tumor response measurement categorized according to the AJCC/CAP TRG schema and ypTN stage. This is a single-center retrospective review of 90 consecutive patients with stage II-III rectal cancer receiving neoadjuvant chemoradiation (nCRT), total mesorectal excision (TME) and adjuvant chemotherapy (ACT) between 2007 and 2018. Two pathologists re-examined each case to assign a consensus AJCC TRG. A Cox proportional hazards ratio model assessed the effect of patient, tumor, and treatment factors on disease-free survival (DFS). Median follow-up after surgery was 46 months (95% CI: 41-50 months). Median age at diagnosis was 55 years (range: 27-80). Most patients were male (58%) and Caucasian (92%) with clinical stage III disease (68%). Seventy-three patients (81%) underwent low anterior resection (LAR), 17 (19%) underwent abdominoperineal resection (APR). The median interval from completion of nCRT to surgery was 62 days (IQR: 56-70 days). The 4-year OS, DFS, and LC was 92.4%, 74.4%, and 90.2%, respectively. In the multivariate analysis, ypTN downstaging was not prognostically significant; however, AJCC TRG score 3 (minimal tumor response to treatment) was strongly predictive for inferior DFS (3-year DFS 79% vs. 25%, P<0.001). Patients with TRG 3 had a significantly higher risk of both local (75% vs. 5%) and distant failure (75% vs. 19%). Minimal tumor response to neoadjuvant therapy, AJCC TRG 3, irrespective of ypTN downstaging, is a pattern of residual disease that is at highest risk for recurrence. Response categorization discrepancies may be partly explained by alternative patterns of residual disease, including tumor fragmentation, and may be best reflected by TRG. The optimal tumor response categorization method requires further study to best stratify patient risk and management.

Abstract LB032: High tumor mutational burden subtends better response in locally advanced rectal adenocarcinoma undergoing neoadjuvant chemoradiotherapy and is associated with mutations in DNA damage repair genes

Abstract Tumor mutational burden (TMB), defined as the number of somatic mutations per Megabase (Mb) in a neoplastic specimen measured by next-generation sequencing (NGS), is shown to increase its rate in association with higher genomic instability. Neoadjuvant chemoradiotherapy (NACRT) in locally advanced rectal adenocarcinoma (LARC) may be more active in genomically unstable tumors. However, response to NACRT is highly variable and pathological complete response is observed in only 15-25% of cases. Currently, the association of TMB with response to NACRT in LARC is unknown. The aim of this study was to investigate the role of TMB as a potential predictive biomarker of response in NACRT-treated LARC. We collected a set of pre-treatment biopsies from LARC cases, clinical stage II or III, undergoing NACRT with capecitabine combined with radiotherapy (N = 22). After DNA extraction and quality and concentration evaluation, we performed mutational profiling and assessed TMB through multigene sequencing of 409 cancer-related genes by NGS (OncomineTM Tumor Mutation Load Assay, Thermo Fisher Scientific). The most common mutations were, as expected, in KRAS, TP53 and PIK3CA. We compared our results with those of The Cancer Genome Atlas (TCGA) database and proportion was similar, with no statistically significant differences. We found three cases with TMB over 10 mutations/Mb, a threshold above which neoantigen formation occurs with higher likelihood and response to NACRT may be more efficient due to the stimulation of immune system through radiotherapy. Two cases harbored deleterious mutations in genes involved in DNA damage repair (PMS2 and NBN). Furthermore, cases with mutations in the transcription factor FBXW7 (N = 4/22) and in the receptor tyrosine kinase ERBB3 (N = 3/22) had significantly higher TMB than non-mutated ones (p-value = 0.0090 and 0.0107 respectively, false discovery rate &amp;lt; 0.1 for both genes). We compared TMB in major/complete responders vs. minor/non-responders as evaluated by post-surgical pathological examination (responders if Dworak tumor regression grading score, TRG, was equal to 3-4 and non-responders if TRG was equal to 0-1) and investigated the possible association of higher TMB with better response to NACRT. In our cohort we found a trend for higher TMB in responder vs. non-responder cases (5.08 mutations/Mb, IQR 3.40-9.29 vs. 4.25 mutations/Mb, IQR 2.54-5.5) (p-value = 0.05, one-sided). In conclusion, our study shows that higher TMB is correlated with a better response to NACRT in LARC and can be found in cases presenting with defects in DNA damage repair as well as enriched for specific gene mutations, notably in the receptor tyrosine kinase ERBB3. Citation Format: Martina Dameri, Gabriella Cirmena, Anna Garuti, Lorenzo Ferrando, Maurizio Gallo, Shirin Djahandideh Sheijani, Ester Cecchella, Edoardo Isnaldi, Roberto Murialdo, Valentina Barbero, Lucia Tixi, Federica Grillo, Luca Mastracci, Ciro Marrone, Andrea Massobrio, Stefano Scabini, Alberto Ballestrero, Gabriele Zoppoli. High tumor mutational burden subtends better response in locally advanced rectal adenocarcinoma undergoing neoadjuvant chemoradiotherapy and is associated with mutations in DNA damage repair genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB032.

