SARS-CoV-2 Vaccine Immunogenicity in Patients with Gastrointestinal Cancer Receiving Systemic Anti-Cancer Therapy.

INTRODUCTION: Patients with gastrointestinal (GI) cancers have an increased risk of serious complications and death from SARS-CoV-2 infection. The immunogenicity of vaccines in patients with GI cancers receiving anti-cancer therapies is unclear. We conducted a prospective study to evaluate the prevalence of neutralizing antibodies in a cohort of GI cancer patients receiving chemotherapy following SARS-CoV-2 vaccination. MATERIALS AND METHODS: Between September 2020 and April 2021, patients with cancer undergoing chemotherapy were enrolled. At baseline (day 0), days 28, 56, and 84, we assessed serum antibodies to SARS-CoV-2 spike (anti-S) and anti-nucleocapsid (anti-NP) and concomitantly assessed virus neutralization using a pseudovirus neutralization assay. Patients received either the Pfizer/BioNTech BNT162b2, or the Oxford/AstraZeneca ChAdOx1 vaccine. RESULTS: All 152 patients enrolled had a prior diagnosis of cancer; colorectal (n = 80, 52.6%), oesophagogastric (n = 38, 25.0%), and hepato pancreatic biliary (n = 22, 12.5%). Nearly all were receiving systemic anti-cancer therapy (99.3%). Of the 51 patients who did not receive a vaccination prior to, or during the study, 5 patients had detectable anti-NP antibodies. Ninety-nine patients received at least one dose of vaccine prior to, or during the study. Within 19 days following the first dose of vaccine, 30.0% had anti-S detected in serum which increased to 70.2% at days 20-39. In the 19 days following a second dose, anti-S positivity was 84.2% (32/38). However, pseudovirus neutralization titers (pVNT80) decreased from days 20 to 39. CONCLUSION: Despite the immunosuppressive effects of chemotherapy, 2 doses of SARS-CoV-2 vaccines are able to elicit a protective immune response in patients' ongoing treatment for gastrointestinal cancers. Decreases in pseudoviral neutralization were observed after 20-39 days, re-affirming the current recommendation for vaccine booster doses. CLINICAL TRIAL REGISTRATION NUMBER: NCT04427280.

ctDNA guided adjuvant chemotherapy versus standard of care adjuvant chemotherapy after curative surgery in patients with high risk stage II or stage III colorectal cancer: a multi-centre, prospective, randomised control trial (TRACC Part C).

BACKGROUND: Circulating tumour DNA (ctDNA) to detect minimal residual disease (MRD) is emerging as a biomarker to predict recurrence in patients with curatively treated early stage colorectal cancer (CRC). ctDNA risk stratifies patients to guide adjuvant treatment decisions. We are conducting the UK's first multi-centre, prospective, randomised study to determine whether a de-escalation strategy using ctDNA to guide adjuvant chemotherapy (ACT) decisions is non-inferior to standard of care (SOC) chemotherapy, as measured by 3-year disease free survival (DFS) in patients with resected CRC with no evidence of MRD (ctDNA negative post-operatively). In doing so we may be able to spare patients unnecessary chemotherapy and associated toxicity and achieve significant cost savings for the National Health Service (NHS). METHODS: We are recruiting patients with fully resected high risk stage II and stage III CRC who are being considered for ACT into the study which uses results from a plasma-only ctDNA assay to guide treatment decisions. Eligible patients are randomised 1:1 to receive ctDNA-guided chemotherapy versus SOC chemotherapy. The primary endpoint is the difference in DFS at 3 years between the trial arms. Secondary endpoints include the proportion of patients in the ctDNA-guided arm who are ctDNA negative post-operatively and receive de-escalated ACT compared to the standard arm, the difference in overall survival (OS), neurotoxicity and quality of life between the arms, and the cost-effectiveness of ctDNA-guided therapy compared to SOC treatment. We hypothesise that using a ctDNA-guided approach to ACT decisions is non-inferior to SOC. Target accrual is 1621 patients over 4 years, which will provide a power of 80% with an alpha of 0.1 to demonstrate non-inferiority with a margin of 1.25 in survival of the ctDNA-guided approach compared to SOC. We anticipate approximately 50 UK centres will participate. The study opened with the Guardant Reveal plasma-only ctDNA assay in August 2022. DISCUSSION: The trial will determine whether ctDNA guided ACT is non-inferior to SOC ACT in patients with fully resected high risk stage II and stage III resected CRC, with the potential to significantly reduce unnecessary ACT and the toxicity associated with it. TRIAL REGISTRATION: NCT04050345.

