Targeting circadian transcriptional programs in triple negative breast cancer through a cis-regulatory mechanism.

Circadian clock genes are emerging targets in many types of cancer, but their mechanistic contributions to tumor progression are still largely unknown. This makes it challenging to stratify patient populations and develop corresponding treatments. In this work, we show that in breast cancer, the disrupted expression of circadian genes has the potential to serve as biomarkers. We also show that the master circadian transcription factors (TFs) BMAL1 and CLOCK are required for the proliferation of metastatic mesenchymal stem-like (mMSL) triple-negative breast cancer (TNBC) cells. Using currently available small molecule modulators, we found that a stabilizer of cryptochrome 2 (CRY2), the direct repressor of BMAL1 and CLOCK transcriptional activity, synergizes with inhibitors of proteasome, which is required for BMAL1 and CLOCK function, to repress a transcriptional program comprising circadian cycling genes in mMSL TNBC cells. Omics analyses on drug-treated cells implied that this repression of transcription is mediated by the transcription factor binding sites (TFBSs) features in the cis-regulatory elements (CRE) of clock-controlled genes. Through a massive parallel reporter assay, we defined a set of CRE features that are potentially repressed by the specific drug combination. The identification of cis -element enrichment may serve as a new way of defining and targeting tumor types through the modulation of cis -regulatory programs, and ultimately provide a new paradigm of therapy design for cancer types with unclear drivers like TNBC.

Genomic Profiling of Rare Undifferentiated Sarcomatoid Subtypes of Pancreatic Carcinomas: In Search of Therapeutic Targets.

PURPOSE: The highly aggressive undifferentiated sarcomatoid carcinoma (USC) subtype of pancreatic ductal adenocarcinoma (PDAC) remains poorly characterized because of its rarity. Previous case reports suggest that immune checkpoint inhibitors could be a promising treatment strategy, but the prevalence of established predictive biomarkers of response is largely unknown. The objective of this study was to leverage comprehensive genomic profiling of USC PDAC tumors to determine the prevalence of biomarkers associated with potential response to targeted therapies. METHODS: USC tumors (n = 20) underwent central pathology review by a board-certified gastrointestinal pathologist to confirm the diagnosis. These samples were compared with non-USC PDAC tumors (N = 5,562). Retrospective analysis of DNA and RNA next-generation sequencing data was performed. RESULTS: USC PDACs were more frequently PD-L1+ by immunohistochemistry than non-USC PDAC (63% v 16%, respectively, P < .001). Furthermore, USC PDAC had an increase in neutrophils (8.99% v 5.55%, P = .005) and dendritic cells (1.08% v 0.00%, q = 0.022) and an increased expression of PDCD1LG2 (4.6% v 1.3%, q = 0.001), PDCD1 (2.0% v 0.8%, q = 0.060), and HAVCR2 (45.9% v 21.7%, q = 0.107) than non-USC PDAC. Similar to non-USC PDAC, KRAS was the most commonly mutated gene (86% v 90%, respectively, P = 1). CONCLUSION: To our knowledge, this work represents the largest molecular analysis of USC tumors to date and showed an increased expression of immune checkpoint genes in USC tumors. These findings provide evidence for further investigation into immune checkpoint inhibitors in USC tumors.

Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

q-Diffusion leverages the full dimensionality of gene coexpression in single-cell transcriptomics.

Unlocking the full dimensionality of single-cell RNA sequencing data (scRNAseq) is the next frontier to a richer, fuller understanding of cell biology. We introduce q-diffusion, a framework for capturing the coexpression structure of an entire library of genes, improving on state-of-the-art analysis tools. The method is demonstrated via three case studies. In the first, q-diffusion helps gain statistical significance for differential effects on patient outcomes when analyzing the CALGB/SWOG 80405 randomized phase III clinical trial, suggesting precision guidance for the treatment of metastatic colorectal cancer. Secondly, q-diffusion is benchmarked against existing scRNAseq classification methods using an in vitro PBMC dataset, in which the proposed method discriminates IFN-γ stimulation more accurately. The same case study demonstrates improvements in unsupervised cell clustering with the recent Tabula Sapiens human atlas. Finally, a local distributional segmentation approach for spatial scRNAseq, driven by q-diffusion, yields interpretable structures of human cortical tissue.

