bluuebunny/crossref_metadata_2025 huggingface dataset:
AB1, a novel protein targeting TP53 mutated GI tumors.
Authors: Epenetos Agamemnon A. , Karagussova Karima , Deonarain Mahendra
Published: January 2022

96

Background: The tumor suppressor gene TP53 is one of the most frequently deleted or mutated genes in gastrointestinal cancers. Normal p53 regulates several important proteins that control cell cycle, cell death, DNA damage/repair, stemness, differentiation and other key cellular functions. If the TP53 gene is damaged, tumor suppression is severely compromised. On the other hand, and downstream of p53, p21 a potent cyclin-dependent kinase inhibitor (CKI) protein binds and inhibitsthe activity of cyclin - CDK2, - CDK1, and - CDK4/6, thus functioning as a regulator ofcell cycle progression at G 1 and S phase. It can act as de facto p53 repair/ replacement mechanism. We have thus hypothesised that, if we were able to deliver wild type p21 into all p53 mutated cancer cells, it would have a possible therapeutic effect. Methods: We have constructed recombinantly a new fusion protein, named AB1, composed of a cell penetrating protein (antennapedia) ANTP and wild type p21 and tested it in in vitro and in vivo preclinical models prior to clinical studies. AB1 could also be constructed semi-synthetically by conjugating recombinant ANTP chemically to p21 protein. Results: AB1 penetrated and killed p53 mutated cancer cells but did not kill cells that did not have p53 mutations AB1 penetrated but did not kill p53- or p21- wild-type cells. AB1 was not immunogenic in normal New Zealand White rabbits. AB1 was more cytotoxic when administered with conventionally-used chemotherapeutic agents. Conclusions: We have generated a selectively cytotoxic fusion protein against p53 mutated GI cancers which is effective when used as a single agent but more so when used in combination with chemotherapy. The phaseI/II clinical trial will include eligible patients who have p53 mutated GI cancers
Ezabenlimab (BI 754091), an anti-PD-1 antibody, in combination with BI 836880, a VEGF/Ang2-blocking nanobody, in patients (pts) with advanced colorectal cancer (CRC).
Authors: Ulahannan Susanna Varkey , Percent Ivor John , Arrowsmith Edward , Hussein Maen A. , Bhanderi Viralkumar K. , Hamm John , Durm Greg Andrew , Erzen Damijan , Mohanty Prasant , Spigel David R.
Published: January 2022

98

Background: Anti-PD-1 antibodies may have synergistic effects with other immunomodulatory or targeted agents. This open-label, Phase II platform trial is investigating ezabenlimab, an anti-PD-1 antibody, combined with other agents. Module C of the platform is assessing ezabenlimab plus BI 836880, a humanized bispecific nanobody targeting VEGF/Ang2. Pts are being enrolled into 5 advanced solid tumor cohorts: gastric/gastroesophageal adenocarcinoma; solid tumors (except non-squamous NSCLC or melanoma) with secondary resistance to anti-PD-(L)1 treatment (progression after at least SD for ≥4 months); solid tumors with primary resistance to anti-PD-(L)1 treatment; microsatellite stable (MSS) CRC; mismatch repair-proficient/MSS endometrial carcinoma. Here, we report data from the CRC cohort which has completed recruitment. Methods: Pts with locally advanced, unresectable or metastatic, MSS CRC were enrolled. Patients had received ≥1 line of prior systemic therapy for metastatic disease but were anti-PD-(L)-1 therapy-naïve. Prior anti-angiogenic therapy was permitted. Pts received BI 836880 720 mg plus ezabenlimab 240 mg iv q3w for 1 year or until disease progression, consent withdrawal or undue toxicity. Primary endpoint: investigator-assessed OR (CR or PR per RECIST v1.1). Secondary endpoints: duration of response, disease control, and PFS; safety is also being assessed. Results: 30 pts have been treated: 57% male; median age 61.5 years. All pts had received prior chemotherapy; most pts (23 [77%]) had received prior bevacizumab. At data cut-off (Sep 2021), median duration (range) of treatment was 115.5 (28–295) days; 6 pts remain on treatment. 1 (3%) pt (who had not received prior bevacizumab) achieved a confirmed PR; 16 (53%) pts had SD. Median duration (range) of SD was 128.5 (42–242) days. 29/17/2 (97/57/7%) pts had an AE (any/G3/G4). The most frequent AEs (any/G3) were nausea (40/10%), fatigue (30/3%), peripheral edema (30/0%), vomiting (27/7%), and hypertension (27/17%). There were two G4 AEs (hypertension; platelet count decreased) and no G5 AEs. 24/10/2 (80/33/7%) pts had a drug-related AE (any/G3/G4); most commonly (any/G3) nausea (33/7%), fatigue (27/3%) and hypertension (27/17%). 3 (10%) pts had an infusion-related reaction (G1, n = 1; G2, n = 2). 2 (7%) pts had an AE leading to discontinuation (G3 bile duct stone and G2 peripheral edema). Immune-related AEs were reported in 6 (20%) pts and serious AEs occurred in 13 (43%) pts. Conclusions: BI 836880 plus ezabenlimab had a manageable safety profile in pts with advanced MSS CRC; however, anti-tumor activity was limited in these pts, the majority of whom had received prior bevacizumab. Clinical trial information: NCT03697304.
QoL analysis: A randomized phase 3 study of sequential versus combination treatment in first-line chemotherapy for metastatic colorectal cancer—The C-cubed study.
Authors: Noura Shingo , Inada Ryo , Ojima Hitoshi , Nagasaka Takeshi , Tanioka Hiroaki , Munemoto Yoshinori , Shimada Yasuhiro , Ishibashi Keiichiro , Shindo Yoshiaki , Shimokawa Mototsugu , Mishima Hideyuki , Yamaguchi Yoshiyuki , Okajima Masazumi ,   The C-cubed study group
Published: January 2022

