COVID-19 epidemic strongly affected cancer research in Italy: a survey of the Italian Cancer Society (SIC).

BACKGROUND: Italy was among the first countries hit by the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The application of strict lockdown measures disproportionately affected both cancer patient care as well as basic and translational cancer research. MATERIALS AND METHODS: The Italian Cancer Society (SIC) conducted a survey on the effect of lockdown on laboratories involved in cancer research in Italy. The survey was completed by 570 researchers at different stages of their career, working in cancer centers, research institutes and universities from 19 Italian regions. RESULTS: During the lockdown period, the impact of the COVID-19 pandemic emergency on face-to-face research activities was high, with a complete (47.7%) or partial (36.1%) shutdown of the laboratories. In the post-lockdown period, research activities were resumed in most of the respondents' institutions (80.4%), though with some restrictions (77.2%). COVID-19 testing was offered to research personnel only in ~50% of research institutions. Overall, the response to the pandemic was fragmented as in many cases institutions adopted different strategies often aimed at limiting possible infections without a clearly defined contingency plan. Nevertheless, research was able to provide the first answers and possible ways out of the pandemic, also with the contribution of many cancer researchers that sacrificed their research programs to help overcome the pandemic by offering their knowledge and technologies. CONCLUSIONS: Given the current persistence of an emergency situation in many European countries, a more adequate organization of research centers will be urgent and necessary to ensure the continuity of laboratory activities in a safe environment.

Temozolomide and irinotecan (TEMIRI regimen) as salvage treatment of irinotecan-sensitive advanced colorectal cancer patients bearing MGMT methylation.

Background: Non-randomized studies showed that temozolomide (TMZ) achieves an average 10% response rate in heavily pretreated metastatic colorectal cancer (mCRC) patients with promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT). In this phase II trial, irinotecan and temozolomide (TEMIRI) combination regimen was assessed in irinotecan-sensitive, MGMT methylated/microsatellite stable (MSS) pretreated mCRC patients. Patients and methods: Key inclusion criteria were centrally confirmed MGMT methylation by methylation-specific PCR, MSS mCRC, progression after at least two prior chemotherapy regimens for advanced disease and irinotecan-free interval >3 months. TEMIRI (TMZ 150 mg/m2 on days 1-5 plus irinotecan 100 mg/m2 on days 1, 15 q28 days) was administered for six cycles, followed by maintenance with TMZ. The primary end point was overall response rate (ORR). Exploratory translational analyses included MGMT immunohistochemistry (IHC) and methyl-BEAMing (MB). Results: Between December 2014 and June 2017, 25 patients were enrolled. The primary end point was met, since six patients achieved a partial response [ORR 24%, 95% confidence interval (CI) 11% to 43%]. At a median follow-up of 15.6 months, median progression-free survival (mPFS) and overall survival (mOS) were 4.4 and 13.8 months, respectively. Only four (16%) patients had ≥ grade 3 (CTCAE 4.0) adverse events. All patients whose cancer was MGMT-positive IHC were non-responders. Consistently, patients with MGMT-negative/low tumors had a significantly longer mPFS than others (6.9 versus 2.0 months; hazard ratio = 0.29, 95% CI 0.02-0.41; P = 0.003) and a non-significant trend for longer mOS. MB testing showed similar accuracy. Conclusions: TEMIRI regimen is a safe and active option in pre-treated, irinotecan-sensitive mCRC patients with MGMT methylation.

RET fusions in a small subset of advanced colorectal cancers at risk of being neglected.