Differential landscape of immune evasion in oncogenic RAS-driven primary and metastatic colorectal cancers.

Oncogenic drivers such as KRAS extensively modulate the tumor inflammatory microenvironment (TIME) of colorectal cancer (CRC). The influence of KRAS on modulating immune cell composition remains unclear. The objective of this study was to identify signatures of infiltrative immune cells and distinctive patterns that differ between RAS wild-type (WT) and oncogenic mutant (MT) CRC that explain immune evasion in MT tumors. A total of 7,801 CRC specimens were analyzed using next-generation DNA sequencing, whole-exome sequencing, and/or whole transcriptome sequencing. Deficiency of mismatch repair (dMMR)/microsatellite instability (MSI) and tumor mutation burden (TMB) were also assessed. KRAS mutations were present in 48% of CRC, similarly distributed in patients younger than vs. 50 years and older. In microsatellite stable (MSS) KRAS MT tumors, composition of the TIME included higher neutrophil infiltration and lower infiltration of B cells. MSI-H/dMMR was significantly more prevalent in RAS WT (9.1%) than in KRAS MT (2.9%) CRC. In MSS CRC, TMB-high cases were significantly higher in RAS MT (3.1%) than in RAS WT (2.1%) tumors. KRAS and NRAS mutations are associated with increased neutrophil infiltration, with codon-specific differences. These results demonstrate significant differences in the TIME of RAS mutant CRC that match previous reports of immunoevasive characteristics of such tumors.

Gene expression biomarkers differentiate overall survival of colorectal cancer upon targeted therapies.

While monoclonal antibody-based targeted therapies have substantially improved progression-free survival in cancer patients, the variability in individual responses poses a significant challenge in patient care. Therefore, identifying cancer subtypes and their associated biomarkers is required for assigning effective treatment. In this study, we integrated genotype and pre-treatment tissue RNA-seq data and identified biomarkers causally associated with the overall survival (OS) of colorectal cancer (CRC) patients treated with either cetuximab or bevacizumab. We performed enrichment analysis for specific consensus molecular subtypes (CMS) of colorectal cancer and evaluated differential expression of identified genes using paired tumor and normal tissue from an external cohort. In addition, we replicated the causal effect of these genes on OS using validation cohort and assessed their association with the Cancer Genome Atlas Program data as an external cohort. One of the replicated findings was WDR62, whose overexpression shortened OS of patients treated with cetuximab. Enrichment of its over expression in CMS1 and low expression in CMS4 suggests that patients with CMS4 subtype may drive greater benefit from cetuximab. In summary, this study highlights the importance of integrating different omics data for identifying promising biomarkers specific to a treatment or a cancer subtype.

Modified FOLFOX6 plus bevacizumab with and without nivolumab for first-line treatment of metastatic colorectal cancer: phase 2 results from the CheckMate 9X8 randomized clinical trial.