102

Background: The C-cubed (C3) study demonstrated a sequential approach start from ﬂuoropyrimidines (FP) plus bevacizumab (Bmab) followed by oxaliplatin (OX) adding significantly improved a median Treatment failure of strategy (TFS) for a combination approach start from FP+OX+Bmab [15.2 months vs. 7.6 months, HR, 0.475; 95% CI, 0.362 to 0.623; p < 0.0001] in first-line metastatic colorectal cancer (mCRC). In this congress, we focus on the quality of life (QOL) assessments as a pre-planned analysis. (Study information: UMIN000015405). Methods: The C3 study was a randomized phase III study which evaluated the time to discontinuation of OX-containing therapy (sequential approach [Capecitabine/5-FU (FP)+Bmab followed by OX-FP+Bmab] vs. combination approach [OX-FP+Bmab]. The primary endpoint was TFS and secondary endpoints were ORR, OS, PFS, Safety and QoL. QOL assessments included European Organisation for Research and Treatment of Cancer (EORTC) quality-of-life questionnaire for cancer (QLQ-C30) (EORTC QLQ C-30), EuroQol 5D 5L (EQ5D) and the Patient Neurotoxicity Questionnaire (PNQ) in both arm as a pre-planned analysis. Each questionnaire was collected at the time of enrolment, 6, 12, 18 months and end of treatment. QOL scores were compared using a mixed-effects models for repeated measures (MMRM). Results: A total 292 patients participated in QoL part (arm A: n =148; arm B: n =144). The returned questionnaire sheets were 292 (reply rate: 97%), 206 (68%), 199 (65%), 61 (20%) at baseline, 6, 12, 18 months, respectively. Sequential approach was statistically improved than combination approach as follows: Physical functioning (p<0.001), Cognitive functioning (p=0.012), Social functioning (p=0.0004), and Fatigue (p=0.013) in EORTC QLQ C-30. In addition, at 6 months (after which attrition in the combination arm was more than 50%) after randomization, the mean change rate from baseline of EQ5D score in the sequential approach versus combination approach were: –1.91 (SD 27.57) versus –9.62 (29.60). In contract, PNQ sensory score showed that sequential approach was not statistically improved for combination approach (0.22 [SD: 0.89] vs. 0.61 [SD: 0.95], p=0.115). Conclusions: The further clarification of patients’ characteristics are needed, but this sequential approach can be advocated as a valuable treatment option in first-line mCRC for current guideline based on these QoLs and main efficacy data.
Evaluating the technical feasibility of novel salvage endoscopic submucosal dissection after chemoradiotherapy for locally advanced rectal adenocarcinoma.
Authors: Leung Galen , Nishimura Makoto , Hingorani Neha , Lin I-Hsin , Schattner Mark
Published: January 2022

103

Background: According to the National Comprehensive Cancer Network guideline, locally advanced rectal cancer is treated with chemotherapy with or without radiation, followed by transabdominal resection. However, there has been an emerging role for nonoperative management after chemoradiotherapy, and there is significant morbidity and risks associated with surgical resection. Endoscopic submucosal dissection (ESD) after chemoradiotherapy (CRT) for rectal cancer, which is termed “salvage ESD,” may be a viable option for patients, in terms of evaluating pathological response and providing potential curative resection. We aim to evaluate the feasibility and safety of salvage ESD. Methods: Retrospective chart review of cases of salvage ESD after CRT for locally advanced rectal cancer (salvage group) and standard ESD cases for rectal tumors without prior CRT (standard group) from July 2018 to August 2020 at our institution was performed. Clinical factors, imaging, procedural, and pathology results were collected and compared between the two groups. Results: 12 salvage ESD cases were compared to 27 standard ESD cases. 83.3% of the lesions in the salvage ESD group were initially staged as T3 prior to CRT. 100% of the lesions in the salvage ESD group were scarred down, compared to 33.3% in the standard ESD group (p <0.01). Technical outcomes are shown in the table. The en bloc resection rates were 92.7% and 91.7% (p=1.00) and R0 resection rates were 66.7% and 75.0% (p=0.55) for the standard and salvage groups, respectively. In the salvage ESD group, no adverse events were observed. 66.7% of the adenocarcinomas in the salvage ESD group had morphologically changed to hyperplasia or adenoma after CRT and there were no identifiable lesions greater than T1 staging. Conclusions: Salvage ESD after CRT for locally advanced rectal cancer is technically feasible and with comparable low complication rates. There may be a diagnostic role in salvage ESD in assessing pathological response to CRT, as well as a possibly therapeutic role in resection of residual lesions with the potential to avoid surgery. Further studies are underway comparing the clinical outcomes of salvage ESD with standard surgical resection after CRT for locally advanced rectal cancer.[Table: see text]
Efficacy and safety of anlotinib plus XELOX regimen as first-line treatment followed by maintenance monotherapy of anlotinib for patients with mCRC: A single-arm, multicenter, phase II clinical trial—Update results from ALTER-C001.
Authors: Yan Jin , Han Yunwei , Zhang Li , Jin Yongdong , Sun Hao
Published: January 2022