SO-30 Efficacy and safety of neoadjuvant short-course radiation followed by mFOLFOX-6 plus avelumab for locally-advanced rectal adenocarcinoma: Averectal study

Total neoadjuvant therapy (TNT) for locally advanced rectal cancer is becoming an accepted approach over the last few years with increasing pathologic complete response (pCR) and compliance with chemotherapy in comparison with the current standard of care i.e., fluoropyrimidine-based chemoradiation followed by surgery and adjuvant chemotherapy. Sequential use of anti–PD-1/PD-L1 antibody after radiation therapy has demonstrated a synergistic effect in in vivo models leading to a decrease in size of irradiated and non-irradiated secondary tumors outside the radiation field (abscopal effect). Open-label, single-arm, multicenter, 2-stage phase II study. This is an investigator-initiated, open-label, single-arm, multicenter, phase II study, adopting Simon’s two-stage aiming at evaluating the pCR rate and safety of using short-course radiation therapy (25 Grays in 5 fractions), followed by 6 cycles of mFOLFOX-6 (oxaliplatin, 5-FU and leucovorin) plus avelumab, then total mesorectal excision (TME) 3-4 weeks after last dose in patients with locally-advanced, potentially resectable rectal adenocarcinoma. From July 2018 to October 2020, 44 patients were accrued and completed the 2 stages of the study. They all met the inclusion criteria and were consented. 40 patients completed at least 1 treatment cycle and TME and entered the current analysis with a median follow-up period of 13.2 (range from 3.6 to 31.9) months. 1 of the 44 patients had disease progression shortly after finishing chemotherapy, so surgery was aborted, 2 patients voluntarily withdrew from the study directly before surgery after completing all their treatment cycles and 1 patient died after the 4th cycle of chemo-immunotherapy. The study population consisted of 26 (65%) males and 14 (35%) females with median age of 58.5 (31.0, 74.0) years. The primary endpoint results revealed that 15/40 patients (37.5%) achieved pCR (tumor regression grade (TRG) =0) as compared to the historical control group with pCR of 16% (p=0.025). In addition, 12/40 (30%) had a near complete response with TRG =1 (< 10% viable cells in tumor bed). In total, 27/40 patients (67.5%) had major pathologic response rate (mpRR) (TRG 0 and 1). As for safety, 43 out of 242 (17.7%) adverse events were serious adverse events (SAEs). Of the total SAEs, 25/43 (58.1%), 5/43 (11.6%) and 1/43 (2.3%) had a toxicity grade of 3,4 and 5, respectively. In addition, none of the 31 reported grade 3-5 SAEs were related to avelumab; 11 (35%) were related to TME (including 6 anastomosis leaks), 3 (10%) to post-ileostomy closure, and 17 (55%) were miscellaneous. In terms of SAE outcomes, only 1 (3%) treatment-unrelated SAE resulted in death (cardiopulmonary arrest) while 27 (87%) resolved without sequalae and 3 (10%) are still ongoing. Based on this analysis, the primary endpoint was successfully met with significant improvement in pCR (37.5%, p=0.025) and mpRR rates in the setting of an acceptable safety profile. 3-year disease-free survival and overall survival will be reported later in the final study manuscript.

Treatment-induced evolutionary dynamics in nonmetastatic locally advanced rectal adenocarcinoma.

Multi-modal treatment of non-metastatic locally advanced rectal adenocarcinoma (LARC) includes chemotherapy, radiation, and life-altering surgery. Although highly effective for local cancer control, metastatic failure remains significant and drives rectal cancer-related mortality. A consistent observation of this tri-modality treatment paradigm is that histologic response of the primary tumor to neoadjuvant treatment(s), which varies across patients, predicts overall oncologic outcome. In this chapter, we will examine this treatment response heterogeneity in the context of evolutionary dynamics. We hypothesize that improved understanding of eco-evolutionary pressures rendering small cancer cell populations vulnerable to extinction may influence treatment strategies and improve patient outcomes. Applying effective treatment(s) to cancer populations causes a "race to extinction." We explore principles of eco-evolutionary extinction in the context of these small cancer cell populations, evaluating how treatment(s) aim to eradicate the cancer populations to ultimately result in cure. In this chapter, we provide an evolutionary rationale for limiting continuous treatment(s) with the same agent or combination of agents to avoid selection of resistant cancer subpopulation phenotypes, allowing "evolutionary rescue." We draw upon evidence from nature demonstrating species extinction rarely occurring as a single event phenomenon, but rather a series of events in the slide to extinction. We posit that eradicating small cancer populations, similar to small populations in natural extinctions, will usually require a sequence of different external perturbations that produce negative, synergistic dynamics termed the "extinction vortex." By exploiting these unique extinction vulnerabilities of small cancer populations, the optimal therapeutic sequences may be informed by evolution-informed strategies for patients with LARC.