BACKGROUND: Standard first-line therapies for metastatic colorectal cancer (mCRC) include fluoropyrimidine-containing regimens with oxaliplatin and/or irinotecan and a biologic agent. Immunotherapy may enhance antitumor activity in combination with standard therapies in patients with mCRC. Here, we present phase 2 results of nivolumab plus standard-of-care therapy (SOC; 5-fluorouracil/leucovorin/oxaliplatin/bevacizumab) versus SOC in the first-line treatment of patients with mCRC (CheckMate 9X8). METHODS: CheckMate 9X8 was a multicenter, open-label, randomized, phase 2/3 trial. Eligible patients were at least 18 years of age with unresectable mCRC and no prior chemotherapy for metastatic disease. Patients were randomized 2:1 to receive nivolumab 240 mg plus SOC or SOC alone every 2 weeks. The primary endpoint was progression-free survival (PFS) by blinded independent central review (BICR) per Response Evaluation Criteria in Solid Tumors V.1.1. Secondary endpoints included PFS by investigator assessment; objective response rate (ORR), disease control rate, duration of response, and time to response, all by BICR and investigator assessments; overall survival; and safety. Preplanned exploratory biomarker analyses were also performed. RESULTS: From February 2018 through April 2019, 310 patients were enrolled, of which 195 patients were randomized to nivolumab plus SOC (n=127) or SOC (n=68). At 21.5-month minimum follow-up, PFS with nivolumab plus SOC versus SOC did not meet the prespecified threshold for statistical significance; median PFS by BICR was 11.9 months in both arms (HR, 0.81 (95% CI, 0.53 to 1.23); p=0.30). Higher PFS rates after 12 months (18 months: 28% vs 9%), higher ORR (60% vs 46%), and durable responses (median 12.9 vs 9.3 months) were observed with nivolumab plus SOC versus SOC. Grade 3-4 treatment-related adverse events were reported in 75% versus 48% of patients; no new safety signals were identified. CONCLUSIONS: The CheckMate 9X8 trial investigating first-line nivolumab plus SOC versus SOC in patients with mCRC did not meet its primary endpoint of PFS by BICR. Nivolumab plus SOC showed numerically higher PFS rates after 12 months, a higher response rate, and more durable responses compared with SOC alone, with acceptable safety. Further investigation to identify subgroups of patients with mCRC that may benefit from nivolumab plus SOC versus SOC in the first-line setting is warranted. TRIAL REGISTRATION NUMBER: NCT03414983.

HER2 Gene Expression Levels Are Predictive and Prognostic in Patients With Metastatic Colorectal Cancer Enrolled in CALGB/SWOG 80405.

PURPOSE: The phase III Cancer and Leukemia Group B (CALGB)/SWOG 80405 trial found no difference in overall survival (OS) in patients with metastatic colorectal cancer receiving first-line chemotherapy in combination with either bevacizumab or cetuximab. We investigated the potential prognostic and predictive value of HER2 amplification and gene expression using next-generation sequencing (NGS) and NanoString data. PATIENTS AND METHODS: Primary tumor DNA from 559 patients was profiled for HER2 amplification by NGS (FoundationOne CDx). Tumor tissue from 925 patients was tested for NanoString gene expression using an 800-gene panel. OS and progression-free survival (PFS) were the time-to-event end points. RESULTS: High HER2 expression (dichotomized at median) was associated with longer PFS (11.6 v 10 months, P = .012) and OS (32 v 25.3 months, P = .033), independent of treatment. An OS benefit for cetuximab versus bevacizumab was observed in the high HER2 expression group (P = .02), whereas a worse PFS for cetuximab was seen in the low-expression group (P = .019). When modeled as a continuous variable, increased HER2 expression was associated with longer OS (hazard ratio [HR], 0.83 [95% CI, 0.75 to 0.93]; adjusted P = .0007) and PFS (HR, 0.82 [95% CI, 0.74 to 0.91]; adjusted P = .0002), reaching a plateau effect after the median. In patients with HER2 expression lower than median, treatment with cetuximab was associated with worse PFS (HR, 1.38 [95% CI, 1.12 to 1.71]; adjusted P = .0027) and OS (HR, 1.28 [95% CI, 1.02 to 1.59]; adjusted P = .03) compared with that with bevacizumab. A significant interaction between HER2 expression and the treatment arm was observed for OS (Pintx = .017), PFS (Pintx = .048), and objective response rate (Pintx = .001). CONCLUSION: HER2 gene expression was prognostic and predictive in CALGB/SWOG 80405. HER2 tumor expression may inform treatment selection for patients with low HER2 favoring bevacizumab- versus cetuximab-based therapies.

Differential Responses to Immune Checkpoint Inhibitors are Governed by Diverse Mismatch Repair Gene Alterations.