104

Background: The combination of anti-VEGF or anti-EGFR targeted drugs with chemotherapy is the standard first-line therapy for metastatic colorectal cancer (mCRC), and the followed maintenance treatment is an optional approach to balance the efficacy and toxicity. However, studies regarding the maintenance strategies based on antiangiogenic TKIs are limited currently. Anlotinib, a novel oral multi-target TKI which can inhibit both tumor angiogenesis and tumor cell proliferation simultaneously, substantially prolonged the PFS with manageable toxicity for refractory mCRC in the phase III ALTER0703 clinical trial (NCT02332499). We present updated data from the study of anlotinib plus XELOX as first-line treatment followed by anlotinib monotherapy for mCRC. Methods: In this open label, single-arm, multicenter phase II clinical trial, 29 mCRC patients without prior systemic treated, aged 18-75 and an ECOG performance status of 0 or 1 were planned to recruit. Eligible patients received capecitabine (1000 mg/m2, po, d1-14, q3w) and oxaliplatin (130 mg/m2, iv, d1, q3w) plus anlotinib (10mg, po, d1̃14, q3w) treatment for 6 cycles. After 6 cycles of inducing therapy, patients would receive anlotinib (12mg, po, d1̃14, q3w) as maintenance therapy until disease progression or intolerable adverse events (AEs). The primary endpoint was PFS; Secondary endpoints included ORR, DCR, DOR and safety. Results: At updated analysis (cutoff: Sep 10, 2021), a total of 27 patients were enrolled, of which 19 patients were available for efficacy assessment. In best overall response assessment, there were 63.2% PR (12/19), 26.3% SD (5/19) and 10.5% PD (2/19). The ORR was 63.2% (95% CI, 38.4-83.7%) and DCR was 89.5% (95% CI, 66.9-98.7%). One patient experienced the longest duration of treatment which was 17.5 months. The median PFS was not reached. The most common treatment related adverse events (TRAEs) of any grade (≥25%) were leukopenia, nausea/vomiting, hypertension, neutropenia, hypohemoglobinemia, hypertriglyceridemia. Grade 3/4 TRAEs (≥10%) were neutropenia (14.8%), hypertension (14.8%) and hypertriglyceridemia (11.1%). One grade 5 TRAE was pancytopenia that occurred at 2.7 months. Conclusions: The update results suggested that anlotinib combined with XELOX as first line regimen followed by anlotinib monotherapy showed promising anti-tumor activity and manageable safety for patients with mCRC. And the conclusions needed to be confirmed in randomized studies. Clinical trial information: ChiCTR1900028417.
A phase II study of pembrolizumab, binimetinib, and bevacizumab in patients with microsatellite-stable, refractory, metastatic colorectal cancer (mCRC).
Authors: Friedrich Tyler , Blatchford Patrick Jud , Lentz Robert William , Davis S. Lindsey , Kim Sunnie S. , Leal Alexis Diane , Van De Voorde Zoe , Lee Matthew R , Waring Meredith , Cull Tiffany , Martin Anne , Eckhardt S. Gail , Messersmith Wells A. , Lieu Christopher Hanyoung
Published: January 2022

118

Background: To date, immune-checkpoint inhibition for microsatellite stable (MSS) mCRC has been ineffective, though targeted therapy combination strategies may be promising. This phase II, investigator-initiated trial (NCT03475004) was designed to evaluate the efficacy and safety of the three-drug combination of pembrolizumab (pembro), binimetinib, and bevacizumab in patients with advanced, MSS treatment-refractory colorectal cancer. Methods: Patients with mCRC locally determined to be MSS and whom have progressed on two prior lines of therapy were enrolled. Treatment consists of pembro (200 mg every 3 weeks), binimetinib (45 mg BID) and bevacizumab (7.5 mg/kg every 3 weeks) until disease progression or unacceptable toxicity. The primary endpoint is PFS using RECIST v1.1 by investigator review. Additional endpoints include objective response rate, disease control rate at time of first re-staging (2 mo), duration of response, and safety and tolerability. Results: 50 patients have been enrolled (accrual is completed). 53% of patients are male and the mean age is 53.6 (range 31-79). The mean number of prior therapies is 5.3. At the time of preliminary data review, 39 patients are evaluable for response data. The median PFS was 5.8 mo (95% CI 4.2 to 8.9). The objective response rate was 13% with 5 partial responses. 24 patients (62%) had stable disease and 10 (26%) had progressive disease as the best response. The disease control rate at the time of first re-staging was 74%. Median duration of response was 6.5 mo. 19 (40%) patients experienced serious adverse events; the most common grade ≥3 adverse events included transaminase elevation (15%), diarrhea (11%), acneiform rash (9%), hypertension (9%), and anemia (9%). Conclusions: Preliminary results from this phase II study indicate that this regimen of pembrolizumab, binimetinib, and bevacizumab has promising activity and acceptable tolerability in this heavily pre-treated population of patients with MSS metastatic colorectal cancer. Final results will be presented as well as ongoing correlative studies. Clinical trial information: NCT03475004.
Tumor volume regression of rectal cancer in daily MRI during preoperative chemoradiotherapy with capecitabine.
Authors: Kwak Yoo-Kang , Lee Sea-Won , Jung Yoon Ju
Published: January 2022

