Preclinical efficacy of a HER2 synNotch/CEA-CAR combinatorial immunotherapy against colorectal cancer with HER2 amplification.

HER2 amplification occurs in about 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined HER2/EGFR targeted treatment. An alternative approach based on adoptive cell therapy (ACT) using T-cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to "on-target off-tumor" activity. Here we describe a combinatorial strategy to safely target HER2 amplification and CEA expression in CRC using a synNotch-CAR based artificial regulatory network. The natural killer cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2amp/CEA+ CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable and safe off-the shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.

Talniflumate abrogates mucin immune suppressive barrier improving efficacy of gemcitabine and nab-paclitaxel treatment in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. This is due to its aggressive course, late diagnosis and its intrinsic drugs resistance. The complexity of the tumor, in terms of cell components and heterogeneity, has led to the approval of few therapies with limited efficacy. The study of the early stages of carcinogenesis provides the opportunity for the identification of actionable pathways that underpin therapeutic resistance. METHODS: We analyzed 43 Intraductal papillary mucinous neoplasms (IPMN) (12 Low-grade and 31 High-grade) by Spatial Transcriptomics. Mouse and human pancreatic cancer organoids and T cells interaction platforms were established to test the role of mucins expression on T cells activity. Syngeneic mouse model of PDAC was used to explore the impact of mucins downregulation on standard therapy efficacy. RESULTS: Spatial transcriptomics showed that mucin O-glycosylation pathway is increased in the progression from low-grade to high-grade IPMN. We identified GCNT3, a master regulator of mucins expression, as an actionable target of this pathway by talniflumate. We showed that talniflumate impaired mucins expression increasing T cell activation and recognition using both mouse and human organoid interaction platforms. In vivo experiments showed that talniflumate was able to increase the efficacy of the chemotherapy by boosting immune infiltration. CONCLUSIONS: Finally, we demonstrated that combination of talniflumate, an anti-inflammatory drug, with chemotherapy effectively improves anti-tumor effect in PDAC.

Targeted knock-in of the polymorphism rs61764370 does not affect KRAS expression but reduces let-7 levels.

Understanding the role of single-nucleotide polymorphisms (SNPs) in the pathological process represents a unique experimental challenge especially when the variants occur outside of coding regions. The noncoding SNP rs61764370 located in the 3'-untranslated region of Kirsten rat sarcoma viral oncogene homolog (KRAS) has been implicated as a risk factor for the development of cancer and the response to targeted therapies. This cancer-associated variant is thought to affect the binding of the microRNA let-7, which allegedly modulates KRAS expression. Using site-specific homologous recombination, we inserted the rs61764370:T>G KRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. We observed a significant increase in cellular proliferation, as well as a reduction in the levels of the microRNA let-7a, let-7b, and let-7c. Transcriptional and biochemical analysis showed no concomitant change in the KRAS protein expression or modulation of the downstream mitogen activated kinase or PI3K/AKT signaling. These results suggest that the cancer-associated rs61764370 variant exerts a biological effect not through transcriptional modulation of KRAS but rather by tuning the expression of the microRNA let-7.

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Novel frontiers in urogenital cancers: from molecular bases to preclinical models to tailor personalized treatments in ovarian and prostate cancer patients.

Over the last few decades, the incidence of urogenital cancers has exhibited diverse trends influenced by screening programs and geographical variations. Among women, there has been a consistent or even increased occurrence of endometrial and ovarian cancers; conversely, prostate cancer remains one of the most diagnosed malignancies, with a rise in reported cases, partly due to enhanced and improved screening efforts.Simultaneously, the landscape of cancer therapeutics has undergone a remarkable evolution, encompassing the introduction of targeted therapies and significant advancements in traditional chemotherapy. Modern targeted treatments aim to selectively address the molecular aberrations driving cancer, minimizing adverse effects on normal cells. However, traditional chemotherapy retains its crucial role, offering a broad-spectrum approach that, despite its wider range of side effects, remains indispensable in the treatment of various cancers, often working synergistically with targeted therapies to enhance overall efficacy.For urogenital cancers, especially ovarian and prostate cancers, DNA damage response inhibitors, such as PARP inhibitors, have emerged as promising therapeutic avenues. In BRCA-mutated ovarian cancer, PARP inhibitors like olaparib and niraparib have demonstrated efficacy, leading to their approval for specific indications. Similarly, patients with DNA damage response mutations have shown sensitivity to these agents in prostate cancer, heralding a new frontier in disease management. Furthermore, the progression of ovarian and prostate cancer is intricately linked to hormonal regulation. Ovarian cancer development has also been associated with prolonged exposure to estrogen, while testosterone and its metabolite dihydrotestosterone, can fuel the growth of prostate cancer cells. Thus, understanding the interplay between hormones, DNA damage and repair mechanisms can hold promise for exploring novel targeted therapies for ovarian and prostate tumors.In addition, it is of primary importance the use of preclinical models that mirror as close as possible the biological and genetic features of patients' tumors in order to effectively translate novel therapeutic findings "from the bench to the bedside".In summary, the complex landscape of urogenital cancers underscores the need for innovative approaches. Targeted therapy tailored to DNA repair mechanisms and hormone regulation might offer promising avenues for improving the management and outcomes for patients affected by ovarian and prostate cancers.