Functional imaging and circulating biomarkers of response to regorafenib in treatment-refractory metastatic colorectal cancer patients in a prospective phase II study.

OBJECTIVE: Regorafenib demonstrated efficacy in patients with metastatic colorectal cancer (mCRC). Lack of predictive biomarkers, potential toxicities and cost-effectiveness concerns highlight the unmet need for better patient selection. DESIGN: Patients with RAS mutant mCRC with biopsiable metastases were enrolled in this phase II trial. Dynamic contrast-enhanced (DCE) MRI was acquired pretreatment and at day 15 post-treatment. Median values of volume transfer constant (Ktrans), enhancing fraction (EF) and their product KEF (summarised median values of Ktrans× EF) were generated. Circulating tumour (ct) DNA was collected monthly until progressive disease and tested for clonal RAS mutations by digital-droplet PCR. Tumour vasculature (CD-31) was scored by immunohistochemistry on 70 sequential tissue biopsies. RESULTS: Twenty-seven patients with paired DCE-MRI scans were analysed. Median KEF decrease was 58.2%. Of the 23 patients with outcome data, >70% drop in KEF (6/23) was associated with higher disease control rate (p=0.048) measured by RECIST V. 1.1 at 2 months, improved progression-free survival (PFS) (HR 0.16 (95% CI 0.04 to 0.72), p=0.02), 4-month PFS (66.7% vs 23.5%) and overall survival (OS) (HR 0.08 (95% CI 0.01 to 0.63), p=0.02). KEF drop correlated with CD-31 reduction in sequential tissue biopsies (p=0.04). RAS mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS (HR 0.21 (95% CI 0.06 to 0.71), p=0.01) and OS (HR 0.28 (95% CI 0.07-1.04), p=0.06). CONCLUSIONS: Combining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.

Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing.

BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to >95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing. CONCLUSIONS: This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution. CLINICALTRIALSGOV IDENTIFIER: NCT02112357.

Ibrutinib in c-MYC and HER2 Amplified Oesophagogastric Carcinoma: Results of the Proof-of-Concept iMYC Study.

Oesophagogastric (OG) cancer is a highly lethal disease requiring novel treatment options. c-MYC and/or HER-2 amplified oesophageal cancer models have demonstrated sensitivity to BTK inhibition with ibrutinib. We evaluated the safety and efficacy of ibrutinib in patients with c-MYC and/or HER2 amplified pre-treated advanced OG cancer. c-MYC and HER2 amplification status were determined by FISH. The primary endpoint was overall response rate (ORR). Secondary endpoints were disease control rate (DC) at 8 weeks, safety, progression-free survival (PFS) and overall survival (OS). Eleven patients were enrolled. Eight patients had c-MYC amplified tumours, six were HER2 amplified and three were c-MYC and HER2 co-amplified. Grade ≥ 3 adverse events were fever, neutropenia, and vomiting. Grade ≥ 3 gastrointestinal haemorrhage occurred in three patients and was fatal in two cases. Among seven evaluable patients, three patients (43%) achieved a best response of SD at 8 weeks. No PR or CR was observed. Disease control was achieved for 32 weeks in one patient with a dual c-MYC and HER2 highly co-amplified tumour. The median PFS and OS were 1.5 (95% CI: 0.8-5.1) and 5.1 (95% CI: 0.8-14.5) months, respectively. Ibrutinib had limited clinical efficacy in patients with c-MYC and/or HER2 amplified OG cancer. Unexpected gastrointestinal bleeding was observed in 3 out of 8 treated patients which was considered a new safety finding for ibrutinib in this population.

MRI Tumor Regression Grade and Circulating Tumor DNA as Complementary Tools to Assess Response and Guide Therapy Adaptation in Rectal Cancer.