Short-course radiation followed by mFOLFOX-6 plus avelumab for locally-advanced rectal adenocarcinoma

BackgroundCurrent standard practice for locally advanced rectal cancer (LARC) entails a multidisciplinary approach that includes preoperative chemoradiotherapy, followed by total mesorectal excision, and then adjuvant chemotherapy. The latter has been accompanied by low compliance rates and no survival benefit in phase III randomized trials, so the strategy of administering neoadjuvant, rather than adjuvant, chemotherapy has been adapted by many trials, with improvement in pathologic complete response. Induction chemotherapy with oxaliplatin has been shown to have increased efficacy in rectal cancer, while short-course radiation therapy with consolidation chemotherapy increased short-term overall survival rate and decreased toxicity levels, making it cheaper and more convenient than long-course radiation therapy.This led to recognition of total neoadjuvant therapy as a valid treatment approach in many guidelines despite limited available survival data. With the upregulation (PDL-1) expression in rectal tumors after radiotherapy and the increased use of in malignant melanoma, the novel approach of combining immunotherapy with chemotherapy after radiation may have a role in further increasing pCR and improving overall outcomes in rectal cancer.MethodsThe study is an open label single arm multi- center phase II trial. Forty-four recruited LARC patients will receive 5Gy x 5fractions of SCRT, followed by 6 cycles of mFOLFOX-6 plus avelumab, before TME is performed. The hypothesis is that the addition of avelumab to mFOLFOX-6, administered following SCRT, will improve pCR and overall outcomes. The primary outcome measure is the proportion of patients who achieve a pCR, defined as no viable tumor cells on the excised specimen. Secondary objectives are to evaluate 3-year progression-free survival, tumor response to treatment (tumor regression grades 0 & 1), density of tumor-infiltrating lymphocytes, correlation of baseline Immunoscore with pCR rates and changes in PD-L1 expression.DiscussionRecent studies show an increase in PD-L1 expression and density of CD8+ TILs after CRT in rectal cancer patients, implying a potential role for combinatory strategies using PD-L1- and programmed-death- 1 inhibiting drugs. We aim through this study to evaluate pCR following SCRT, followed by mFOLFOX-6 with avelumab, and then TME procedure in patients with LARC.Trial registrationTrial Registration Number and Date of Registration: ClinicalTrials.gov NCT03503630, April 20, 2018.

Development of a long non-coding RNA signature for prediction of response to neoadjuvant chemoradiotherapy in locally advanced rectal adenocarcinoma.

Standard treatment for locally advanced rectal adenocarcinoma (LARC) includes a combination of chemotherapy with pyrimidine analogues, such as capecitabine, and radiation therapy, followed by surgery. Currently no clinically useful genomic predictors of benefit from neoadjuvant chemoradiotherapy (nCRT) exist for LARC. In this study we assessed the expression of 8,127 long noncoding RNAs (lncRNAs), poorly studied in LARC, to infer their ability in classifying patients’ pathological complete response (pCR). We collected and analyzed, using lncRNA-specific Agilent microarrays a consecutive series of 61 LARC cases undergoing nCRT. Potential lncRNA predictors in responders and non-responders to nCRT were identified with LASSO regression, and a model was optimized using k-fold cross-validation after selection of the three most informative lncRNA. 11 lncRNAs were differentially expressed with false discovery rate < 0.01 between responders and non-responders to NACT. We identified lnc-KLF7-1, lnc-MAB21L2-1, and LINC00324 as the most promising variable subset for classification building. Overall sensitivity and specificity were 0.91 and 0.94 respectively, with an AUC of our ROC curve = 0.93. Our study shows for the first time that lncRNAs can accurately predict response in LARC undergoing nCRT. Our three-lncRNA based signature must be independently validated and further analyses must be conducted to fully understand the biological role of the identified signature, but our results suggest lncRNAs may be an ideal biomarker for response prediction in the studied setting.

A Retrospective Analysis Comparing Outcomes Following Total Neoadjuvant Therapy and Following Neoadjuvant Chemoradiation Therapy in Patients with Locally Advanced Rectal Cancer