Transcriptome-wide gene expression outlier analysis pinpoints therapeutic vulnerabilities in colorectal cancer.

Multiple strategies are continuously being explored to expand the drug target repertoire in solid tumors. We devised a novel computational workflow for transcriptome-wide gene expression outlier analysis that allows the systematic identification of both overexpression and underexpression events in cancer cells. Here, it was applied to expression values obtained through RNA sequencing in 226 colorectal cancer (CRC) cell lines that were also characterized by whole-exome sequencing and microarray-based DNA methylation profiling. We found cell models displaying an abnormally high or low expression level for 3533 and 965 genes, respectively. Gene expression abnormalities that have been previously associated with clinically relevant features of CRC cell lines were confirmed. Moreover, by integrating multi-omics data, we identified both genetic and epigenetic alternations underlying outlier expression values. Importantly, our atlas of CRC gene expression outliers can guide the discovery of novel drug targets and biomarkers. As a proof of concept, we found that CRC cell lines lacking expression of the MTAP gene are sensitive to treatment with a PRMT5-MTA inhibitor (MRTX1719). Finally, other tumor types may also benefit from this approach.

Tolerance to colibactin correlates with homologous recombination proficiency and resistance to irinotecan in colorectal cancer cells.

The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC.

Modeling tumor progression by the sequential introduction of genetic alterations into the genome of human normal cells.

Cancer genomes display a complex blend of genetic lesions affecting oncogenes and tumor suppressor genes. Multiple modeling approaches indicate that 5-15 driver oncogenic events are required to achieve tumor progression in common epithelial cancers. In vitro, a lower number (2-3) of events is typically sufficient to achieve full transformation. We developed cellular models that closely resemble the occurrence of multiple genetic lesions to understand their role in tumor progression. Homologous recombination and transcriptional downregulation were used to recapitulate the co-occurrence of driver mutations targeting oncogenes and inactivation of tumor suppressor genes in human nontransformed epithelial cells. Knockdown of the tumor suppressor genes PTEN or RB1 was combined with mutagenic activation of individual oncogenes (EGFR, KRAS, BRAF, or PIK3CA), thus generating a combinatorial model. The simultaneous presence of oncogenic and tumor suppressive events resulted in distinct biochemical properties and anchorage-independent growth abilities. Notably, however, we found that even when up to four individual alterations were concomitantly present they were not sufficient to fully transform the target cells. Our results suggest that the close recapitulation of cancer lesions in not-transformed cells is essential to unveil their oncogenic potential and raise questions concerning the minimal requirements for neoplastic transformation of epithelial cells.

Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT).

Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.

Case report: Preclinical efficacy of NEDD8 and proteasome inhibitors in patient-derived models of signet ring high-grade mucinous colorectal cancer from a Lynch syndrome patient.