PURPOSE: Response to preoperative chemo-radiotherapy (CRT) varies. We assessed whether circulating tumor DNA (ctDNA) might be an early indicator of tumor response or progression to guide therapy adaptation in rectal cancer. EXPERIMENTAL DESIGN: A total of 243 serial plasma samples were analyzed from 47 patients with localized rectal cancer undergoing CRT. Up to three somatic variants were tracked in plasma using droplet digital PCR. RECIST and MRI tumor regression grade (mrTRG) evaluated response. Survival analyses applied Kaplan-Meier method and Cox regression. RESULTS: ctDNA detection rates were: 74% (n = 35/47) pretreatment, 21% (n = 10/47) mid CRT, 21% (n = 10/47) after completing CRT, and 13% (n = 3/23) after surgery. ctDNA status after CRT was associated with primary tumor response by mrTRG (P = 0.03). With a median follow-up of 26.4 months, metastases-free survival was shorter in patients with detectable ctDNA after completing CRT [HR 7.1; 95% confidence interval (CI), 2.4-21.5; P < 0.001], persistently detectable ctDNA pre and mid CRT (HR 3.8; 95% CI, 1.2-11.7; P = 0.02), and pre, mid, and after CRT (HR 11.5; 95% CI, 3.3-40.4; P < 0.001) compared with patients with undetectable or nonpersistent ctDNA. In patients with detectable ctDNA, a fractional abundance threshold of ≥0.07% mid CRT or ≥0.13% after completing CRT predicted for metastases with 100% sensitivity and 83.3% specificity for mid CRT and 66.7% for CRT completion. All 3 patients with detectable ctDNA post-surgery relapsed compared with none of the 20 patients with undetectable ctDNA (P = 0.001). CONCLUSIONS: ctDNA identified patients at risk of developing metastases during the neoadjuvant period and post-surgery, and could be used to tailor treatment.

Detecting and Tracking Circulating Tumour DNA Copy Number Profiles during First Line Chemotherapy in Oesophagogastric Adenocarcinoma.

DNA somatic copy number aberrations (SCNAs) are key drivers in oesophagogastric adenocarcinoma (OGA). Whether minimally invasive SCNA analysis of circulating tumour (ct)DNA can predict treatment outcomes and reveal how SCNAs evolve during chemotherapy is unknown. We investigated this by low-coverage whole genome sequencing (lcWGS) of ctDNA from 30 patients with advanced OGA prior to first-line chemotherapy and on progression. SCNA profiles were detectable pretreatment in 23/30 (76.7%) patients. The presence of liver metastases, primary tumour in situ, or of oesophageal or junctional tumour location predicted for a high ctDNA fraction. A low ctDNA concentration associated with significantly longer overall survival. Neither chromosomal instability metrics nor ploidy correlated with chemotherapy outcome. Chromosome 2q and 8p gains before treatment were associated with chemotherapy responses. lcWGS identified all amplifications found by prior targeted tumour tissue sequencing in cases with detectable ctDNA as well as finding additional changes. SCNA profiles changed during chemotherapy, indicating that cancer cell populations evolved during treatment; however, no recurrent SCNA changes were acquired at progression. Tracking the evolution of OGA cancer cell populations in ctDNA is feasible during chemotherapy. The observation of genetic evolution warrants investigation in larger series and with higher resolution techniques to reveal potential genetic predictors of response and drivers of chemotherapy resistance. The presence of liver metastasis is a potential biomarker for the selection of patients with high ctDNA content for such studies.

Clonal diversity of MYC amplification evaluated by fluorescent in situ hybridisation and digital droplet polymerase chain reaction in oesophagogastric cancer: Results from a prospective clinical trial screening programme.

