Elisa Sottotetti

DPD and UGT1A1 deficiency in colorectal cancer patients receiving triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan.

AIMS Triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan is a standard therapy for metastatic colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) in DPYD and UGT1A1 influence fluoropyrimdines and irinotecan adverse events (AEs). Low frequency DPYD variants (c.1905 + 1G > A, c.1679 T > G, c.2846A > T) are validated but more frequent ones (c.496A > G, c.1129-5923C > G and c.1896 T > C) are not. rs895819 T > C polymorphism in hsa-mir-27a is associated with reduced DPD activity. In this study, we evaluated the clinical usefulness of a pharmacogenetic panel for patients receiving triplet combinations. METHODS Germline DNA was available from 64 CRC patients enrolled between 2008 and 2013 in two phase II trials of capecitabine, oxaliplatin and irinotecan plus bevacizumab or cetuximab. SNPs were determined by Real-Time PCR. We evaluated the functional variants in DPYD (rare: c.1905 + 1G > A, c.1679 T > G, c.2846A > T; most common: c.496A > G, c.1129-5923C > G, c.1896 T > C), hsa-mir-27a (rs895819) and UGT1A1 (*28) genes to assess their association with grade 3-4 AEs. RESULTS None of the patients carried rare DPYD variants. We found DPYD c.496A > G, c.1129-5923C > G, c.1896 T > C in heterozygosity in 19%, 5% and 8%, respectively, homozygous rs895819 in hsa-mir-27a in 9% and homozygous UGT1A1*28 in 8%. Grade 3-4 AEs were observed in 36% patients and were associated with DPYD c.496A > G (odds ratio (OR) 4.93, 95% CI 1.29, 18.87; P = 0.021) and homozygous rs895819 in hsa-mir-27a (OR 11.11, 95% CI 1.21, 102.09; P = 0.020). Carriers of DPYD c.1896 T > C and homozygous UGT1A1*28 showed an OR of 8.42 (95% CI 0.88, 80.56; P = 0.052). Multivariate analysis confirmed an independent value for DPYD c.496A > G and c.1896 T > C. CONCLUSIONS Concomitant assessment of DPYD variants and the UGT1A1*28 allele is a promising strategy needing further validation for dose personalization.

Circulating tumor cells as a longitudinal biomarker in patients with advanced chemorefractory, RAS-BRAF wild-type colorectal cancer receiving cetuximab or panitumumab.

A still relevant number of patients with RAS‐BRAF wild‐type colorectal cancer (CRC) do not respond to treatment with antiepidermal growth factor receptor (EGFR) monoclonal antibodies cetuximab and panitumumab, suggesting that additional biomarkers to guide patient selection are urgently needed. Circulating tumor cells (CTCs) may represent such a biomarker. In this prospective study, 38 patients with advanced RAS‐BRAF‐wild‐type CRC received third‐line therapy with cetuximab‐irinotecan or panitumumab. Peripheral blood samples for CTC status determination were collected at baseline, during treatment at early (2–4 weeks) and at later (8–10 weeks) times. CTC enrichment was done with the AdnaTest ColonCancerSelect kit, whereas CTC detection was done with the AdnaTest ColonCancerDetect kit. CTC status positivity was defined according to the kit manufacturers thresholds. Fifty percent of patients were defined as CTC positive at baseline and the overall RECIST response rate was 26%. CTC baseline status was not associated with treatment response, whereas early CTC status and CTC status changes during treatment were significantly associated with tumor response. Kaplan‐Meier analysis showed a significantly shorter progression‐free survival (median, 2.0 versus 4.0 months, p = 0.004) and overall survival (4.7 versus11.4, p = 0.039) in patients with early CTC + status compared with CTC ‐ ones. In multivariable analysis including classical prognostic factors, the CTC status changes profile during treatment was an independent predictor of both progression‐free survival (p < 0.001) and overall‐survival (p = 0.001). CTC status assessed early during treatment with anti‐EGFR monoclonal antibodies may predict treatment failure in advance compared to imaging‐based tools.

Circulating Biomarkers in Advanced Colorectal Cancer Patients Randomly Assigned to Three Bevacizumab-Based Regimens

The need to identify biomarkers for bevacizumab-based treatment in advanced colorectal cancer is imperative. The aim of this study was to investigate the prognostic role of circulating VEGF, PDGF, SDF-1, osteopontin and CEA in patients randomly assigned to three bevacizumab-based regimens. Plasma samples from 50 patients treated at a single Institution were analysed using the multiplex assay BioPlex™ 2200 (Bio-Rad Laboratories, Inc, Berkeley, CA, USA) at baseline, before first three cycles and subsequently every three cycles until disease progression. Prognostic analyses of baseline values were performed using multivariable Cox models, including disease extension >10 cm or ≤10 cm (measured as the sum of the diameters for all target lesions) as adjustment factor. The association between progression-free and overall survival and biomarkers modulation during treatment was studied using multivariable Cox models, which included summary statistics synthesizing during-treatment modulation together with disease extension. The biomarkers significantly associated with disease extension were baseline CEA (p = 0.012) and SDF-1 (p = 0.030). High values of VEGF and SDF-1 tended to be associated with worse prognosis, especially in terms of overall survival. The negative prognostic trend was more marked for baseline CEA as compared to other biomarkers; increasing values during treatment was significantly related to worse prognosis independently of disease extension (p = 0.007 and 0.016 for progression-free and overall survival, respectively). VEGF is related to bevacizumab pharmacodynamics and is associated to other angiogenic cytokines; some of the proposed biomarkers such as SDF-1 and CEA should be further validated for prognosis assessment and monitoring of bevacizumab-based treatment of advanced colorectal cancer.

