Overcoming the Challenge: A Comprehensive Review of Neoadjuvant Treatment Resistance in Rectal Cancer.

Tumor regression grading as prognostic factor in patients with locally advanced rectal cancer treated with preoperative radiochemotherapy

The interactions between neoplastic cells and their tumor microenvironment (TME) have become increasingly evident. Cancer-associated fibroblasts (CAFs) and M2 macrophages (M2 Mθ) are important components of the TME that have been recognized to orchestrate tumor-promoting inflammation and thereby tumor growth and progression. Here, we aim to determine the presence and role of CAFs and M2 Mθ in high grade cervical intraepithelial neoplasia (CIN 2/3) and invasive cervical carcinomas (SCCx). We optimized immunohistochemistry (IHC) markers for M2 Mθ (CD163, ARG1), CAFs (αSMA) and Th1 T cells (TBX21) in paraffin-embedded tissue sections from CIN2/3 (n=6) and SCCx cases (n=4). We found an increased infiltration of TBX21+ cells in the epithelium and stroma of CIN2/3 and SCCx cases compared to their normal adjacent stroma. This suggests that despite increased Th1 T cells, the progression from normal to CIN2/3 to SCCx might be facilitated by M2 Mθ and CAFs. Moreover, by performing double staining Immunofluorescence (IF) we found that M2 Mθ (CD163+) were the cells responsible for ARG1 production, an immunesuppressive mechanism utilized by these cells. We recently stained by IHC a cervical tissue microarray (TMA) of normal, CIN2/3 and SCCx cases (n=63) with CD163, αSMA and TBX21, and we are currently analyzing the correlation of these markers with clinicopathological data available for these cases (HPV status, tumor stage, therapy response, overall survival). With the hypothesis in mind that CAFs induce the polarization of macrophages into a M2 Mθ phenotype, we isolated fibroblasts from normal (n=2), CIN2/3 (n=1) and SCCx (n=2) fresh tissue explants obtained from surgery. We performed Immunocytochemistry (ICC) with αSMA, and observed a progressive increase in αSMA+ fibroblasts from normal to CIN2/3 and from CIN2/3 to SCCx fibroblasts, suggesting that CAFs might play an important role in the transformation of normal cervix into CIN2/3 and SCCx. Furthermore, we are currently performing co-culture experiments with normal, CIN2/3 and SCCx isolated fibroblasts and peripheral blood monocytes obtained from healthy donors. We will perform triple staining IF with vimentin, αSMA and CD163 in the slides generated from the co-culture, to evaluate if monocytes acquired a M2 Mθ phenotype with the presence of any of these 3 subsets of fibroblasts. Moreover, we will perform a cytokine analysis in the supernatants of these co-cultures to evaluate for potential cytokines produced by CAFs, implicated in M2 Mθ polarization. In this study we provide evidence regarding the role of CAFs in SCCx initiation and progression. Although the mechanisms that regulate fibroblast activation have not been elucidated, it is possible that CAFs might serve as novel therapeutic targets in cancer, either alone or in combination with existing anti-cancer therapies. Citation Format: Leonel Maldonado, Teresa Diaz-Montes, Abdulrahman Sinno, Edward J. Tanner, Christopher VandenBussche, Richard Roden, Iveta Yotova, Benjamin Tycko, Cornelia L. Trimble. Role and presence of cancer associated fibroblasts and M2 macrophages in high grade cervical intraepithelial neoplasia and invasive cervical carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3672. doi:10.1158/1538-7445.AM2014-3672