INTRODUCTION: The MYC proto-oncogene is among the most commonly dysregulated genes in human cancers. We report screening data from the iMYC trial, an ongoing phase II study assessing ibrutinib monotherapy in advanced pretreated MYC- and/or HER2-amplified oesophagogastric cancer, representing the first attempt to prospectively identify MYC amplifications in this tumour type for the purposes of therapeutic targeting. METHODS: Screening utilising a fluorescent in situ hybridisation (FISH) assay for assessment of tumour MYC amplification has been instituted. An experimental digital droplet polymerase chain reaction (ddPCR) assay to assess MYC amplification in both tumour and circulating-tumour (ct)DNA has been developed and investigated. RESULTS: One hundred thirty-five archival tumour specimens have undergone successful FISH analysis with 23% displaying evidence of MYC amplification. Intertumour heterogeneity was observed, with the percentage of cancer cells harbouring MYC amplification ranging widely between samples (median 51%, range 11-94%). Intratumoural clonal diversity of MYC amplification was also observed, with a significant degree of variance in amplification ratios (Bartlett's test for equal variance p < 0.001), and an association between greater variance in MYC amplification and improved outcome with prior first-line chemotherapy. ddPCR was most accurate in quantifying MYC amplification in tumour-derived DNA from cases with a high proportion (>70%) of amplified cells within the tumour specimen but was not reliable in samples containing a low proportion of amplified cells or in ctDNA. CONCLUSIONS: Our results illustrate the utility of FISH to assess MYC amplification prospectively for a biomarker-selected trial by providing reliable and reproducible results in real time, with a high degree of heterogeneity of MYC amplification observed. We show that ddPCR can potentially detect high-level MYC amplifications in tumour tissue.

miR-31-3p Expression and Benefit from Anti-EGFR Inhibitors in Metastatic Colorectal Cancer Patients Enrolled in the Prospective Phase II PROSPECT-C Trial.

PURPOSE: Anti-EGFR mAbs are effective in the treatment of metastatic colorectal cancer (mCRC) patients. RAS status and tumor location (sidedness) are predictive markers of patients' response to anti-EGFR mAbs. Recently, low miR-31-3p expression levels have been correlated with clinical benefit from the anti-EGFR mAb cetuximab. Here, we aimed to validate the predictive power of miR-31-3p in a prospective cohort of chemorefractory mCRC patients treated with single-agent anti-EGFR mAbs. EXPERIMENTAL DESIGN: miR-31-3p was tested by in situ hybridization (ISH) in 91 pretreatment core biopsies from metastatic deposits of 45 patients with mCRC. Sequential tissue biopsies obtained before treatment, at the time of partial response, and at disease progression were tested to monitor changes in miR-31-3p expression overtreatment. miR-31-3p expression, sidedness, and RAS status in pretreatment cell-free DNA were combined in multivariable regression models to assess the predictive value of each variable alone or in combination. RESULTS: Patients with low miR-31-3p expression in pretreatment biopsies showed better overall response rate, as well as better progression-free survival and overall survival, compared to those with high miR-31-3p expression. The prognostic effect of miR-31-3p was independent from age, gender, and sidedness. No significant changes in the expression of miR-31-3p were observed when sequential tissue biopsies were tested in long-term or poor responders to anti-EGFR mAbs. miR-31-3p scores were similar when pretreatment biopsies were compared with treatment-naïve archival tissues (often primary colorectal cancer). CONCLUSIONS: Our study validates the role of miR-31-3p as potential predictive biomarker of selection for anti-EGFR mAbs.

Genomic and Transcriptomic Determinants of Therapy Resistance and Immune Landscape Evolution during Anti-EGFR Treatment in Colorectal Cancer.

Despite biomarker stratification, the anti-EGFR antibody cetuximab is only effective against a subgroup of colorectal cancers (CRCs). This genomic and transcriptomic analysis of the cetuximab resistance landscape in 35 RAS wild-type CRCs identified associations of NF1 and non-canonical RAS/RAF aberrations with primary resistance and validated transcriptomic CRC subtypes as non-genetic predictors of benefit. Sixty-four percent of biopsies with acquired resistance harbored no genetic resistance drivers. Most of these had switched from a cetuximab-sensitive transcriptomic subtype at baseline to a fibroblast- and growth factor-rich subtype at progression. Fibroblast-supernatant conferred cetuximab resistance in vitro, confirming a major role for non-genetic resistance through stromal remodeling. Cetuximab treatment increased cytotoxic immune infiltrates and PD-L1 and LAG3 immune checkpoint expression, potentially providing opportunities to treat cetuximab-resistant CRCs with immunotherapy.