To compare outcomes following Total Neoadjuvant Therapy (TNT) and following neoadjuvant chemoradiation therapy (nCRT) with or without adjuvant chemotherapy in patients with locally advanced rectal adenocarcinoma (LARC), defined as T3/4 or node positive disease, using the National Cancer Database. We identified LARC patients diagnosed between 2004 and 2015 who received chemotherapy and radiotherapy (RT) preoperatively and definitive surgery within one year of RT. Patients were considered to have received TNT if multiagent chemotherapy was given ≥2 months before RT and no adjuvant chemotherapy was given. Patients were considered to have received neoadjuvant chemoradiation therapy (nCRT) if preoperative RT and chemotherapy were started within 2 weeks from each other. A 2:1 propensity match was performed to account for selection bias, adjusting for age, gender, diagnosis year, race, comorbidity score, cT, cN, grade, facility type, urban status, insurance, and income. Kaplan-Meier survival analysis with log-rank test was used to compare overall survival (OS). Multivariable Cox regression modeling was performed to determine factors associated with OS. Multivariable logistic regression modeling was used to determine if TNT is associated with pathological complete response (pCR), defined as ypT0N0, and negative circumferential resection margin (CRM). After propensity matching, 372 patients receiving TNT were paired with 707 patients receiving nCRT. In the matched cohort, the median follow-up time was 31.0 months for TNT and 33.7 months for nCRT. Median time to surgery was 8.4 months (IQR: 7.2 to 9.2 months) for TNT compared to 4.4 months (IQR: 3.9 to 5.2 months) for nCRT. OS for TNT was similar to nCRT in both the matched (log-rank p=0.15) and the unmatched cohort (log-rank p=0.85). The 3- and 5-year OS rates for TNT vs. nCRT in the matched cohort were 88.5% vs. 88.9% and 73.6% vs. 78.5%, respectively (p>0.2 for both years). The multivariable analysis showed no significant difference in OS between TNT and nCRT (matched cohort: HR=1.21, 95%CI 0.87-1.69, p=0.25; unmatched cohort: HR=1.17, 95%CI: 0.90-1.51, p=0.24). Sensitivity analysis with 10:1 propensity matching for improved statistical power showed similar OS between TNT and nCRT (HR=1.16, 95% CI: 0.88-1.53, p=0.28). The pCR and negative CRM rates with TNT and nCRT were 16.9% vs. 13.1% (p=0.12) and 95.3% vs. 93.1% (p=0.30), respectively. TNT was not found to be significantly associated with pCR (OR=1.36, p=0.13) or negative CRM (OR=1.77, p=0.19) in multivariable logistic regression modeling. With results from current clinical trials pending, our data suggested that TNT and nCRT resulted in similar survival, while TNT led to higher pCR and CRM negative rate, albeit not statistically significant.

The prognostic significance of magnetic resonance imaging (MRI) following neoadjuvant concurrent chemoradiation (NCCR) in patients with locally advanced rectal adenocarcinoma (LARA).

e15166 Background: In patients with locally advanced rectal adenocarcinoma (LARA), the response to neoadjuvant concurrent chemoradiation (NCCR) correlates with long-term outcomes. Neoadjuvant rectal (NAR) score ([5 ypN - 3 (cT - ypT) + 12]2/9.61) is a novel short-term surrogate endpoint for disease free survival (DFS) and Overall survival (OS). The prognostic significance of magnetic resonance imaging (MRI) findings following NCCR is yet to be explored. Here, we evaluated the agreement between post NCCR MRI yT and yN and pathological ypT and ypN. In addition, we calculated the post NCCR MRI NAR (mNAR) score using the MRI yT and yN ([5 yN - 3 (cT - yT) + 12]2/9.61) and explored its prognostic significance. Methods: Between 2014 and 2018, all patients with LARA were identified. Among those, 43 patients received NCCR, had post NCCR MRI and underwent surgical resection. Weighted Kappa was used to measure the agreement between post NCCR MRI yT and yN and pathological ypT and ypN. Paired t-test was used to compare the means between NAR and mNAR scores. NAR and mNAR scores were classified as low ( &lt; 8), intermediate (8-16) and high ( &gt; 16). DFS and OS were analyzed using Kaplan-Meier curves. Results: In our cohort, the agreement was slight between post NCCR MRI yT and Pathological ypT (p = 0.111), and fair between post NCCR MRI yN and pathological ypN (0.278). The mean NAR score was 16 and the mean mNAR score was 20 (p = 0.0079). Low-intermediate and high NAR scores were seen in 31 (72%) and 12 (28%) patients respectively. However, low-intermediate and high mNAR scores were seen in 20 (47%) and 23 (53%) patients respectively. The median DFS in patients with low-intermediate and high NAR scores was not reached and 30 months (p = 0.00063) respectively. The median OS in patients with low-intermediate and high NAR scores was not reached and 40 months (p = 0.00056) respectively. The median DFS in patients with low-intermediate and high mNAR scores was not reached in both groups (p = 0.058). The median OS in patients with low-intermediate and high mNAR scores was not reached in both groups (p = 0.15). Conclusions: Post NCCR MRI yT and yN had slight and fair agreements with ypT and ypN respectively. In our cohort, there was a difference between NAR and mNAR. NAR demonstrated prognostic significance for DFS and OS, while mNAR did not at the time of analysis.

MicroRNA molecular profiling identifies potential signaling pathways conferring resistance to chemoradiation in locally-advanced rectal adenocarcinoma.