Background: Recognition of rare molecular subgroups is a challenge for precision oncology and may lead to tissue-agnostic approval of targeted agents. Here we aimed to comprehensively characterize the clinical, pathological and molecular landscape of RET rearranged metastatic colorectal cancer (mCRC). Patients and methods: In this case series, we compared clinical, pathological and molecular characteristics of 24 RET rearranged mCRC patients with those of a control group of 291 patients with RET negative tumors. RET rearranged and RET negative mCRCs were retrieved by systematic literature review and by taking advantage of three screening sources: (i) Ignyta's phase 1/1b study on RXDX-105 (NCT01877811), (ii) cohorts screened at two Italian and one South Korean Institutions and (iii) Foundation Medicine Inc. database. Next-generation sequencing data were analyzed for RET rearranged cases. Results: RET fusions were more frequent in older patients (median age of 66 versus 60 years, P = 0.052), with ECOG PS 1-2 (90% versus 50%, P = 0.02), right-sided (55% versus 32%, P = 0.013), previously unresected primary tumors (58% versus 21%, P < 0.001), RAS and BRAF wild-type (100% versus 40%, P < 0.001) and MSI-high (48% versus 7%, P < 0.001). Notably, 11 (26%) out of 43 patients with right-sided, RAS and BRAF wild-type tumors harbored a RET rearrangement. At a median follow-up of 45.8 months, patients with RET fusion-positive tumors showed a significantly worse OS when compared with RET-negative ones (median OS 14.0 versus 38.0 months, HR: 4.59; 95% CI, 3.64-32.66; P < 0.001). In the multivariable model, RET rearrangements were still associated with shorter OS (HR: 2.97; 95% CI, 1.25-7.07; P = 0.014), while primary tumor location, RAS and BRAF mutations and MSI status were not. Conclusions: Though very rare, RET rearrangements define a new subtype of mCRC that shows poor prognosis with conventional treatments and is therefore worth of a specific management.

Adipocytes sustain pancreatic cancer progression through a non-canonical WNT paracrine network inducing ROR2 nuclear shuttling.

BACKGROUND: Solid epidemiological evidences connect obesity with incidence, stage and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions. METHODS: We studied the role of factors secreted by two adipogenic model systems from primary human bone marrow stromal cells (hBMSCs) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS),Results:We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells. CONCLUSIONS: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer.

Tracking a CAD-ALK gene rearrangement in urine and blood of a colorectal cancer patient treated with an ALK inhibitor.

BACKGROUND: Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. PATIENTS AND METHODS: We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying a CAD-ALK translocation during treatment with an ALK inhibitor. RESULTS: Using a custom next generation sequencing panel we identified the genomic CAD-ALK rearrangement and a TP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of the CAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient's clinical course. Detection of the CAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identified ALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. CONCLUSION: We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.

Tumor MGMT promoter hypermethylation changes over time limit temozolomide efficacy in a phase II trial for metastatic colorectal cancer.

BACKGROUND: Objective response to dacarbazine, the intravenous form of temozolomide (TMZ), in metastatic colorectal cancer (mCRC) is confined to tumors harboring O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter hypermethylation. We conducted a phase II study of TMZ enriched by MGMT hypermethylation in archival tumor (AT), exploring dynamic of this biomarker in baseline tumor (BT) biopsy and plasma (liquid biopsy). PATIENTS AND METHODS: We screened 150 mCRC patients for MGMT hypermethylation with methylation-specific PCR on AT from FFPE specimens. Eligible patients (n = 29) underwent BT biopsy and then received TMZ 200 mg/m(2) days 1-5 q28 until progression. A Fleming single-stage design was used to determine whether progression-free survival (PFS) rate at 12 weeks would be ≥35% [H0 ≤ 15%, type I error = 0.059 (one-sided), power = 0.849]. Exploratory analyses included comparison between MGMT hypermethylation in AT and BT, and MGMT methylation testing by MethylBEAMing in solid (AT, BT) and LB with regard to tumor response. RESULTS: The PFS rate at 12 weeks was 10.3% [90% confidence interval (CI) 2.9-24.6]. Objective response rate was 3.4% (90% CI 0.2-15.3), disease control rate 48.3% (90% CI 32.0-64.8), median OS 6.2 months (95% CI 3.8-7.6), and median PFS 2.6 months (95% CI 1.4-2.7). We observed the absence of MGMT hypermethylation in BT in 62.7% of tumors. CONCLUSION: Treatment of mCRC with TMZ driven by MGMT promoter hypermethylation in AT samples did not provide meaningful PFS rate at 12 weeks. This biomarker changed from AT to BT, indicating that testing BT biopsy or plasma is needed for refined target selection.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

Therapeutic implications of resistance to molecular therapies in metastatic colorectal cancer.