123

Background: Although cone beam computed tomography (CBCT) has been available for over a decade, the pattern of tumor volume change in rectal dancer during preoperative concurrent chemoradiotherapy (CCRT) has been under the veil due to poor soft tissue contrast of registration CBCT. This study was conducted to investigate daily rectal tumor volume change using registration magnetic resonance imaging (MRI). Methods: Patients diagnosed with cT3-4 and/or cN+ rectal adenocarcinoma undergoing preoperative CCRT with capecitabine on the pelvis up to 50 Gy in 25 daily fractions from November 2018 to June 2019 were consecutively included. Rectal tumor volume was uniformly measured by single physician (YKK) in daily 0.35T MRI obtained with ViewRay MRIdian Linac (ViewRay Inc., Oakwood, USA). The mean +/- standard deviation (SD) of daily tumor volume (cc), difference of tumor volume between first fraction and daily fraction (daily tumor volume – baseline tumor volume at first fraction), and tumor volume reduction rate (%, (daily tumor volume – baseline tumor volume at first fraction)/baseline tumor volume at first fraction x 100) were calculated. Statistical significance of differences were tested using the Wilcoxon’s signed rank test and paired t-test. Results: Thirteen patients were included. Median age was 65 and majority of the patients were male (92.3%). Most tumors were T3 (76.9%) and N1-2 (92.3%). Tumors were located median 6 cm (range: 2.4 – 9) above anal verge. Median follow-up was 27.3 months (range: 11.7 – 30.6) and 2 patients had recurred at the time of analysis (1 local and 1 distant). Tumor volume steadily regressed with daily administration of CCRT (mean 2.06 +/-1.73 cc; Figure 1). Significant tumor volume reduction compared to baseline was observed from fourth fraction (mean -12.37 +/- 16.72 cc; P < 0.0005). The difference of tumor volume between baseline and the last fraction was mean -49.33 +/- 68.13 cc ( P < 0.0002). The tumor volume reduction rate was significantly increased since fourth fraction (mean - 17.2 +/- 11.78 %; P < 0.0002). The tumor volume reduction rate at the last fraction was - 70.62 +/- 14.01 % ( P < 0.0001). Conclusions: For the first time, this study demonstrated daily tumor volume regression in preoperative rectal CCRT with capecitabine using daily MRI. Steady pattern of tumor regression may be explained in part by daily administration of capecitabine. Based on the hypothesis-generating observation, this study may warrant initiation of further investigations such as analysis and comparison with daily tumor volume regression with CCRT using leucovorin and 5-FU.
Clinical significance and biomarker potential of MGMT protein measurement in colorectal cancer.
Authors: Zhang Janie Yue , Cecchini Michael , Desai Shruti S. , Schalper Kurt A.
Published: January 2022

136

Background: O6-methylguanine DNA methyltransferase (MGMT) is the principal repair mechanism of DNA damage created by alkylating agents. Approximately 40% of colorectal cancers (CRC) exhibit MGMT promoter hypermethylation, expected to result in gene silencing. The role of MGMT as a biomarker is poorly understood, and quantitative measurement of MGMT protein has not been performed in CRC. We hypothesized that altered tumor MGMT would affect DNA damage response, modulate adaptive anti-tumor immune responses, and influence survival in CRC. Methods: We used multiplexed quantitative immunofluorescence (QIF) to study 4 clinically annotated retrospective patient cohorts treated from 2000 to 2017 at Yale, consisting of 400 formalin-fixed paraffin-embedded CRC cases represented in tissue microarray format. These included paired tumors and adjacent non-tumor colonic mucosa (n = 112 pairs in Cohort 1), paired primary CRC and lymph node metastases (n = 31 pairs in Cohort 2), and 2 cohorts with primary stage I-IV tumors (n = 250 in Cohort 3; n = 150 in Cohort 4). We established a QIF panel for simultaneous, localized measurement of DAPI (all cells), cytokeratin (CK; tumor epithelial cells), MGMT, γH2AX (DNA damage response), and CD8. The measurement of fluorescent signals in CK-positive tumor cells or CK-negative stromal cells was achieved using co-localization strategies and the AQUA QIF platform. Results: We identified lower MGMT protein levels in CRC cells than in non-tumor colonic epithelium (p < 0.0001), and in lymph node metastases compared to paired primary CRC tumors (p = 0.0411). Using the visual detection threshold, tumor selective MGMT protein downregulation was identified in 20% of cases in Cohorts 3 and 4. Microsatellite instability-high (MSI-H) cases showed decreased tumoral MGMT protein compared to microsatellite stable (MSS) cases (p = 0.002). In Cohorts 3 and 4, low tumoral MGMT was consistently associated with increased CD8+ tumor infiltrating lymphocytes (p = 0.0472; p = 0.0002). The association between MGMT and γH2AX was inconsistent. The association between MGMT and CD8 was significant only in MSS cases (p = 0.0001) but not in MSI-H cases (p = 0.0979), supporting that the effect is not driven by MSI-H status. Tumor-selective MGMT downregulation evidenced by a low tumor-to-stroma ratio was associated with improved progression-free survival and overall survival in both Cohorts 3 and 4, but statistical significance was only seen in Cohort 3. Conclusions: MGMT protein downregulation occurs in 20% of CRCs and is associated with increased adaptive anti-tumor immune responses, mismatch repair (MMR) deficiency and better prognosis. MGMT deficiency may alter DNA repair in tumor cells and mediate the accumulation of antigenic mutations or neopeptides, independent from MMR status. Our results support a biomarker role of MGMT protein and suggest a role for immunotherapy combinations in MGMT deficient tumors.
Clinical significance of Bacteroides fragilis as potential prognostic factor in colorectal cancer patients.
Authors: Matsumiya Yuriko , Suenaga Mitsukuni , Ishikawa Toshiaki , Hanaoka Marie , Iwata Noriko , Masuda Taiki , Yamauchi Shinichi , Tokunaga Masanori , Kinugasa Yusuke
Published: January 2022