PurposeThere has been growing interest in using chemoradiation (CRT) for non-operative management of rectal cancer, and identifying patients who might benefit most from this approach is crucial. This study identified miRNAs (miRs) associated with clinical outcomes and treatment resistance by evaluating both pre- and post-CRT expression profiles.MethodsForty patients, 9 with pathologic complete response (pCR) and 31 with pathologic incomplete response (pIR) were included. MicroRNA was extracted from 40 pre-therapy tumor samples and 31 post-chemoradiation surgical samples with pathologic incomplete response (pIR). A generalized linear model was used to identify miRs associated with pCR. A linear mixed effects model was used to identify miRs differentially expressed before and after treatment. miR expression was dichotomized at the mean and clinical outcomes were evaluated using Cox proportional hazard modeling.ResultsNine miRs were associated with pCR (p<0.05), but none were significant after false discovery rate correction. Among patients with pIR, 68 miRs were differentially expressed between the pre and post-CRT groups (FDR p<0.05). Ingenuity pathway analysis (IPA) demonstrated multiple signaling networks associated with pIR, including p38MAPK, TP53, AKT, IL-6, and RAS. Increased let-7b was correlated with increased distant metastasis (DM), worse relapse-free survival (RFS), and worse overall survival (OS) (p<0.05).ConclusionsNo miRs were significantly correlated with pCR. We identified miRs that were differentially expressed between pre- and post-CRT tumor samples, and these miRs implicated multiple signaling pathways that may confer resistance to CRT. In addition, we identified an association between increased let-7b and worse clinical outcomes (DM, DFS, OS).

Abstract CT088: A phase I trial combining MEK-1/2 inhibition in combination with 5-fluorouracil and radiation for locally-advanced rectal adenocarcinoma

Abstract Introduction: 5-fluorouracil (5-FU)-based chemoradiation (CRT) is a standard neoadjuvant treatment strategy for the treatment of locally-advanced rectal adenocarcinomas (LARC). MEK-1/2 inhibition has been shown to radiosensitize Ras mutant tumors in preclinical models, including colorectal cancer. In this phase 1 trial, we are testing MEK-1/2 inhibition in combination with neoadjuvant CRT for LARC. Methods: This trial is a standard 3+3 design testing 3 dose levels of the MEK-1/2 inhibitor trametinib in combination with CRT: 0.5 mg, 1.0 mg, and 2.0 mg daily, followed by an expansion cohort. All patients first receive a one week lead-in dosing of trametinib monotherapy, and undergo a tumor biopsy at the end of that week to assess pharmacodynamic inhibition by immunohistochemistry (IHC). Then, patients receive standard CRT (5-FU 225 mg/m2 per day by continuous infusion during radiotherapy, and 50.4 Gy in 28 fractions, 5 days/ week) in combination with trametinib (5 days/week). Patients have total mesorectal excision (TME) 6-10 weeks after completion of CRT. Results: 17 patients have been enrolled to date, with current enrollment occurring on the trametinib 2 mg expansion cohort (total 18 patients planned). Of these, 15 patients (10 males, 5 females) have toxicity and surgical response data available. Median age is 53 years (range 35-74 years), and 6 patients have Stage II disease, while 9 patients with Stage III disease. There have been no grade 4-5 toxicities. One dose-limiting toxicity (DLT) of diarrhea occurred in the 2mg dose cohort, attributed mainly to 5FU CRT, but led to trametinib discontinuation. Generalized skin rash led to trametinib discontinuation after 2 weeks of therapy for 1 patient and was held for 3 days for 2 patients. All patients completed TME, and 3 patients had wound infection at 2 weeks after TME, while 2 patients had perineal wound infection at 4 weeks after TME. Three out of 9 (33%) patients at the 2mg dose cohort had a pathologic complete response (pCR), and 2/9 (22%) patients had a near pCR. Adjuvant chemotherapy was given to 10 patients, 4 patients did not receive (3 refused), and 1 patient is pending. Correlative research studies have been performed in the first 12 patients enrolled. Examination of phospho-ERK levels using Vectra quantitative immunohistochemistry in pre-treatment versus post-lead-in trametinib tumor tissue reveals dose-dependent decreases in p-ERK expression in both tumor and stromal tissue as trametinib is increased from 0.5 to 2.0 mg (range 18-46% absolute reduction), with maximal reduction observed in the 2 mg cohort. Additionally, we have performed whole exome sequencing of a custom panel of 279 cancer associated genes (IGNITE platform) in tumor and normal tissue, and have identified high frequency mutations in KRAS, BRAF, and TP53, as well as additional lower frequency mutations in every tumor. One patient was noted to have microsatellite instability. Finally, we will present data correlating degree of change in p-ERK staining, mutational analysis, and mismatch repair status with pathologic response. Conclusions: The combination of trametinib with 5-FU-based CRT for LARC is feasible with promising signs of clinical activity and tolerability. Initial analysis of tumor tissue confirms dose-dependent pharmacodynamic inhibition of ERK-1/2 within 5 days of starting trametinib. Citation Format: Terence M. Williams, Christina Wu, Lai Wei, Emily Chan, Ryan Robb, Sameek Roychowdhury, Amy Webb, Cynthia Timmers, Tanios Bekaii-Saab, Evan Wuthrick. A phase I trial combining MEK-1/2 inhibition in combination with 5-fluorouracil and radiation for locally-advanced rectal adenocarcinoma [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 CT088. doi:10.1158/1538-7445.AM2017-CT088

Molecular profiling of locally-advanced rectal adenocarcinoma using microRNA expression (Review).