PURPOSE: The response to immune checkpoint inhibitors (ICI) in deficient mismatch repair (dMMR) colorectal cancer and endometrial cancer is variable. Here, we explored the differential response to ICIs according to different mismatch repair alterations. EXPERIMENTAL DESIGN: Colorectal cancer (N = 13,701) and endometrial cancer (N = 3,315) specimens were tested at Caris Life Sciences. Median overall survival (mOS) was estimated using Kaplan-Meier. The prediction of high-, intermediate-, and low-affinity epitopes by tumor mutation burden (TMB) values was conducted using R-squared (R2). RESULTS: Compared with mutL (MLH1 and PMS2) co-loss, the mOS was longer in mutS (MSH2 and MSH6) co-loss in all colorectal cancer (54.6 vs. 36 months; P = 0.0.025) and endometrial cancer (81.5 vs. 48.2 months; P < 0.001) patients. In ICI-treated patients, the mOS was longer in mutS co-loss in colorectal cancer [not reached (NR) vs. 36 months; P = 0.011). In endometrial cancer, the mOS was NR vs. 42.2 months; P = 0.711]. The neoantigen load (NAL) in mutS co-loss compared with mutL co-loss was higher in colorectal cancer (high-affinity epitopes: 25.5 vs. 19; q = 0.017, intermediate: 39 vs. 32; q = 0.004, low: 87.5 vs. 73; q < 0.001) and endometrial cancer (high-affinity epitopes: 15 vs. 11; q = 0.002, intermediate: 27.5 vs. 19; q < 0.001, low: 59 vs. 41; q < 0.001), respectively. R2 ranged from 0.25 in mutS co-loss colorectal cancer to 0.95 in mutL co-loss endometrial cancer. CONCLUSIONS: Patients with mutS co-loss experienced longer mOS in colorectal cancer and endometrial cancer and better response to ICIs in colorectal cancer. Among all explored biomarkers, NAL was higher in mutS co-loss and may be a potential driving factor for the observed better outcomes. TMB did not reliably predict NAL.

Predictive value of CDC37 gene expression for targeted therapy in metastatic colorectal cancer.

BACKGROUND: CDC37 is a key determinant of client kinase recruitment to the HSP90 chaperoning system. We hypothesized that kinase-specific dependency on CDC37 alters the efficacy of targeted therapies for metastatic colorectal cancer (mCRC). MATERIAL AND METHODS: Two independent mCRC cohorts were analyzed to compare the survival outcomes between CDC37-high and CDC37-low patients (stratified by the median cutoff values): the CALGB/SWOG 80405 trial (226 and 207 patients receiving first-line bevacizumab- and cetuximab-containing chemotherapies, respectively) and Japanese retrospective (50 refractory patients receiving regorafenib) cohorts. A dataset of specimens submitted to a commercial CLIA-certified laboratory was utilized to characterize molecular profiles of CDC37-high (top quartile, N = 5055) and CDC37-low (bottom quartile, N = 5055) CRCs. RESULTS: In the bevacizumab-treated group, CDC37-high patients showed significantly better progression-free survival (PFS) (median 13.3 vs 9.6 months, hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.44-0.79, p < 0.01) than CDC37-low patients. In the cetuximab-treated group, CDC37-high and CDC37-low patients had similar outcomes. In the regorafenib-treated group, CDC37-high patients showed significantly better overall survival (median 11.3 vs 6.0 months, HR 0.24, 95% CI 0.11-0.54, p < 0.01) and PFS (median 3.5 vs 1.9 months, HR 0.51, 95% CI 0.28-0.94, p = 0.03). Comprehensive molecular profiling revealed that CDC37-high CRCs were associated with higher VEGFA, FLT1, and KDR expressions and activated hypoxia signature. CONCLUSIONS: CDC37-high mCRC patients derived more benefit from anti-VEGF therapies, including bevacizumab and regorafenib, but not from cetuximab. Molecular profiles suggested that such tumors were dependent on angiogenesis-relating pathways.

A phase II study of guadecitabine combined with irinotecan vs regorafenib or TAS-102 in irinotecan-refractory metastatic colorectal cancer patients.