High-grade mucinous colorectal cancer (HGM CRC) is particularly aggressive, prone to metastasis and treatment resistance, frequently accompanied by "signet ring" cancer cells. A sizeable fraction of HGM CRCs (20-40%) arises in the context of the Lynch Syndrome, an autosomal hereditary syndrome that predisposes to microsatellite instable (MSI) CRC. Development of patient-derived preclinical models for this challenging subtype of colorectal cancer represents an unmet need in oncology. We describe here successful propagation of preclinical models from a case of early-onset, MSI-positive metastatic colorectal cancer in a male Lynch syndrome patient, refractory to standard care (FOLFOX6, FOLFIRI-Panitumumab) and, surprisingly, also to immunotherapy. Surgical material from a debulking operation was implanted in NOD/SCID mice, successfully yielding one patient-derived xenograft (PDX). PDX explants were subsequently used to generate 2D and 3D cell cultures. Histologically, all models resembled the tumor of origin, displaying a high-grade mucinous phenotype with signet ring cells. For preclinical exploration of alternative treatments, in light of recent findings, we considered inhibition of the proteasome by bortezomib and of the related NEDD8 pathway by pevonedistat. Indeed, sensitivity to bortezomib was observed in mucinous adenocarcinoma of the lung, and we previously found that HGM CRC is preferentially sensitive to pevonedistat in models with low or absent expression of cadherin 17 (CDH17), a differentiation marker. We therefore performed IHC on the tumor and models, and observed no CDH17 expression, suggesting sensitivity to pevonedistat. Both bortezomib and pevonedistat showed strong activity on 2D cells at 72 hours and on 3D organoids at 7 days, thus providing valid options for in vivo testing. Accordingly, three PDX cohorts were treated for four weeks, respectively with vehicle, bortezomib and pevonedistat. Both drugs significantly reduced tumor growth, as compared to the vehicle group. Interestingly, while bortezomib was more effective in vitro, pevonedistat was more effective in vivo. Drug efficacy was further substantiated by a reduction of cellularity and of Ki67-positive cells in the treated tumors. These results highlight proteasome and NEDD8 inhibition as potentially effective therapeutic approaches against Lynch syndrome-associated HGM CRC, also when the disease is refractory to all available treatment options.

Efficacy of Retreatment with Oxaliplatin-Based Regimens in Metastatic Colorectal Cancer Patients: The RETROX-CRC Retrospective Study.

BACKGROUND: oxaliplatin with fluoropyrimidine is a "mainstay" regarding the upfront treatment of metastatic colorectal cancer (mCRC). In contrast, the efficacy and safety of oxaliplatin-based regimens in late-care settings have been poorly reported. METHODS: we identified a real-world mCRC patient cohort who were re-treated with oxaliplatin, and in which clinicopathological features were retrospectively analyzed to identify efficacy-predictive determinants (RETROX-CRC study). RESULTS: of 2606 patients, 119 fulfilled the eligibility criteria. Oxaliplatin retreatment response rate (RR) and disease control rate (DCR) were 21.6% (CI 14.4-31.0%), and 57.8% (CI 47.7-67.4). A trend towards better RR and DCR was observed among patients who had first oxaliplatin in an adjuvant setting; a poorer outcome was observed if two or more intervening treatments were delivered. Median progression-free survival (PFS) was 5.1 months (95%CI 4.3-6.1), reducing to 4.0 months (95%CI 3.07-5.13) if oxaliplatin was readministered beyond third-line (HR 2.02; 1.25-3.25; p = 0.004). Safety data were retrieved in 65 patients (54.6%); 18.5% (12/65) and 7.7% (5/65) had G3-4 toxicities. Toxicities led to discontinuation in 34/119 (28.6%). CONCLUSIONS: oxaliplatin retreatment produced further RR in around one-fifth of patients and DCR 57.8%. Efficacy decreased in more pre-treated patients and around one-third of patients discontinued treatment due to adverse events. Translational studies improving patient selection are warranted.

Targeting the DNA Damage Response Pathways and Replication Stress in Colorectal Cancer.

PURPOSE: Genomic instability is a hallmark of cancer and targeting DNA damage response (DDR) is emerging as a promising therapeutic strategy in different solid tumors. The effectiveness of targeting DDR in colorectal cancer has not been extensively explored. EXPERIMENTAL DESIGN: We challenged 112 cell models recapitulating the genomic landscape of metastatic colorectal cancer with ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors, in parallel with chemotherapeutic agents. We focused then on ATR inhibitors (ATRi) and, to identify putative biomarkers of response and resistance, we analyzed at multiple levels colorectal cancer models highly sensitive or resistant to these drugs. RESULTS: We found that around 30% of colorectal cancers, including those carrying KRAS and BRAF mutations and unresponsive to targeted agents, are sensitive to at least one DDR inhibitor. By investigating potential biomarkers of response to ATRi, we found that ATRi-sensitive cells displayed reduced phospho-RPA32 foci at basal level, while ATRi-resistant cells showed increased RAD51 foci formation in response to replication stress. Lack of ATM and RAD51C expression was associated with ATRi sensitivity. Analysis of mutational signatures and HRDetect score identified a subgroup of ATRi-sensitive models. Organoids derived from patients with metastatic colorectal cancer recapitulated findings obtained in cell lines. CONCLUSIONS: In conclusion, a subset of colorectal cancers refractory to current therapies could benefit from inhibitors of DDR pathways and replication stress. A composite biomarker involving phospho-RPA32 and RAD51 foci, lack of ATM and RAD51C expression, as well as analysis of mutational signatures could be used to identify colorectal cancers likely to respond to ATRi.