137

Background: Bacteroides fragilis ( B. fragilis) is an obligate anaerobe and generally acts as anti-inflammatory manner on the intestinal tract. Enterotoxigenic Bacteroides fragilis (ETBF), a subtype of B. fragilis, produces Bacteroides fragilis toxin (BFT) leading to either asymptomatic chronic colonic inflammation or colonic tumor formation. However, the impact of B. fragilis and ETBF on colorectal cancer (CRC) prognoses still remains unclear. We tested whether the presence of B. fragilis and ETBF affect clinical outcome in CRC patients who underwent curative surgery. Methods: We obtained 197 pairs of matched FFPE samples from colorectal cancerous and adjusted non-cancerous tissues of patients with stage II and III CRC who underwent curative resection between 2014 and 2016. Quantitative analyses of B. fragilis and ETBF in the colon tissues were performed using quantitative PCR with primers, 16S rRNA for B. fragilis and bft DNA, respectively. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using Kaplan-Meier curves, log-rank test, and Cox proportional hazards regression. Results: Among 197 patients, 16S rRNA for B. fragilis DNA and bft DNA were detected in 120 patients (60.9%) and 12 patients (6.1%), respectively. B. fragilis-positive patients had better RFS (5-y RFS rate: 81.4% vs. 73.4%, HR0.59, 95% CI: 0.31-1.12, p =0.10) and OS (5-y OS rate: 88.9% vs 78.3%, HR0.53, 95% CI: 0.26-1.11, p =0.091) compared with B. fragilis-negative patients though statistically not significant. In multivariable analysis for RFS, B. fragilis-positive remained as an independent prognostic factor (HR0.53, 95% CI: 0.28-0.99, p =0.049) along with tumor depth T4 and Stage III, while there was no significance in OS. No significant differences were observed between ETBF and nontoxigenic B. fragilis in patients' characteristics and clinical outcomes. Conclusions: Our findings suggest that the presence of B. fragilis may predict outcome especially RFS in patients with curatively resected stage II and III CRC. Further research are warranted to explore whether B. fragilis status could be involved in a novel prediction model for outcome in early-stage CRC and develop probiotics treatments to prevent recurrence.
Deep learning analysis of the adipose tissue and the prediction of prognosis in colorectal cancer.
Authors: Lin Anqi , Zhang Jian , Luo Peng
Published: January 2022

139

Background: Colorectal cancer (CRC) is the third most common type of cancer and has a poor prognosis and high recurrence rate. Research has shown that the lipid microenvironment surrounding tumors is closely associated with the occurrence, development, and metastasis of CRC. Recently, advances in artificial intelligence have greatly improved the accuracy of models for CRC prognosis and survival analysis. Methods: According to pathological images from the National Center for Tumor diseases(NCT), the University Medical Center Mannheim(UMM) database and the ImageNet data set, a model called VGG19 was pre-trained. A deep convolutional neural network(CNN), VGG19CRC, was trained by the migration learning method. According to the VGG19CRC model, adipose tissue scores were calculated for TCGA-CRC hematoxylin and eosin(H&E) images and images from patients at Zhujiang Hospital of Southern Medical University and First People’s Hospital of Chenzhou. Kaplan-Meier(KM) analysis was used to compare the overall survival(OS) of patients. The XCell and MCP-Counter algorithms were used to evaluate the immune cell scores of the patients. Gene set enrichment analysis(GSEA) and single-sample GSEA(ssGSEA) were used to analyze upregulated and downregulated pathways. Results: In TCGA-CRC, patients with high-adipocytes(high-ADI) CRC had significantly shorter OS times than those with low-ADI CRC. In a validation queue from Zhujiang Hospital of Southern Medical University(Local-CRC1), patients with high-ADI had worse OS than CRC patients with low-ADI. In another validation queue from First People's Hospital of Chenzhou(Local-CRC2), patients with low-ADI CRC had significantly longer OS than patients with high-ADI CRC. In subgroup analysis, ADI could be used as a prognostic marker for patients with colon adenocarcinoma(COAD) and rectum adenocarcinoma(READ), as well as male and female CRC patients. Among these subgroups, patients with lower ADI also had significantly improved OS. Compared with the low-ADI group, high-ADI patients had significantly decreased CD8+ T cells, T cells,and monocytes in the tumor immune microenvironment(TIME), while M2 macrophages were significantly increased. According to the GSEA and ssGSEA analyses, pathways mediating anti-tumor immunity were significantly downregulated in the high-ADI group, while some oncogenic signaling pathways were significantly upregulated. Conclusions: We developed a deep convolution network to segment various tissues from pathological H&E images of CRC and automatically quantify ADI. This allowed us to further analyze and predict the survival of CRC patients according to information from their segmented pathological tissue images, such as tissue components and the tumor microenvironment. Furthermore, we found that ADI may also predict OS in CRC patients and among the subgroups.
The role of germline polymorphisms in genes involved in the antioxidant system to predict the efficacy of cetuximab for patients with metastatic colorectal cancer (mCRC) enrolled in FIRE-3 trial.
Authors: Arai Hiroyuki , Millstein Joshua , Stintzing Sebastian , Wang Jingyuan , Battaglin Francesca , Kawanishi Natsuko , Jayachandran Priya , Soni Shivani , Wu Zhang , Heinemann Volker , Lenz Heinz-Josef
Published: January 2022