Treatment for locally-advanced rectal cancer (LARC) typically consists of neoadjuvant chemoradiation followed by total mesorectal excision. Recently, there has been growing interest in non-operative management for patients who are medically-inoperable or wish to avoid surgical morbidity and permanent colostomy. Approximately 50% of patients who receive pre-operative neoadjuvant chemoradiation develop some degree of pathologic response. Approximately 10-20% of patients are found to have a complete pathologic response, a finding which has frequently been shown to predict better clinical outcomes, including local-regional control, distant metastasis and survival. Many recent studies have evaluated the role of molecular biomarkers in predicting response to neoadjuvant therapy. MicroRNAs (miRNAs) are an emerging class of biomarkers that have the potential to predict which patients are most likely to benefit from pre-operative therapy and from a selective surgical approach. Here, we review the published literature on microRNAs as prognostic and predictive biomarkers in rectal cancer after pre-operative therapy. In the future, the development of prospectively validated miRNA signatures will allow clinical implementation of miRNAs as prognostic and predictive signatures in LARC.

Association of T-cell infiltration assessed in pretherapeutic biopsies (PTB) of patients with locally advanced rectal adenocarcinoma (LARC) with tumor response and relapse after chemoradiotherapy (CRT) and rectal surgery.

3599 Background: Pre-operative CRT followed by total mesorectal excision (TME) is nowadays the standard of care for patient with LARC (cT3-T4N0 or cTxN+). Currently, pathologic complete response occurs in +/- 15% after CRT. Colorectal cancer T-cell infiltration is a strong prognostic factor for survival after primary tumor resection. Our aim was to determine whether T-cell infiltration in PTB could be predictive of tumor response and relapse after CRT + TME. Methods: Between 1999 and 2012, patients with LARC who underwent CRT + TME and with available clinical follow-up and PTB (with sufficient tumor cells density) were identified at the Cliniques universitaires St-Luc. The density of CD3 (T cells) and CD8 (cytotoxic) was quantified on immunostained PTB slides and analyzed with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Cumulative disease-free survival (DFS) was performed using the Kaplan-Meier estimator and compared by log-rank tests. Cox regression we used for uni- and multi-variate analysis. P value of less than 0.05 was considered statistically significant. Results: 154 patients (sex ratio M/F 1.8; mean age 65 years-old; upper (20%), mid (29%) and low rectum (51%), synchronous metastases (11%)) were analyzed. High CD3 and CD8 PTB densities were significantly associated with a higher pathological response (Dworak 3-4) and lower ypTNM stage after CRT +TME (p &lt; 0,05). Higher CD3 and CD8 PTB densities were associated with higher patient DFS (CD3: HR = 2,30 (CI95%:1,15-4,59) p = 0,02; CD8: HR = 1,95 (CI95%: 1,01-3,75) p = 0,04). These results were confirmed in uni and multivariate analysis. CD3 and CD8 PTB densities added to pathological response (ypTNM/Dworak) but also clinical response (ycTNM) after CRT + TME increases significantly the accuracy prediction of tumor relapse. Conclusions: Pretherapeutic T-cell infiltration of LARC is predictive of tumor response and relapse after CRT +TME. This biomarker could be helpful for patient treatment decision. It must be validated in larger patient cohorts.

Abstract CT025: A phase I trial combining MEK-1/2 inhibition in combination with 5-fluorouracil and radiation for KRAS, BRAF, and NRAS mutant locally advanced rectal adenocarcinoma