Metastatic colorectal cancer (mCRC) patients carrying KRAS mutated tumors do not benefit from epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies. Indeed, the mutational status of KRAS is currently a validated predictive biomarker employed to select mCRC patients for EGFR targeted drugs. When patients fail standard 5-fluorouracil-, oxaliplatin-, irinotecan- and bevacizumab-based therapies, EGFR-targeted salvage therapy can be prescribed only for those individuals with KRAS wild-type cancer. Thus, clinicians are now facing the urgent issue of better understanding the biology of KRAS mutant disease, in order to devise novel effective therapies in such defined genetic setting. In addition to KRAS, recent data point out that BRAF and PIK3CA exon 20 mutations hamper response to EGFR-targeted treatment in mCRC, potentially excluding from treatment also patients with these molecular alterations in their tumor. This review will focus on current knowledge regarding the molecular landscape of mCRC including and beyond KRAS, and will summarize novel rationally-developed combinatorial regimens that are being evaluated in early clinical trials.

Combination chemotherapy in advanced gastrointestinal cancers: ex vivo sensitivity to gemcitabine and mitomycin C.

Advanced or metastatic disease is common in both oesophagogastric and colorectal cancers, with poor 5-year survival despite palliative chemotherapy. We have investigated the sensitivity of gastrointestinal tumours to gemcitabine in combination with mitomycin C (GeM), using a modified ex vivo ATP-based tumour chemosensitivity assay (ATP-TCA). Tumour material from 41 colorectal and 22 oesophagogastric cancers were assessed. The GeM combination showed variable but definite activity in most of the samples tested. The results show that GeM achieves >95% inhibition at concentrations within the range achievable clinically in 60% of colorectal tumours (21 out of 35) and 38% of oesophagogastric tumours (five out of 13) tested. We did not identify any significant difference in sensitivity using concurrent or sequential exposure of tumour-derived cells to these two drugs. The results from this study suggest that GeM may be a useful combination in the treatment of advanced gastrointestinal malignancy.

Absence of BRAF gene mutations in uveal melanomas in contrast to cutaneous melanomas.

The recent discovery of activating mutations in the BRAF gene in many cutaneous melanomas led us to screen the genomic sequence of BRAF exons 11 and 15 in a series of 48 intraocular (uveal) melanomas, together with control samples from three cutaneous melanomas and the SK-Mel-28 cell line, which has a BRAF mutation. The same mutation was detected in two-thirds of our cutaneous melanoma samples, but was not present in any uveal melanomas. This finding further underlines the distinction between uveal and cutaneous melanomas, and suggests that BRAF inhibitors are unlikely to benefit patients with uveal melanoma.

Modulation of cerebellar and hepatic nitric oxide synthase by exogenous arginine and endotoxin.

We have studied in mice the effect of treatment with exogenous arginine and/or LPS by monitoring serum nitrite/nitrate levels and by investigating the response of cerebellar and liver nitric oxide synthase (NOS). We measured NOS activity in cerebellar extracts while changes in iNOS mRNA were followed in the liver since direct assay of NOS activity proved unreliable with this tissue. In fact, liver and cerebellum extracts were both very active in converting arginine into a citrulline-like metabolite, but only cerebellum conversion was dependent on addition of NADPH and inhibitable by N(G)-methyl-l-arginine. Treatment with LPS, on its own, increased serum nitrite/nitrate levels at 5 and 20 h after injection, while treatment with LPS and arginine produced nitrite/nitrate levels in the serum even greater at 5 h, but significantly lower at 20 h. Liver iNOS mRNA levels were markedly increased by LPS, and this effect was significantly decreased when mice were also given exogenous arginine. A stimulatory effect of LPS was also found on NOS activity in the cerebellum, where a very small stimulation may have also been caused by arginine feeding. These findings indicate that LPS stimulates NOS expression/activity both in the cerebellum and in the liver and suggest a complex pattern of modulation of iNOS by arginine, with NO being first produced in excess and then downregulating iNOS expression.