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Background: Reactive oxygen species activate RAS/MAPK signaling either through inactivation of phosphatases or by direct oxidation of kinases. We hypothesized that functional genetic variants in genes involved in the antioxidant system may be associated with the efficacy of cetuximab in mCRC patients. Methods: We analyzed genomic and clinical data from FIRE-3, a phase III trial which compared cetuximab and bevacizumab in combination with FOLFIRI in untreated mCRC patients. Genomic DNA extracted from blood samples was genotyped using an OncoArray (Illumina, Inc., San Diego, CA, USA). Candidate 13 functional single nucleotide polymorphisms (SNPs) ( TXN rs2301242, TXN rs2301241, TXN2 rs4821494, TXN2 rs9619611, TXN2 rs59841625, CAT rs7943316, CAT rs564250, CAT rs11604331, CAT rs1001179, CAT rs769217, GPX4 rs757229, GPX4 rs4807542, and GPX4 rs713041) were tested for association with progression-free survival and overall survival (OS), using log-rank test and Cox proportional hazards model. To confirm the predictive value, the treatment-by-SNP interaction was tested. Results: A total of 236 patients were available for the SNP analyses (cetuximab arm, n = 129; bevacizumab arm, n = 107). In the cetuximab arm, two SNPs were significantly associated with clinical outcomes in univariate analyses: TXN2 rs4821494 (T/T vs any G allele, hazard ratio [HR] on OS = 2.17, 95% confidence interval [CI] = 1.04–4.56, log-rank p = 0.04) and GPX4 rs4807542 (G/G vs any A allele, HR on OS = 2.04, 95% CI = 1.05–3.98, log-rank p = 0.03). Multivariate analysis confirmed the significance in TXN2 rs4821494 (T/T vs any G allele, HR on OS = 2.47, 95% CI = 1.06–5.72, p = 0.03). In the bevacizumab arm, univariate analyses did not detect any significant associations between the SNPs and clinical outcomes. Treatment-by-SNP interaction test showed the significant predictive value of TXN2 rs4821494 in terms of OS ( p = 0.03). Conclusions: TXN2 rs4821494 involved in the antioxidant system may predict the efficacy of cetuximab, in comparing with bevacizumab, in the first-line treatment of mCRC. Our novel findings warrant further validation studies.
Encorafenib, cetuximab, and cytotoxic chemotherapy combinations in BRAFV600E CRC murine models.
Authors: Woods Melanie Nicole , De Falco Vincenzo , Prisco Claudia , Martini Giulia , Guerrera Luigi Pio , Belli Valentina , Troiani Teresa , Morris Van K. , Shen John Paul Y.C. , Lee Hey Min , Villarreal Oscar , Sorokin Alexey , Kanikarla Marie Preeti , Ciardiello Fortunato , Kopetz Scott , Napolitano Stefania
Published: January 2022

145

Background: Based on promising results from latest trials, a crucial point is to evaluate whether encorafenib (E) plus cetuximab (C), alone or in combination with chemotherapy, can improve clinical outcomes relative to current standard of care in previously untreated BRAFV600E mutant mCRC. Considering the high number of BRAFV600E mutant mCRC patients who will never receive a second-line treatment, the rationale of this strategy is to maximize treatment outcome within the first-line setting. Methods: We performed an in vivo study using human BRAFV600E CRC cell line-derived xenografts in nude mice. We evaluated the efficacy of encorafenib (E) + cetuximab (C), FOLFOX, and FOLFIRI, both as individual regimens and in combinations. Mice were treated for 3 weeks and followed for an additional 8 weeks to evaluate durability of tumor control. Additionally, we validated our findings using 3 BRAFV600E mutated patient derived xenografts. Tumors progressing on single agent and combined treatment were profiled by RNA sequencing, protein extraction for RPPA/Western blot, and establishment of in vitro primary cell cultures for further analyses. Results: Our study showed across all 4 models both FOLFOX and FOLFIRI, each in combination with encorafenib plus cetuximab, having greater efficacy than encorafenib plus cetuximab or either chemotherapy alone. No significant change in toxicity was seen with the addition of chemotherapy. Interestingly, in the one model with long term treatment, FOLFOX + E +/- C performed greatest over the long-term, with significant endpoint separation against all other treatment arms (P < 0.05). Conclusions: Taken together, results from our study suggest that the addition of chemotherapy to BRAF+EGFR targeted therapy can further increase the magnitude of response in BRAFV600E mCRC and is a promising combination now being explored clinically. Additionally, this research will substantially contribute to our understanding of the genetic and molecular bases of resistance to target therapies and chemo-based approach in BRAFV600Econtext.
Gene expression-based personalized prescription of targeted drugs in colorectal cancer.
Authors: Poddubskaya Elena , Sorokin Maxim , Zolotovskaya Marianna , Suntsova Maria , Garazha Andrew , Moisseev Alexey , Bondarenko Alexey , Skrijalin Vasiliy , Baranova Madina , Sekacheva Marina , Seryakov Alexander , Buzdin Anton
Published: January 2022

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Background: Colorectal cancer (CRC) is the fourth most common cancer worldwide with relatively poor patient survival. Transcriptome assay could be used to personalize CRC treatment thus complementing standard mutation analysis. Methods: We performed retrospective hybrid experimental and meta-analysis of CRC patient gene expression data with available progression-free survival (PFS) information and/or targeted drug response status. In total we analyzed 243 gene expression profiles from four publicly available (TCGA and three datasets from Gene Expression Omnibus GSE19860, GSE19862, GSE104645), and one experimental (PRJNA663280) patient cohorts. Each gene expression profile was analyzed using bioinformatic second-opinion platform Oncobox to calculate balanced drug efficiency scores (BES) to build personalized ratings of potentially effective targeted drugs. Area under the ROC curve (AUC) metric and Cox regression analysis were used to assess Oncobox capacity to predict tumor response and PFS, respectively. Results: Patients from GSE19860 (n = 12), GSE19862 (n = 14), GSE104645 (n = 81) received bevacizumab as monotherapy or in combination with chemotherapy as the nearest line of treatment after biopsy collection. Oncobox correctly classified treatment responders vs non-responders with AUC 0.94, 0.90 and 0.84, respectively. BES value was strongly associated with PFS (HR = 0.53, CI 0.33-0.84, log-rank test p-value 0.0057) in the GSE104645 cohort. However, BES was ineffective for predicting response and PFS after second-line (after biopsy collection) treatment with cetuximab. BES also predicted treatment response with AUC 0.74 in the TCGA cohort (n = 17) treated with 4 different targeted drugs. Thirty clinical outcomes were collected for 14 patients from our experimental cohort PRJNA663280. Patients were treated with 10 different targeted drugs. BES was an effective biomarker that could predict treatment outcomes with AUC 0.74 for all lines of therapy and 0.94 for the first line therapy (after biopsy), and could predict PFS after first-line treatment (HR 0.14, CI 0.027-0.73, log-rank test p-value 0.0091). Conclusions: Our results suggest that RNA profiling in tumor samples may be helpful for personalizing prescriptions of targeted therapeutics in CRC. Using recent biopsies is essential to obtain robust estimates of targeted drugs efficacy.
Transcriptomic profiling to identify subsets of immune hot locally advanced rectal adenocarcinomas with favorable outcomes after neoadjuvant treatment.
Authors: Sanchez Vega Francisco , Chatila Walid Khaled , Kim Jin Ki , Walch Henry S. , Marco Michael , Chen Ching-Tung , Wu Fan , Khalil Danny , Ganesh Karuna , Qu Xuan , Luthra Anisha , Choi Seo-Hyun , Ho Yu-jui , Omer Dana , Shia Jinru , Romesser Paul Bernard , Schultz Nikolaus , Yaeger Rona , Smith Jesse Joshua , Garcia-Aguilar Julio
Published: January 2022