Cancer associated fibroblasts (CAFs) are thought to play an essential role in cancer invasion, migration, metastasis and resistance to anticancer drugs. CAFs were originally defined as fibroblasts expressing α-SMA in cancer tissue; however, fibroblast activation protein (FAP) has been recently identified as a more functional and specific surface protein of CAFs. The photoimmunotherapy (PIT) is a new molecular targeted therapy, which is based on a near-infrared (NIR) photosensitizer IR700, conjugated to monoclonal antibody (mAb) targeting particular molecules. PIT induces the selective destruction of targeted cells. The aim of this study is to analyze the influence of CAFs on human esophageal cancer and to develop a novel therapeutic strategy to eliminate CAFs by using FAP-targeting PIT. We produced the IR700-FAP antibody for this study. Human esophageal cancer cell lines (TE-4 and OE-19) and FEF3 human fibroblasts were used in this study. TE-4 and OE-19 cells stimulated with conditioned medium (CM) of CAFs exhibited malignant phenotypes confirmed by invasion assay, migration assay, and colony formation assay. Tumor cells were significantly more malignant when directly co-cultured with CAFs. We further examined whether tumor cells stimulated by CM (CAFs) could acquire the resistance to chemotherapy or radiotherapy. Cell viability assay showed that tumor cells stimulated with CAFs was more resistant to conventional chemotherapy or radiation than unstimulated tumor cells. Western blot analysis demonstrated that E-cadherin expression was decreased and that of vimentin was increased in CAF-stimulated tumor cells, indicating the epithelial mesenchymal transition (EMT) induction. Moreover, OE-19 cells stimulated with CAFs contained more CD133-positive cancer stem-like cells. In vivo experiments demonstrated that subcutaneous TE4 tumors were more refractory to chemotherapy in the presence of co-inoculated CAFs as observed in vitro. Finally, we investigated whether elimination of CAFs by FAP-targeting PIT could affect the resistance to chemotherapy in vitro and in vivo. In vivo PIT following intratumoral anti-FAP mAb-conjugated IR700 suppressed the growth of subcutaneous tumors co-inoculated with CAFs. These results demonstrated that human tumor cells became more malignant and resistant in the presence of CAFs, which could be reversed by using CAF-specific PIT. Targeting fibroblast itself is a unique strategy and can be clinically promising as combination targeting cancer cells and their fundamental microenvironment. Citation Format: Ryoichi Katsube, Kazuhiro Noma, Shinichiro Watanabe, Shinichi Urano, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Hisataka Kobayashi, Toshiyoshi Fujiwara. A novel photoimmunotherapy targeting cancer-associated fibroblasts (CAFs) overcomes therapeutic resistance in human esophageal 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 401. doi:10.1158/1538-7445.AM2015-401

Immune checkpoint blockade (ICB) treatment improves the prognosis of several types of solid tumors, however, responsiveness to ICB therapy remains low in pancreatic ductal adenocarcinoma (PDACs), which has a rich tumor microenvironment (TME). The TME is composed of various stromal cells, including cancer-associated fibroblasts (CAFs), which contribute to the establishment of an immunosuppressive microenvironment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an innate immune pathway that results in the upregulation of immune cell recruiting-cytokines and anti-tumor efficacy. In this study, we aimed to investigate the impact of cGAS-STING expression and the presence of CAFs upon immune cell infiltration in PDACs. cGAS and STING co-expressing PDAC cases showed favorable survival, with many cytotoxic CD8 + T cell infiltrations from the stromal component adjacent to the cancer cells toward cancer cells, but not in cGAS-STING signaling defected PDAC cases. The signatures of tumor-restrain CAFs were expressed in tumors with cGAS-STING signaling. Finally, transwell co-culture experiments demonstrated that immune cell infiltration was impeded by the presence of CAFs, but not by activation of cGAS-STING signaling. In conclusion, pro-infiltration signals, such as cGAS-STING, and characterization of CAFs are crucial in defeating CAF barricades and encouraging immune cell infiltration in PDACs.

Patient derived organoids are becoming a popular 3D in vitro model that more accurately recapitulate in vivo conditions compared to standard 2D culture systems. Organoids consist of epithelial cells and lack stroma and mesenchyme, allowing cells of interest to be studied as an isolated system. However, the tunability of the organoid model also permits the re-addition of microenvironmental factors, i.e. cancer associated fibroblasts (CAFs), resulting in a wide range of experimental capabilities. Organoids are easily scalable, making them more efficient and cost-effective than traditional animal models. Despite the popularity of animal models for pre-clinical drug testing, there still remain issues with translating results from animal testing to patient outcomes; even patient-derived xenograph models experience non-physiological mouse-human interactions. Therefore, studying the impact of microenvironmental perturbations, such as the presence of CAFs, or nutrient and drug gradients, on organoids may reduce translational error. Using our biorepository of patient derived colon cancer (primary and liver metastases) organoids, we can observe changes in tumor architecture and cell growth or death, allowing us to more accurately assess the efficacy of drug therapies and observe how interpatient heterogeneity impacts their efficacy. Here we focus on the histopathology of different patient derived organoids to quantitatively study cell growth or death under drug treatments (+/- CAFs) which could ultimately become a standard procedure for drug screening. Here we investigate the effects of CAF co-culture in patient derived organoids in response to standard chemotherapies such as Irinotecan and Oxaliplatin. Following treatment, the organoids are paraffin fixed to retain their structural integrity, then sectioned. The samples are stained with H&E to show the tumor architecture, and immunofluorescent antibodies (IF) to identify cells that are proliferating (Ki67) or apoptotic (TUNEL). As a result, we are able to investigate how tumor microenvironmental factors affect the tumor architecture of an individual patient tumor. We also examine the cytotoxic or cytostatic effects of standard chemotherapies by quantitating the number of proliferative and apoptotic cells, as well as their location within the tumor, in response to the presence of both CAFs and therapy. Ultimately, we envision the method outlined here will further personalize medicine by allowing physicians to more rapidly assess an individual patient’s response to therapy and adjust treatment accordingly. Additionally, this method could prove to be a more inexpensive and faster means of screening new drug compounds. By creating a system that more closely recapitulates patient outcomes, we hope to move therapies towards faster approval while still retaining results representative of in vivo outcomes. Citation Format: Sarah J. Choung, Erin Spiller, Roy Lau, Shannon Mumenthaler. Histopathology of patient derived organoids for the quantitative analysis of response to microenvironmental perturbations [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 5786. doi:10.1158/1538-7445.AM2017-5786