DNA methyltransferase inhibitors (DNMTi) have demonstrated benefit in reversing resistance to systemic therapies for several cancer types. In a phase II trial of guadecitabine and irinotecan compared to regorafenib or TAS-102 in pts with advanced mCRC refractory to irinotecan. Patients with mCRC refractory to irinotecan were randomized 2:1 to guadecitabine and irinotecan (Arm A) vs standard of care regorafenib or TAS-102 (Arm B) on a 28-day cycle. Between January 15, 2016 and October 24, 2018, 104 pts were randomized at four international sites, with 96 pts undergoing treatment, 62 in Arm A and 34 in Arm B. Median overall survival was 7.15 months for Arm A and 7.66 months for Arm B (HR 0.93, 95% CI: 0.58-1.47, P = .75). The Kaplan-Meier rates of progression free survival at 4 months were 32% in Arm A and 26% in Arm B. Common ≥Grade 3 treatment related adverse events in Arm A were neutropenia (42%), anemia (18%), diarrhea (11%), compared to Arm B pts with neutropenia (12%), anemia (12%). Guadecitabine and irinotecan had similar OS compared to standard of care TAS-102 or regorafenib, with evidence of target modulation. Clinical trial information: NCT01896856.

Onvansertib in Combination with FOLFIRI and Bevacizumab in Second-Line Treatment of KRAS-Mutant Metastatic Colorectal Cancer: A Phase Ib Clinical Study.

PURPOSE: Onvansertib is a highly specific inhibitor of polo-like kinase 1 (PLK1), with demonstrated safety in solid tumors. We evaluated, preclinically and clinically, the potential of onvansertib in combination with chemotherapy as a therapeutic option for KRAS-mutant colorectal cancer. PATIENTS AND METHODS: Preclinical activity of onvansertib was assessed (i) in vitro in KRAS wild-type and -mutant isogenic colorectal cancer cells and (ii) in vivo, in combination with irinotecan, in a KRAS-mutant xenograft model. Clinically, a phase Ib trial was conducted to investigate onvansertib at doses 12, 15, and 18 mg/m2 (days 1-5 and 14-19 of a 28-day cycle) in combination with FOLFIRI/bevacizumab (days 1 and 15) in patients with KRAS-mutant metastatic colorectal cancer who had prior oxaliplatin exposure. Safety, efficacy, and changes in circulating tumor DNA (ctDNA) were assessed. RESULTS: In preclinical models, onvansertib displayed superior activity in KRAS-mutant than wild-type isogenic colorectal cancer cells and demonstrated potent antitumor activity in combination with irinotecan in vivo. Eighteen patients enrolled in the phase Ib study. Onvansertib recommended phase II dose was established at 15 mg/m2. Grade 3 and 4 adverse events (AE) represented 15% of all treatment-related AEs, with neutropenia being the most common. Partial responses were observed in 44% of patients, with a median duration of response of 9.5 months. Early ctDNA dynamics were predictive of treatment efficacy. CONCLUSIONS: Onvansertib combined with FOLIFRI/bevacizumab exhibited manageable safety and promising efficacy in second-line treatment of patients with KRAS-mutant metastatic colorectal cancer. Further exploration of this combination therapy is ongoing. See related commentary by Stebbing and Bullock, p. 2005.

Evidence for a causal link between intra-pancreatic fat deposition and pancreatic cancer: A prospective cohort and Mendelian randomization study.

Prior observational studies suggest an association between intra-pancreatic fat deposition (IPFD) and pancreatic ductal adenocarcinoma (PDAC); however, the causal relationship is unclear. To elucidate causality, we conduct a prospective observational study using magnetic resonance imaging (MRI)-measured IPFD data and also perform a Mendelian randomization study using genetic instruments for IPFD. In the observational study, we use UK Biobank data (N = 29,463, median follow-up: 4.5 years) and find that high IPFD (>10%) is associated with PDAC risk (adjusted hazard ratio [HR]: 3.35, 95% confidence interval [95% CI]: 1.60-7.00). In the Mendelian randomization study, we leverage eight out of nine IPFD-associated genetic variants (p < 5 × 10-8) from a genome-wide association study in the UK Biobank (N = 25,617) and find that genetically determined IPFD is associated with PDAC (odds ratio [OR] per 1-standard deviation [SD] increase in IPFD: 2.46, 95% CI: 1.38-4.40) in the Pancreatic Cancer Cohort Consortium I, II, III (PanScan I-III)/Pancreatic Cancer Case-Control Consortium (PanC4) dataset (8,275 PDAC cases and 6,723 non-cases). This study provides evidence for a potential causal role of IPFD in the pathogenesis of PDAC. Thus, reducing IPFD may lower PDAC risk.