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Background: Understanding the role of the tumor microenvironment in the response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II-III) can lead to the identification of novel immunologic biomarkers to preselect patients who can avoid surgery and benefit from watch-and-wait strategies. Methods: We performed DNA and RNA sequencing of pre-treatment biopsies from 89 LARC patients who received neoadjuvant therapy, including 5 microsatellite unstable (MSI) and 84 microsatellite stable (MSS) patients. We computed single-sample gene set enrichment analysis (ssGSEA) scores for immune infiltrates and signaling pathways implicated in tumor progression. Immunofluorescence and hematoxylin-eosin staining of tumor slides was performed to confirm significant correlations with RNA-Seq estimates of immune markers. Other genomic variables were also included in the analysis, such as tumor mutational burden (TMB), fraction of genome altered by copy number changes, whole genome duplication events and somatic mutations in rectal cancer driver genes and pathways. Results were largely replicated using an independent cohort of 42 LARC samples with publicly available data from The Cancer Genome Atlas (TCGA). Results: Since MSI tumors are known to have a distinct immunologic profile, we separated them into their own group and performed unsupervised hierarchical clustering on the MSS tumors. We identified a set of immune hot MSS tumors (n = 7) with extensive immune infiltration. These tumors had low TMB and were predominantly classified as CMS4 (5/7). None of the 12 patients in the combined MSI and immune hot MSS groups recurred during the length of our study and they had response rates > 50% (vs. < 25% in the rest of MSS patients). MSI and immune hot MSS tumors had lower frequency of TP53 and APC mutations, and they exhibited increased levels of T cell infiltration. In particular, we observed overexpression of markers for Th1 cells, which produce inflammatory cytokines (e.g., IFN-gamma) and are associated with antitumor immunity. Genes encoding protein targets of immune checkpoint blockade, such as PDCD1 (PD-1), CD274 (PD-L1), CTLA4, HAVCR2 (TIM3) and LAG3, were also overexpressed in the immune hot MSS and - to a lesser extent – the MSI tumors, suggesting that these patients might benefit from the use of immune checkpoint inhibitors. Conclusions: Our results uncover a unique LARC tumor immune profile evident in the pre-treatment setting that could be used to better prognosticate rectal cancer patients and develop novel therapeutic strategies.
CLK2 upregulation and outcomes for males with colorectal adenocarcinoma.
Authors: Siddiqui Zain I. , Sun Chenyu , Tuason John Pocholo W. , Lising Jannell F. , Paudel Arpana , Ahmed Marwan K. , Kim Na Hyun , Bhan Chandur , Memon Burhan , Mohammed Abdul Humaed , Prasad Apurwa
Published: January 2022

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Background: Colorectal adenocarcinoma (COAD) is a common cancer in gastrointestinal tract. CDC-like kinase (CLK) family, containing four characterized isoforms (CLK1-4), have been reported for their roles in precursor-mRNA splicing. However, studies on their roles in COAD are limited. Thus, this study was performed to explore the roles of CLK family members in COAD. Methods: RNA-sequencing FPKM data and corresponding clinical information of 41 normal tissues and 480 tumor tissues of COAD were obtained from The Cancer Genome Atlas (TCGA). Then expression differences of CLK family between the normal tissues and COAD cancer tissues were compared with Wilcoxon rank-sum test via ‘limma’ package. Overall survival (OS) of upregulated CLK family members was analyzed by Kaplan–Meier (K–M) method via ‘survminer’ package. Subgroup analyses of different genders were also conducted. The best discriminate cut-off point between the high and low expression groups was assessed by the receiver operating characteristic (ROC) curve and area under the curve (AUC) values for upregulated CLK family members via ‘pROC’ package to assess their diagnostic values. Results: The expression of CLK1 and CLK2 were upregulated in tumors compared with normal tissues (p < 0.001), while CLK3 were downregulated in tumor tissues (p = 0.02) and no statically significant difference was found regarding CLK4 (p = 0.25). Higher expression of CLK1 was not associated with a change of OS (HR: 1.24, 95%CI: 0.84-1.83, p = 0.279), while higher expression of CLK2 is associated with worse OS (HR: 2.03, 95%CI: 1.34-3.06, p = 0.001). Subgroup analysis found that higher expression of CLK2 was associated with worse OS (HR: 2.28, 95%CI 1.27-4.08, p = 0.006) in males, but not in females (HR: 1.72, 95%CI 0.95-3.10, P = 0.072). Further analysis of ROC curve shown that AUC of CLK1 and CLK2 were 0.748 (0.687-0.809) and 0.884 (0.840-0.928), respectively. Conclusions: CLK1 and CLK2 are upregulated in COAD tumor samples, and higher expression of CLK2 in male COAD patients is associated worse OS. This demonstrated the potential therapeutic value of CLK2 in male patients with COAD. ROC curve indicated the potential diagnostic value of CLK1 and CLK2.
Plasma-informed minimal residual disease (MRD) assay: A multicenter prospective study in Japanese patients with stage II colorectal cancer.
Authors: Oki Eiji , Ando Koji , Hisamatsu Yuichi , Nakanishi Ryota , Nakaji Yu , Kudou Kensuke , Kusumoto Tetsuya , Kagawa Yoshinori , Ung Weng Chit , Niiro Hayato , Tada Sachiyo , Hirose Takashi
Published: January 2022