Objective To explore the predictive factors of tumor regression grading (TRG) after neoadjuvant chemoradiotheraphy of patients with locally advanced rectal cancer. Methods A retrospective analysis was performed on clinical and molecular biological data of 68 patients with rectal cancer, who underwent neoadjunant chemoradiotheraphy followed by radical surgery from April 2007 to December 2016. The clinical factors associated with TRG were analyzed by Logistic regression. Results Overall, there were 3 cases of TRG 0 (4.4%), 14 cases of TRG 1 (20.1%), 30 cases of TRG 2 (44.1%), 13 cases of TRG 3 (19.1 %), and 8 cases of TRG 4 (11.8%). Univariate analysis showed pre-neoadjuvant chemoradiotheraphy stage cT3 (P=0.008), cN0 (P=0.000), and the K-ras mutation status of wide-type (P=0.007) were significantly correlated with TRG after neoadjuvant chemoradiotheraphy. Logistic regression revealed that cT, cN and K-ras were independent predictive factors of TRG after neoadjuvant chemoradiotheraphy in rectal cancer. Conclusion Early stage of T and N, and the K-ras mutation status of wide-type may be predictive factors of high degree of TRG for rectal cancer after neoadjuvant chemoradiotherapy. Key words: Rectal cancer; Neoadjuvant chemoradiotherapy; Tumor regression grading

3610 Background: Neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision has become a standard therapy for patients with rectal cancer. However, not all patients have a good response to CRT. Therefore, it is useful to know what the predictor of chemoradiotherapeutic response is. Cumulative evidence has shown that COX-2 has an important role in the progression of colorectal cancer. The aim of this study was to clarify whether pretreatment COX-2 protein expression was a predictor of histopathologic response in patients with rectal cancer treated with preoperative short-term chemoradiotherapy. Methods: Fifty-two patients with lower rectal cancer received short-term preoperative chemoradiotherapy (20 Gy given in 5 daily doses of 4 Gy and administration of Tegafur/Uracil 400mg/day during the same period), followed by total mesorectal excision at National Defense Medical College from 2001 to 2005. COX-2 expression before and after CRT was measured by immunoshistochemistry. The specimens were graded based on the intensity and extent of staining. Tumor regression grading (TRG) was also evaluated (TRG 4, complete regression; TRG 3, regression > 50%; TRG 2, 25% to 50%; TRG 1, < 25%; and TRG 0, no regression). The correlation between COX-2 expression and tumor regression was analyzed. Results: The patient characteristics were as follows: the male:female ratio was 36:16; median age was 62. The Median postoperative follow-up period was 30 months. Two patients had pathological complete response. Good response (TRG 3 + 4) was found in 57.7% of the resected specimens. COX-2 was positive in 80.8% of patients before CRT while in 100% after CRT. The rates of good response (TRG 3 + 4) in patients with and without COX-2 expression were 50% and 90%, respectively (p = 0.021). Conclusions: Patients without pretreatment COX-2 expression were more likely to demonstrate good response to treatment than those with COX-2 expression. Pretreatment COX-2 protein expression can predict response of CRT in patients with rectal cancer. [Table: see text]