A revised nomenclature for the lemur family of protein kinases.

The lemur family of protein kinases has gained much interest in recent years as they are involved in a variety of cellular processes including regulation of axonal transport and endosomal trafficking, modulation of synaptic functions, memory and learning, and they are centrally placed in several intracellular signalling pathways. Numerous studies have also implicated role of the lemur kinases in the development and progression of a wide range of cancers, cystic fibrosis, and neurodegenerative diseases. However, parallel discoveries and inaccurate prediction of their kinase activity have resulted in a confusing and misleading nomenclature of these proteins. Herein, a group of international scientists with expertise in lemur family of protein kinases set forth a novel nomenclature to rectify this problem and ultimately help the scientific community by providing consistent information about these molecules.

CCR5 and CCL5 gene expression in colorectal cancer: comprehensive profiling and clinical value.

BACKGROUND: The C-C motif chemokine receptor 5 (CCR5)/C-C motif chemokine ligand 5 (CCL5) axis plays a major role in colorectal cancer (CRC). We aimed to characterize the molecular features associated with CCR5/CCL5 expression in CRC and to determine whether CCR5/CCL5 levels could impact treatment outcomes. METHODS: 7604 CRCs tested with NextGen Sequencing on DNA and RNA were analyzed. Molecular features were evaluated according to CCR5 and CCL5 tumor gene expression quartiles. The impact on treatment outcomes was assessed in two cohorts, including 6341 real-world patients and 429 patients from the Cancer and Leukemia Group B (CALGB)/SWOG 80405 trial. RESULTS: CCR5/CCL5 expression was higher in right-sided versus left-sided tumors, and positively associated with consensus molecular subtypes 1 and 4. Higher CCR5/CCL5 expression was associated with higher tumor mutational burden, deficiency in mismatch repair and programmed cell death ligand 1 (PD-L1) levels. Additionally, high CCR5/CCL5 were associated with higher immune cell infiltration in the tumor microenvironment (TME) of MMR proficient tumors. Ingenuity pathway analysis revealed upregulation of the programmed cell death protein 1 (PD-1)/PD-L1 cancer immunotherapy pathway, phosphatase and tensin homolog (PTEN) and peroxisome proliferator-activated receptors (PPAR) signaling, and cytotoxic T-lymphocyte antigen 4 (CTLA-4) signaling in cytotoxic T lymphocytes, whereas several inflammation-related pathways were downregulated. Low CCR5/CCL5 expression was associated with increased benefit from cetuximab-FOLFOX treatment in the CALGB/SWOG 80405 trial, where significant treatment interaction was observed with biologic agents and chemotherapy backbone. CONCLUSIONS: Our data show a strong association between CCR5/CCL5 gene expression and distinct molecular features, gene expression profiles, TME cell infiltration, and treatment benefit in CRC. Targeting the CCR5/CCL5 axis may have clinical applications in selected CRC subgroups and may play a key role in developing and deploying strategies to modulate the immune TME for CRC treatment.

A Randomized Phase III Study of Arfolitixorin versus Leucovorin with 5-Fluorouracil, Oxaliplatin, and Bevacizumab for First-Line Treatment of Metastatic Colorectal Cancer: The AGENT Trial.