Abstract Introduction: 5-fluorouracil (5-FU)-based chemoradiation (CRT) is a standard neoadjuvant treatment strategy for the treatment of locally-advanced rectal adenocarcinomas (LARC). MEK-1/2 inhibition has been shown to radiosensitize Ras mutant tumors in preclinical models, including colorectal cancer. In this phase 1 trial, we will test MEK-1/2 inhibition in combination with neoadjuvant CRT for LARC. Methods: This trial is a standard 3+3 design testing 3 dose levels of the MEK-1/2 inhibitor trametinib (GSK1120212) in combination with CRT: 0.5 mg, 1.0 mg, and 2.0 mg daily, followed by an expansion cohort. All patients first receive a one week lead-in dosing of trametinib monotherapy, and undergo a tumor biopsy at the end of that week to assess pharmacodynamic inhibition by immunohistochemistry (IHC). Then, patients receive standard CRT (5-FU 225 mg/m2 per day by continuous infusion during radiotherapy, and 50.4 Gy in 28 fractions, 5 days/ week) in combination with trametinib (5 days/week). Patients have total mesorectal excision (TME) 6-10 weeks after completion of CRT. Results: The trial opened in 2013, and 31 patients have been screened for KRAS/BRAF/NRAS activating mutations, of which 11 (35.4%) tested positive for a mutation. Of these, 9 patients (8 male, 1 female) have been enrolled so far, with 3 patients accrued in each dose level. Regarding mutation status, six patients have KRAS mutations (five G12V, one G13D), two patients have BRAF mutations (one V600E, one D594N), and one patient is unknown (in dose level 1). Age of patients ranges from 38-64 years old (median 55). All patients have successfully completed neoadjuvant therapy and TME (5 low anterior resection, 4 abdominal-perineal resection). No dose-limiting toxicities were observed on dose levels 1 and 2, and additional patients are currently being accrued onto dose level 3 (2.0 mg). One patient in dose level 3 had pathologic complete response (pCR) at the time of TME. Examination of pre-treatment tumor tissue by IHC reveals baseline hyperactivation of ERK-1/2 (p42/44 MAPK) in most tumors with some heterogeneity in levels of phospho-ERK staining, reflecting hyperactivation of the RAS/RAF-MAPK pathway. Qualitatively, ERK-1/2 activity is reduced from pre-treatment to post-trametinib lead-in tumor tissue, particularly in patients on dose level 3. The patient who experienced a pCR had the most significant reduction in ERK-1/2 activity in tumor tissue and tumor-associated stroma. We will also present quantitative data demonstrating ERK-1/2 inhibition using Vectra quantitative immunohistochemistry. Conclusions: The combination of trametinib with 5-FU-based CRT for LARC is feasible with promising signs of clinical activity and tolerability. Ongoing enrollment will further assess safety, tolerability, and determine the maximally-tolerated dose of trametinib. Initial analysis of tumor tissue suggests dose-dependent pharmacodynamic inhibition of ERK-1/2 within 5 days of starting trametinib. Citation Format: Terence M. Williams, Evan Wuthrick, Christina Wu, Ryan Robb, Cynthia Timmers, Tanios Bekaii-Saab. A phase I trial combining MEK-1/2 inhibition in combination with 5-fluorouracil and radiation for KRAS, BRAF, and NRAS mutant locally advanced rectal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT025.

Predicting outcomes in locally advanced rectal cancer using pretreatment FDG-PET imaging.

495 Background: FDG-PET/CT imaging has been shown to have clinical utility in the management of rectal cancers. The purpose of this study was to investigate multiple FDG-PET/CT parameters to predict for outcome after neoadjuvant chemoradiation (CRT) in patients (pts) with locally advanced rectal adenocarcinoma. Methods: We retrospectively evaluated pts with locally-advanced (T2-4, N0-2, M0) rectal adenocarcinoma treated with neoadjuvant CRT who received FDG-PET/CT scans for radiation therapy planning. We evaluated the impact of SUVmax and metabolic tumor volume (MTV, determined by using PET Edge MimVista), as well as dual-time point PET parameters of retention index (RI, difference in SUVmax between the two PET scans) and RI/time. Endpoints of pathologic complete response (pCR), tumor grade, margin status and pathologic downstaging were assessed using t-test, ANOVA or non-parametric analysis when appropriate. Progression-free survival (PFS) and overall survival (OS) were assessed using Kaplan Meier estimates. Results: Of the 28 consecutive pts with FDG-PET/CT planning scans identified, 25 pts underwent surgical resection. All patients received a 5-FU based concurrent chemotherapy regimen with RT (median RT dose: 50.4 Gy). Median follow-up was 21 months. The median MTV was 45 cc. Compared to pts with a MTV&gt;45 cc, pts with a MTV &lt;45 cc had improved PFS (2-yr PFS: 86% vs. 60%, p=0.04) and improved OS (2-yr OS: 100% vs. 51%, p=0.01). The correlation between MTV and T-stage was not statistically significant (23% of pts with MTV&gt;45 had T4 tumors vs. 0% of pts with MTV&lt;45, p=0.17). MTV did not correlate with the other endpoints (pCR, tumor grade, margin status, pathologic downstaging). SUVmax did not correlate with any of the identified endpoints. Of the patients with dual-time point PET/CT scans (N=19), RI and RI/time did not correlate with any of the identified endpoints. Conclusions: Pretreatment MTV correlates with PFS and OS in patients with locally advanced rectal cancer treated with neoadjuvant chemoradiation. For pts with large MTVs, more aggressive treatment approaches should be considered. Dual-time point FDG-PET imaging does not appear to add clinical value in this patient population.

The influence of seminal events on the transition from adjuvant to neoadjuvant chemoradiation in the treatment of locally advanced rectal adenocarcinoma (LARC).