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

To investigate the association of CD133 expression in rectal cancer tissues with neoadjuvant chemoradiotherapy (nCRT) and tumor regression grading (TRG) after nCRT. Radical resected rectal cancer specimens and clinicopathological data of 105 patients, including 60 men and 45 women with median age of 59 years, diagnosed as locally advanced rectal cancer in Peking Union Medical College Hospital from January 2008 to December 2014 were collected retrospectively. Thirty-nine and 66 cases were histologically classified as good-moderate and poor differentiation respectively. Sixty-eight and 37 cases were clinically graded as stage I(-II( and III(-IIII( in preoperative assessment respectively. NCRT was administered in 61 cases before surgery (nCRT group). The nCRT consisted of preoperative pelvic radiotherapy using 50 Gy (2 Gy once, for 25 sessions) with FOLFOX regimen (5-fluorouracil plus oxaliplatin) for 2-3 cycles or XELOX regimen (capecitabine plus oxaliplatin) for 2 cycles. Patients underwent surgery after 6 courses of nCRT, and then received the same previous chemotherapy regimen. In nCRT group, biopsy specimens before nCRT were obtained in 45 cases. Forty-four cases received surgery alone without nCRT (surgery alone group). CD133 expression was tested by immunohistochemical Envision two-step methods. The histological TRG evaluation was performed in the nCRT group. TRG score 0-2 was defined as insensitivity to nCRT, whereas TRG score 3-4 was defined as sensitivity. CD133 expression in rectal cancer samples before and after nCRT was compared. Association of CD133 expression with TRG after nCRT was examined. No significant differences of baseline parameters were found between nCRT group and surgery alone group (all P>0.05). The positive rate of CD133 in nCRT group was 70.4%(43/61,) which was significantly higher than that in surgery alone group (47.7%, 21/44)(χ(2)=5.566, P=0.018) and that in biopsy samples before nCRT group (44.4%, 20/45)(χ(2)=7.287, P=0.007). Twenty-two cases (36.1%, 22/61) in nCRT group had TRG score of 3-4 . Among these 22 cases, 11 cases were negative CD133, and constituted 61.1% (11/18) of all CD133-low expression cases in nCRT group, whereas the other 11 cases were positive CD133, and constituted 25.6%(11/43) of all CD133-high expression cases in nCRT group (χ(2)=6.974, P=0.008). The CD133 expression up-regulates markedly in rectal cancer after nCRT and nCRT may have potential positive modulation on CD133 expression. CD133-positive cancer reveals lower response to nCRT, suggesting CD133 may be a potential target for improving efficacy of nCRT in rectal cancer.

Prognostic Significance of Tumor Regression Grade after Preoperative Chemoradiotherapy for Rectal Cancer

The relationship of pathologic tumor regression grade (TRG) and outcomes after preoperative therapy in rectal cancer

Purpose: Pre-operative chemoradiation therapy (CRT) is standard treatment in clinical stage T3/4 or node positive rectal cancer, however, there are no established biomarker that can predict pathological response and clinical outcome to CRT. The aim of this study was to evaluate the correlation between expression of molecular markers and pathological response and clinical outcomes in patients with rectal cancer who received 5-FU based chemoradiotherapy. Experiment design: Immunohistochemical stain was performed in tissue arrays constructed from core tissue specimens taken before treatment and from operative specimens from 120 patients who received 5-FU based pre-operative CRT and surgery between June 2003 and Dec. 2008. Expression of Ki67, TS, BAX, EpCAM, p53, p21, EGFR, CD44, CD133, CD166, HIF1a and ALDH1 were assessed and correlated with tumor regression grades and disease free survival. Results Of 120 patients (M/F 77/43, median age:63), 23 (19.2%) patients achieved pathologic complete remission; Dworak grade 1: 18 (15%), grade 2: 51 (42.5%); Grade 3: 28 (23.3%), and Grade 4: 23 (19.2%). In the analysis of association between marker expressions and tumor regression grades, low p21 expression at pretreatment biopsy was significantly associated with grade 4 (total regression) (p=0.039) and better disease free survival (5yr DFS rate - low vs high p21: 89% vs 54%, p=0.001). In the multivariate analysis, low p21 expression level in pre-treatment biopsy was significantly associated with better DFS (p=0.003, HR 0.20; 95% CI 0.72, 0.57) and the score difference of p21 expression between pre- and post treatment tissue was also significant (p=0.002, HR 0.09; 95% CI 0.02, 0.39). Low CD166 expression level at pretreatment biopsy was associated with better DFS (p=0.003; HR 0.19; 95% CI 0.07, 0.58). Low ypN stage and high tumor regression grade also have significantly a good effect on DFS as well (p= 0.001, 0.018 respectively). Conclusion Our results showed low p21 and CD166 expression at pretreatment biopsy was associated with tumor regression and good prognosis in patients treated with 5-FU based chemoradioatherapy. Larger, prospective trials and functional studies are warranted to determine the role of p21 and CD166 as predictive biomarker of response to chemoradiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4525. doi:1538-7445.AM2012-4525