PURPOSE: Suboptimal treatment outcomes with 5-fluorouracil (5-FU)/folate, the standard of care for metastatic colorectal cancer (mCRC), have generated interest in optimizing the folate. Arfolitixorin ([6R]-5,10-methylene-tetrahydrofolate) is an immediately active folate and may improve outcomes over the existing standard of care (leucovorin). EXPERIMENTAL DESIGN: AGENT was a randomized, phase III study (NCT03750786). Patients with mCRC were randomized to arfolitixorin (120 mg/m2 given as two intravenous bolus doses of 60 mg/m2) or leucovorin (400 mg/m2 given as a single intravenous infusion) plus 5-FU, oxaliplatin, and bevacizumab. Assessments were performed every 8 weeks. The primary endpoint was the superiority of arfolitixorin for overall response rate (ORR). RESULTS: Between February 2019 and April 2021, 490 patients were randomized (245 to each arm). After a median follow-up of 266 days, the primary endpoint of superiority for ORR was not achieved (48.2% for arfolitixorin vs. 49.4% for leucovorin, Psuperiority = 0.57). Outcomes were not achieved for median progression-free survival (PFS; 12.8 and 11.6 months, P = 0.38), median duration of response (12.2 and 12.9 months, P = 0.40), and median overall survival (23.8 and 28.0 months, P = 0.78). The proportion of patients with an adverse event of grade ≥3 severity was similar between arms (68.7% and 67.2%, respectively), as was quality of life. BRAF mutations and MTHFD2 expression were both associated with a lower PFS with arfolitixorin. CONCLUSIONS: The study failed to demonstrate clinical benefit of arfolitixorin (120 mg/m2) over leucovorin. However, it provides some useful insights from the first-line treatment setting, including the effect of gene expression on outcomes. SIGNIFICANCE: This phase III study compared arfolitixorin, a direct-acting folate, with leucovorin in FOLFOX plus bevacizumab in mCRC. Arfolitixorin (120 mg/m2) did not improve the ORR, potentially indicating a suboptimal dose.

Sexual dysfunction among early-onset colorectal cancer survivors: Sex-specific correlates of sexual health discussions between patients and providers.

PURPOSE: To examine the prevalence of female sexual dysfunction (FSD), male erectile dysfunction (ED), and the prevalence and correlates of sexual health discussions between early-onset CRC survivors and their health care providers. METHODS: An online, cross-sectional survey was administered in partnership with a national CRC advocacy organization. Respondents (n = 234; diagnosed < 50 years, 6-36 months from diagnosis/relapse) were colon (36.8%) and rectal (63.3%) cancer survivors (62.5% male). The Female Sexual Function Index (FSFI-6) was used to measure FSD, and the International Index of Erectile Function (IIEF-5) was used to measure ED. Survivors reported whether a doctor communicated with them about sexual issues during/after treatment. RESULTS: Among females (n = 87), 81.6% had FSD (mean FSFI-6 score = 14.3 [SD±6.1]). Among males (n = 145), 94.5% had ED (mean IIEF-5 score = 13.6 [SD±3.4]). Overall, 59.4% of males and 45.4% of females reported a sexual health discussion. Among the total sample, older age of diagnosis and relapse were significantly associated with reporting a discussion, while female sex was negatively associated with reporting a sexual health discussion. Among males, older age at diagnosis and relapse, and among females, older age of diagnosis, were significantly associated with reporting a sexual health discussion. CONCLUSION: The prevalence of FSD and ED were high (8 in 10 females reporting FSD, almost all males reporting ED), while reported rates of sexual health discussion were suboptimal (half reported discussion). Interventions to increase CRC provider awareness of patients at risk for not being counseled are needed to optimize long-term health outcomes.

DNA Mutational Profiling in Patients With Colorectal Cancer Treated With Standard of Care Reveals Differences in Outcome and Racial Distribution of Mutations.