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses.

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.

Preclinical models as patients' avatars for precision medicine in colorectal cancer: past and future challenges.

Colorectal cancer (CRC) is a complex and heterogeneous disease, characterized by dismal prognosis and low survival rate in the advanced (metastatic) stage. During the last decade, the establishment of novel preclinical models, leading to the generation of translational discovery and validation platforms, has opened up a new scenario for the clinical practice of CRC patients. To bridge the results developed at the bench with the medical decision process, the ideal model should be easily scalable, reliable to predict treatment responses, and flexibly adapted for various applications in the research. As such, the improved benefit of novel therapies being tested initially on valuable and reproducible preclinical models would lie in personalized treatment recommendations based on the biology and genomics of the patient's tumor with the overall aim to avoid overtreatment and unnecessary toxicity. In this review, we summarize different in vitro and in vivo models, which proved efficacy in detection of novel CRC culprits and shed light into the biology and therapy of this complex disease. Even though cell lines and patient-derived xenografts remain the mainstay of colorectal cancer research, the field has been confidently shifting to the use of organoids as the most relevant preclinical model. Prioritization of organoids is supported by increasing body of evidence that these represent excellent tools worth further therapeutic explorations. In addition, novel preclinical models such as zebrafish avatars are emerging as useful tools for pharmacological interrogation. Finally, all available models represent complementary tools that can be utilized for precision medicine applications.

Werner Helicase Is a Synthetic-Lethal Vulnerability in Mismatch Repair-Deficient Colorectal Cancer Refractory to Targeted Therapies, Chemotherapy, and Immunotherapy.

Targeted therapies, chemotherapy, and immunotherapy are used to treat patients with mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer. The clinical effectiveness of targeted therapy and chemotherapy is limited by resistance and drug toxicities, and about half of patients receiving immunotherapy have disease that is refractory to immune checkpoint inhibitors. Loss of Werner syndrome ATP-dependent helicase (WRN) is a synthetic lethality in dMMR/MSI-H cells. To inform the development of WRN as a therapeutic target, we performed WRN knockout or knockdown in 60 heterogeneous dMMR colorectal cancer preclinical models, demonstrating that WRN dependency is an almost universal feature and a robust marker for patient selection. Furthermore, models of resistance to clinically relevant targeted therapy, chemotherapy, and immunotherapy retain WRN dependency. These data show the potential of therapeutically targeting WRN in patients with dMMR/MSI-H colorectal cancer and support WRN as a therapeutic option for patients with dMMR/MSI-H cancers refractory to current treatment strategies. SIGNIFICANCE: We found that a large, diverse set of dMMR/MSI-H colorectal cancer preclinical models, including models of treatment-refractory disease, are WRN-dependent. Our results support WRN as a promising synthetic-lethal target in dMMR/MSI-H colorectal cancer tumors as a monotherapy or in combination with targeted agents, chemotherapy, or immunotherapy.This article is highlighted in the In This Issue feature, p. 1861.

TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors.

On-target resistance to next-generation TRK inhibitors in TRK fusion-positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. SIGNIFICANCE: In TRK fusion-positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation.This article is highlighted in the In This Issue feature, p. 1.

Expression and functional regulation of myoglobin in epithelial cancers.

Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species. We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development. In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth.

Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab.