To date, there is no uniform consensus on whether tumour regression grade (TRG) is predictive of outcome in rectal cancer. Furthermore, the lack of standardization of TRG grading is a major source of variability in published studies. The aim of this study was to evaluate the prognostic impact of TRG in a cohort of patients with locally advanced rectal cancer treated with neoadjuvant chemoradiation therapy (CRT). In addition to the Mandard TRG, we utilized four TRG systems modified from the Mandard TRG system and applied them to the cohort to assess which TRG system is most informative. One-hundred and fifty-three patients with a T3/T4 and/or a node-positive rectal cancer underwent neoadjuvant 5-fluorouracil-based CRT followed by surgical resection. Thirty-six (23.5%) patients achieving complete pathological response (ypCR) had a 5-year disease-free survival (DFS) rate of 100% compared with a DFS rate of 74% for 117 (76.5%) patients without ypCR (P=0.003). The Royal College of Pathologists (RCPath) TRG best condenses the Mandard five-point TRG by stratifying patients into three groups with distinct 5-year DFS rates of 100%, 86% and 67%, respectively (P=0.001). In multivariate analysis, pathological nodal status and circumferential resection margin (CRM) status, but not TRG, remained significant predictors of DFS (P=0.002, P=0.035 and P= 0.310, respectively). Our findings support the notion that ypCR status, nodal status after neoadjuvant CRT and CRM status, but not TRG, are predictors of long-term survival in patients with locally advanced rectal cancer.

Treatment: the role of contact X-ray brachytherapy (Papillon) in the management of early rectal cancer.

e15534 Background: Preoperative chemoradiotherapy (CRT) is a standard treatment for locally advanced rectal cancer (RC) patients (pts). Despite the benefits of CRT, its use in non-responder pts can be associated with increased toxicities and resection delay. The identification of CRT response biomarkers, such as long non-coding RNAs (lncRNA), could improve the management of these pts. LincRNA-p21 is a lncRNA involved in the p53 pathway and angiogenesis regulation that acts as prognostic marker in several tumors. We aim to study lincRNA-p21 expression in pretreatment samples from RC pts treated with CRT to evaluate whether lincRNA-p21 can act as a predictive biomarker for CRT response. Methods: RNA from pretreatment endoscopy biopsies from 58 RC pts treated with preoperative CRT at Mutua Terrassa University Hospital were analyzed. Pathological response was classified according to the tumor regression grade (TRG) Dworak classification. LincRNA-p21 expression was determined by RTqPCR and was correlated with CRT response and, time to relapse (TTR), disease-free survival (DFS) and overall survival (OS). Results: Samples from 58 pts were analyzed. Most of them males (n = 40, 69%). Median age at diagnosis of 67 (44-82) years. Fifty (86.2%) pts reported stage III and, 42 (72.4%) pts assessed TRG 0-3 with a 70.7% of downstaging reported. LincRNA-p21 was upregulated in stage III tumors (p < 0.0001) and also in tumors with worst response to CRT regarding TRG, TGR0-3 (TRG0-3, n = 42 vs TRG4, n = 16, p = 0.019). In this line, the ROC curve analysis showed that lincRNA-p21 expression had capacity to distinguish pts with maximum response (TRG4) from others (AUC:0.7; p = 0.02). The binary logistic regression analysis validated its independence as response biomarker. LincRNA-p21 levels correlated with pts relapse after surgery. When the pts were classified in 3 groups, high, medium and low, according to lincRNA-p21 levels, RC pts with highest lincRNA-p21 expression had the shortest TTR. TTR for pts with high levels was 74.5 months (m) (95% CI:31-117), while it was 114 m (95% CI:95-133) for those with Medium levels and 123 m (95% CI:111-134) for those with low levels (p = 0.047). Conclusions: LincRNA-p21 is a marker of advanced disease and worse response to CRT. LincRNA-p21 may add valuable information for individualizing pre-operative CRT in locally advanced RC pts.