PURPOSE: CALGB (Alliance)/SWOG 80405 was a randomized phase III trial that in first-line patients with metastatic colorectal cancer (mCRC) treated with bevacizumab or cetuximab with chemotherapy. We aimed to discover novel mutated genes associated with prognosis and differential response to therapy with the biologics. METHODS: Primary tumor DNA from 548 patients was sequenced using FoundationOne. The effect of mutated genes and mutations on overall survival (OS) was tested adjusting for microsatellite instability status, BRAF V600E, all RAS mutations, arm, sex, and age. RESULTS: The median number (lower-upper quartile) of mutated genes was 5 (3-7), 5 (3-6) in microsatellite stable and 12.5 (4.5-32) in microsatellite instability-high tumors. Mutated KRAS and APC were more frequent in Black (53% and 85%) than White (27% and 65%, respectively) patients while BRAF V600E was less frequent in Black (5%) than White (14%) patients. The median OS in patients with BRAF non-V600E (2.2% of patients) was 31.9 months (95% CI, 15.1 to not applicable [NA]) similar to that of BRAF wild-type (WT) patients (31.2 months [95% CI, 29.0 to 33.9]). Mutated LRP1B (10.7% of patients) was associated with improved OS compared with WT LRP1B (hazard ratio, 0.57 [95% CI, 0.40 to 0.80]). RNF43 (5.6% of patients) interacted with treatment arms as, in the cetuximab arm, patients with mutated RNF43 had a median OS of 11.5 (95% CI, 10.8 to NA) months compared with 30.1 (95% CI, 24.9 to 35.3) months in patients with WT RNF43, whereas in the bevacizumab arm, patients with mutated RNF43 had a median OS of 25.0 (95% CI, 14.2 to NA) months compared with 31.3 (95% CI, 29.0 to 34.3) months in patients with WT RNF43. CONCLUSION: These results can provide new tools to predict patient outcome and improve therapeutic decisions and trial participation in patient minorities. The molecular alterations identified in this study may direct biomarker-driven studies.

The role of total neoadjuvant therapy in locally advanced rectal cancer: a survey of specialists attending the All-Ireland Colorectal Cancer Conference 2022 including lead investigators of OPRA, PRODIGE-23 and RAPIDO.

BACKGROUND: The treatment of locally advanced rectal cancer (LARC) has evolved following recent landmark trials of total neoadjuvant therapy (TNT)-the delivery of preoperative chemotherapy sequenced with radiation. AIM: To assess the preferences of colorectal surgery (CRS), radiation oncology (RO) and medical oncology (MO) specialists attending the All-Ireland Colorectal Cancer Conference (AICCC) 2022 regarding the neoadjuvant management of LARC. METHODS: A live electronic survey explored the preferred treatment approach and TNT regimen for early-, intermediate-, bad-, and advanced-risk categories of rectal cancer according to the European Society of Medical Oncology (ESMO) guidelines. The survey was preceded by an update from lead investigators of TNT trials (OPRA, PRODIGE-23 and RAPIDO), who then participated in a multidisciplinary panel discussion. RESULTS: Ten CRS, 7 RO and 15 MO (32 of 45 specialists) participated fully in the survey resulting in a response rate of 71%. Ninety-four percent, 76% and 53% of specialists preferred a TNT approach for patients with advanced, bad, and intermediate-risk rectal cancer, respectively. A consolidation TNT regimen of long-course chemoradiotherapy followed by chemotherapy was the most preferred regimen. Upfront surgery was preferred by 77% for early-risk disease. CONCLUSION: This survey illustrated the general acceptance of TNT by rectal cancer specialists attending the AICCC as a valuable treatment strategy for higher-risk category LARC. Whilst the treatment of LARC changes, it remains best practice to individualize care, incorporating the selective use of TNT as discussed by an MDT and in keeping with the patient's goals of care.

Non-canonical MLL1 activity regulates centromeric phase separation and genome stability.

Epigenetic dysregulation is a prominent feature in cancer, as exemplified by frequent mutations in chromatin regulators, including the MLL/KMT2 family of histone methyltransferases. Although MLL1/KMT2A activity on H3K4 methylation is well documented, their non-canonical activities remain mostly unexplored. Here we show that MLL1/KMT2A methylates Borealin K143 in the intrinsically disordered region essential for liquid-liquid phase separation of the chromosome passenger complex (CPC). The co-crystal structure highlights the distinct binding mode of the MLL1 SET domain with Borealin K143. Inhibiting MLL1 activity or mutating Borealin K143 to arginine perturbs CPC phase separation, reduces Aurora kinase B activity, and impairs the resolution of erroneous kinetochore-microtubule attachments and sister-chromatid cohesion. They significantly increase chromosome instability and aneuploidy in a subset of hepatocellular carcinoma, resulting in growth inhibition. These results demonstrate a non-redundant function of MLL1 in regulating inner centromere liquid condensates and genome stability via a non-canonical enzymatic activity.