CONTEXT: Patients with metastatic colorectal cancer who have KRAS codon 12- or KRAS codon 13-mutated tumors are presently excluded from treatment with the anti-epidermal growth factor receptor monoclonal antibody cetuximab. OBJECTIVE: To test the hypothesis that KRAS codon 13 mutations are associated with a better outcome after treatment with cetuximab than observed with other KRAS mutations. DESIGN, SETTING, AND PATIENTS: We studied the association between KRAS mutation status (p.G13D vs other KRAS mutations) and response and survival in a pooled data set of 579 patients with chemotherapy-refractory colorectal cancer treated with cetuximab between 2001 and 2008. Patients were included in the CO.17, BOND, MABEL, EMR202600, EVEREST, BABEL, or SALVAGE clinical trials or received off-study treatment. Univariate and multivariate analyses, adjusting for possible prognostic factors and data set, were performed. The effect of the different mutations was studied in vitro by constructing isogenic cell lines with wild-type KRAS, p.G12V, or p.G13D mutant alleles and treating them with cetuximab. MAIN OUTCOME MEASURES: The main efficacy end point was overall survival. Secondary efficacy end points were response rate and progression-free survival. RESULTS: In comparison with patients with other KRAS-mutated tumors, patients with p.G13D-mutated tumors (n = 32) treated with cetuximab had longer overall survival (median, 7.6 [95% confidence interval {CI}, 5.7-20.5] months vs 5.7 [95% CI, 4.9-6.8] months; adjusted hazard ratio [HR], 0.50; 95% CI, 0.31-0.81; P = .005) and longer progression-free survival (median, 4.0 [95% CI, 1.9-6.2] months vs 1.9 [95% CI, 1.8-2.8] months; adjusted HR, 0.51; 95% CI, 0.32-0.81; P = .004). There was a significant interaction between KRAS mutation status (p.G13D vs other KRAS mutations) and overall survival benefit with cetuximab treatment (adjusted HR, 0.30; 95% CI, 0.14-0.67; P = .003). In vitro and mouse model analysis showed that although p.G12V-mutated colorectal cells were insensitive to cetuximab, p.G13D-mutated cells were sensitive, as were KRAS wild-type cells. CONCLUSIONS: In this analysis, use of cetuximab was associated with longer overall and progression-free survival among patients with chemotherapy-refractory colorectal cancer with p.G13D-mutated tumors than with other KRAS-mutated tumors. Evaluation of cetuximab therapy in these tumors in prospective randomized trials may be warranted.

Oxaliplatin retreatment in metastatic colorectal cancer: Systematic review and future research opportunities.

BACKGROUND: Oxaliplatin represents a main component of cytotoxic treatment regimens in colorectal cancer (CRC). Given its efficacy, oxaliplatin is frequently re-administered in the context of the continuum of care in metastatic CRC (mCRC). However, efficacy and tolerability of this therapeutic strategy has not been comprehensively assessed. METHODS: We performed a systematic review of the literature on September 19th 2020, according to PRISMA criteria 2009. The research was performed on PubMed, ASCO Meeting Library, ESMO library and ClinicalTrials.gov for citations or ongoing trials. RESULTS: 64 records were retrieved and 13 included in the systematic review: 8 full-text articles, 4 abstracts and 1 ongoing clinical trial. According to readministration timing, studies were classified as rechallenge/reintroduction (n = 8) or stop & go/intermittent therapeutic strategies (n = 4). The studies presented wide heterogeneity in terms of efficacy (Response Rate 6-31%; Disease Control Rate 39-79%; median Progression-Free Survival 3.1-7 months). Those patients who received retreatment after prior adjuvant oxaliplatin or exploiting a stop-&-go strategy appeared to achieve better outcomes. However, no formal comparisons on treatment outcomes were feasible. The most frequent grade 3 or higher adverse events were hematologic toxicities (5-27%), peripheral neuropathy (5-14%) and hypersensitivity reactions (5-20%). CONCLUSIONS: Retreatment with oxaliplatin for mCRC is practiced based on scarce and heterogeneous data indicating efficacy and manageable toxicity. The best strategy to exploit this approach remains to be defined, and the most promising research avenue to improve therapeutic index of oxaliplatin is represented by selection of responder patients whose tumors harbor molecular defects in the DNA damage repair pathway.

High-dose vitamin C enhances cancer immunotherapy.

Vitamin C (VitC) is known to directly impair cancer cell growth in preclinical models, but there is little clinical evidence on its antitumoral efficacy. In addition, whether and how VitC modulates anticancer immune responses is mostly unknown. Here, we show that a fully competent immune system is required to maximize the antiproliferative effect of VitC in breast, colorectal, melanoma, and pancreatic murine tumors. High-dose VitC modulates infiltration of the tumor microenvironment by cells of the immune system and delays cancer growth in a T cell-dependent manner. VitC not only enhances the cytotoxic activity of adoptively transferred CD8 T cells but also cooperates with immune checkpoint therapy (ICT) in several cancer types. Combination of VitC and ICT can be curative in models of mismatch repair-deficient tumors with high mutational burden. This work provides a rationale for clinical trials combining ICT with high doses of VitC.