631 Background: Factors leading to uptake of new oncology treatment innovations are poorly understood. In particular, the degree to which seminal events, such as report of pivotal phase III trials, influence practice is unclear. For example, preoperative (pre-op) 5-fluorouracil + radiation (5-FU + RT) is the current standard of care for definitive treatment of LARC. However, postoperative (post-op) 5-FU + RT was standard before the seminal German Rectal Cancer Study Group (GRCSG) results were published. We investigated the impact of seminal events on the change in practice pattern from pre- to post-op 5-FU + RT at our institution. Methods: Patients with LARC (T2N+; any T3; any T4) treated at our institution between 1994-2010 were identified from the cancer registry. The date of diagnosis was compared to the dates of three seminal events: A) JAMA meta-analysis publication; B) publication of GRCSG results; C) founding of a multidisciplinary clinic at our institution. Pearson Chi square was used for univariate analysis. Results: 334 patients were evaluable. RT +/- 5-FU was delivered pre-op for 207 patients, post-op for 127 patients. The unadjusted odds ratio (OR) for receiving pre-op treatment after vs. before each seminal event was similar: (A) 4.16; (B) 4.08; (C) 3.91. When patients diagnosed prior to (A) or after (C) were excluded, (B) appeared to have a smaller effect, OR 2.07 (p = .053) (Table). Conclusions: All seminal events had similar associated OR, indicating that the process of uptake of the innovation of pre-op 5-FU + RT was gradual. The seminal GRCSG publication, when temporally isolated, had only modest effect. This suggests that report of seminal results is necessary but not sufficient for uptake of a new therapy innovation in LARC at our institution. Whether this pattern of uptake is generalizable is worthy of further investigation. [Table: see text]

Expression of epidermal growth factor receptor as a predictive factor for rectal cancer.

Expression of epidermal growth factor receptor is observed in 50 - 70% of colorectal carcinomas and is associated with poor prognosis. The objective of this study was to analyze whether epidermal growth factor receptor expression predicts tumor response and sphincter preserving in patients treated with preoperative chemoradiation therapy. This study was conducted on 34 patients with locally-advanced rectal adenocarcinoma who were treated with preoperative chemoradiation therapy. The patients had histologically-proven adenocarcinoma of the rectum with the inferior margin of the tumor located no farther than 6 cm from the anal verge. Preoperative radiotherapy was delivered to the pelvis with 60CO to 50.4 Gy. All patients received simultaneous chemotherapy with 5-fluorouracil, 300 mg/m(2) IV infused over 24 hr during radiotherapy on days 1 - 5 every week; 28 patients received oxaliplatin 50 - 60 mg/m(2) weekly during radiotherapy. The patients were restaged by physical examination and pelvic CT, between four and six weeks later. Then, they were referred to a surgeon who was expert in gastrointestinal cancer surgery. Subsequently, postsurgical specimen was histopathologically examined and graded according to the Mandard criteria for assessment of pathologic response after neo-adjuvant chemoradiation. Immunohistochemistry for epidermal growth factor receptor was determined at the preradiation biopsy and was evaluated according to the extension and staining intensity. Fourteen (41%) out of 34 tumors were epidermal growth factor receptor positive. Twenty (59%) patients responded to pelvic preoperative chemoradiation. Sixteen (47%) patients achieved complete response with no residual tumor in the resected specimen; four (12%) were downstaged (partial response). Response to pelvic radiotherapy was observed in 80% of those negative for epidermal growth factor receptor and in 28% of those with positive epidermal growth factor receptor (P = 0.005). Only two of 14 positive epidermal growth factor receptor patients achieved a complete response, while 14 of 20 of the negative epidermal growth factor receptor patients developed complete response. In our study, the sphincter preservation rate was 43% in positive epidermal growth factor receptor patients and 80% in those who did not express epidermal growth factor receptor (P = 0.036). Epidermal growth factor receptor expression in the diagnostic biopsy of locally-advanced rectal cancer treated with chemoradiation therapy may serve as an important predictor of complete response to preoperative treatment.

Phase-2 study of neoadjuvant chemoradiation in locally advanced rectal adenocarcinoma: The Radio-Xelox study

13549 Background: Neoadjuvant Radiation treatment has been shown in many studies to decrease the incidence of loco-regional relapse in pts with locally-advanced Rectal Adenocarcinoma. Capecitabine and Oxaliplatin are both effective antineoplastic drugs in Colorectal Adenocarcinoma. They also have radiosensitizing effects. Hence,it is rational to combine these agents in association with radiotherapy,to achieve local controle and prevent systemic spread of the cancer. Methods: Thirty-eight pts with Rectal Adenocarcinoma (T3-T4 and/or N+) received radiation treatment (2.0 Gy, 5days a wk over 5 wks,total dose 50 Gy) plus oral Capecitabine at 600 mg/m2 twice a day and iv Oxaliplatin at 35 mg/m2 once weekly × 5wks. Surgery was performed 6wks post completion of XRT. Our end points were safety/feasibility and response/efficacy as demonstrable by the rate of pathologic complete remission (path-CR). Results: Seven pts (20%) acheived path-CR. Diarrhea was the most important severe adverse effect (in 25% of pts). fourteen pts (40%) showed good regression (very few cancer cells in fibronecrotic tissue Dworak grade 3). The remaining 17 pts showed less favorable regression or no significant change. Conclusions: Preoperative Radiation treatment plus chemotherapy with XELOX regimen is feasible, safe and effective with an impressive rate of path-CR in locoregionally advanced Rectal Adenocarcinoma. No significant financial relationships to disclose.