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Background: ̃80% of stage II colorectal cancer (CRC) can be cured by surgery alone. However, adjuvant chemotherapy is recommended for patients with high risk features such as bowel obstruction, < 12 lymph nodes examined and T4 tumors. Traditional pathological grading and biomarkers such as carcinoembryonic antigen has limited sensitivity. Several reports indicated circulating tumor DNA (ctDNA) may represent a promising prognostic factor to assess MRD as a factor for prediction of recurrence after surgery. Here, we present a proof-of-concept study for the development of a novel plasma-based highly sensitive Next Generation Sequencing (NGS) panel using SafeSEQ technology in operable CRC Japanese patients. Methods: This multicenter prospective study recruited patients diagnosed as operable clinical stage II CRC (n = 46) with pre- and post- (4̃6 weeks) operative plasma samples collected between Nov, 2019 and Jan, 2021. ctDNA were extracted and a 14-gene NGS panel was used to analyze single nucleotide variants (SNVs) and Indels covered by gene-specific amplicons. MRD variant was defined as same variant detected in both pre- and post- operative plasma samples. Tissue NGS by a 500-gene panel was also performed in a small number of tissue samples (n = 5) to compare the concordance of plasma and tissue variants. Results: Pre- and post-operative ctDNA status of 46 patients were analyzed. ctDNA positive was observed in 69.6% (32/46, 95%CI 55.2, 81.0) pre- and 34.8% (16/46, 95%CI 22.7, 49.3) post- samples. AKT1, CTNNB1, NRAS, POLE and PPP2R1A mutation were not detected in this study. TP53 mutation was most frequently detected in both pre- (22/46) and post- (11/46) samples, whereas APC mutation was ranked 2nd in pre- (15/46) but none in post- samples. A combined 96 variants were detected in all samples, in which 76 of them were < 0.5% mutant allele frequency (MAF). MRD variants were detected in 17.4% (8/46, 95%CI 8.82, 30.99) post- samples. Evaluation of positive percentage agreement between tissue and pre- plasma samples in three patients show that a total 7 variants detected in plasma, and 3 of them were detected in tissue samples. Conclusions: This study assesses the feasibility of a plasma-informed NGS panel by evaluating pre- and post- operative plasma samples. The presence of variants with < 0.5% MAF detected in this study indicate a highly sensitive method is required for accurate MRD detection. Further observation is required to explore the relationship between MRD variant and clinical outcome such as 2-year progression-free survival.
Spatially resolved transcriptomics deconvolutes histological prognostic subgroups in patients with colorectal cancer and synchronous liver metastases.
Authors: Wood Colin , Leslie Holly , Legrini Assya , Melissourgou-Syka Lydia , Pennel Kathryn AF , Edwards Joanne , Steele Colin William , Jamieson Nigel Balfour
Published: January 2022

165

Background: Up to 50% of patients with Colorectal Cancer (CRC) will metastasise to the liver (CRLM). KRAS-mt liver metastases particularly when co-mutated with TP53 are associated with poor prognosis. We have used the Glasgow Microenvironment Score (GMS), a histological score performed on H+E slides utilising immune and stromal components of the microenvironment to robustly stratify outcome in primary CRC. The aim of the current study was firstly to determine the utility of GMS in metastatic CRC and secondly to employ the NanostringTM GeoMx Digital Spatial Profiler (DSP), a state-of-the art analysis platform enabling spatial transcriptomic characterisation while maintaining tumour microenvironment (TME) topographical features, to interrogate the functional biology underlying the GMS. Methods: FFPE specimens from primary and metastatic lesions from 44 patients undergoing synchronous resection of CRLM underwent GMS, IHC and panel genomic assessment. Primary endpoints were recurrence-free survival (RFS) and cancer-specific survival (CSS). In addition to bulk transcriptomic assessment, 4 matched pairs from the cohort were selected for GeoMx analysis: 2 samples were GMS0 (high-immune) and 2 were GMS1 (low-immune) with an equal distribution of KRASmt and wt. After multiplex IF staining (PanCK, CD45, DAPI, αSMA), 48 regions of interest were selected and Cancer Transcriptome Atlas Transcriptomic outputs (2000 genes) were analysed using Pathway enrichment analysis with immune deconvolution of the transcriptome performed. Results: GMS0 (high-immune) was associated with improved RFS (p=0.0048) and CSS (p=0.0012) remaining an independent predictor of survival on multivariate analysis (HR 2.90, 95% C.I 1.18-7.16 P=0.021). GMS0 lesions were enriched for adaptive immune (NES=2.20 p.adj<0.0005) and IL-10 (NES=1.9 p.adj<0.0005) pathways specifically at the invasive edge. In contrast, a poor prognostic KRAS/TP53 lesion demonstrated profound immunosuppression, upregulated NOTCH signalling (NES=2.13 p.adj<0.0005) and neutrophil degranulation (NES=1.99 p.adj<0.0005). Topographical Immune-cell deconvolution demonstrated significantly higher populations of CD4 (p=0.05) and CD8 (p=0.0003) cells in GMS0 leading edges. Conclusions: We have demonstrated that spatial transcriptomic analysis using the Nanostring GeoMx tool can reveal potential novel mechanisms underlying biologically relevant histological and mutational subgroups (KRAS-mt) of CRC, providing potential therapeutic targets requiring further investigation. Future studies will apply this technology to pre and post treatment biopsy samples.