Undetected toxicity risk in pharmacogenetic testing for dihydropyrimidine dehydrogenase.

Fluoropyrimidines, the mainstay agents for the treatment of colorectal cancer, alone or as a part of combination therapies, cause severe adverse reactions in about 10%–30% of patients. Dihydropyrimidine dehydrogenase (DPD), a key enzyme in the catabolism of 5-fluorouracil, has been intensively investigated in relation to fluoropyrimidine toxicity, and several DPD gene (DPYD) polymorphisms are associated with decreased enzyme activity and increased risk of fluoropyrimidine-related toxicity. In patients carrying non-functional DPYD variants (c.1905+1G>A, c.1679T>G, c.2846A>T), fluoropyrimidines should be avoided or reduced according to the patients’ homozygous or heterozygous status, respectively. For other common DPYD variants (c.496A>G, c.1129-5923C>G, c.1896T>C), conflicting data are reported and their use in clinical practice still needs to be validated. The high frequency of DPYD polymorphism and the lack of large prospective trials may explain differences in studies’ results. The epigenetic regulation of DPD expression has been recently investigated to explain the variable activity of the enzyme. DPYD promoter methylation and its regulation by microRNAs may affect the toxicity risk of fluoropyrimidines. The studies we reviewed indicate that pharmacogenetic testing is promising to direct personalised dosing of fluoropyrimidines, although further investigations are needed to establish the role of DPD in severe toxicity in patients treated for colorectal cancer.

Variant alleles in factor V, prothrombin, plasminogen activator inhibitor-1, methylenetetrahydrofolate reductase and risk of thromboembolism in metastatic colorectal cancer patients treated with first-line chemotherapy plus bevacizumab

Single-nucleotide polymorphisms (SNPs) related to hereditary thrombophilia were investigated as risk factors for thromboembolism in cancer patients. Their effect in metastatic colorectal cancer (mCRC) has never been explored so far. Our aim was to analyse the effect of coagulation factor V (FVL G1691A), prothrombin (PT G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and plasminogen activator inhibitor type 1 (PAI-1 5G/4G) allelic variants in this setting. Fifty-two patients treated with first-line chemotherapy plus bevacizumab who developed a thromboembolic event in their lifetime were initially genotyped. A contemporary cohort of 127 patients who did not experience any thromboembolic event was also analysed. DNA was extracted from peripheral blood and genotypes were determined by real-time PCR, using LightSNiP (TIB MOLBIOL) on LightCcler 480 (Roche). The association between thromboembolism and SNPs was investigated by univariable and multivariable analyses. All SNPs were in Hardy–Weinberg equilibrium (χ2 test P>0.20). FVL G1691A and PT G20210A were present only in heterozygosis in 4 (2.2%) and 7 (3.9%) patients, respectively; MTHFR C677T in homozygosis in 29 (16.2%), MTHFR A1298C in homozygosis in 13 (7.3%); PAI-1 5G/4G in 98 (54.7%) and 4G/4G in 41 (23%) patients. At univariable analysis, treatment duration was significantly associated with thromboembolism (P<0.001), whereas gender, age, obesity, platelets count and chemotherapy backbone were not. Similarly, FVL G1691A and PT G20210A as well as MTHFR C677T and PAI-1 4G allele were significantly associated, whereas MTHFR A1298C was not. At multivariable model including PT G20210A, MTHFR C677T and PAI-1 4G (age, obesity, treatment duration and chemotherapy backbone were included as adjustment factors), the three SNPs were significantlty associated with higher risk of thromboembolism (P=0.025, <0.0001 and P=0.033, respectively). Further validation studies are warranted in order to design a prospective trial of thromboprophylaxis in mCRC patients with high-risk genotypes.

Development of a Protocol for Single-Cell Analysis of Circulating Tumor Cells in Patients with Solid Tumors

Genomic characterization of circulating tumor cells (CTCs) enables the monitoring of tumor progression and of adaption occurring during treatment. CTC molecular characterization represents indeed a precious tool to implement in the clinical practice for better dealing with acquired resistance to systemic treatment and tumor evolution. Unfortunately CTCs are very rare and enrichments from blood samples and subsequent identification of these cells are technically very challenging. We describe here the main steps leading to the development of a technical protocol for visualization, enumeration and recovery of single CTCs exploiting the recently developed DEPArray™platform. Our description of the technical workflow starts with evaluation of pre-analytical aspects related to blood sample collection warning about the possible effects on immunoreactivity profiles which may bias the interpretation. Subsequently, other CTC-enrichment approaches are critically discussed and compared in relation to their performances with the DEPArray™. Identification of CTCs represents another critical point due to their heterogeneity and due to the still-to-be clarified role of different subpopulations, typically epithelial, mesenchymal or mixed. Finally, issues related to single cell analysis are illustrated. The chapter ends with an overview of results obtained on real clinical samples which support the reliability of the protocol and its transferability to the daily clinical routine.

Serum soluble urokinase-type plasminogen activator receptor as a serum marker of inflammatory response that leads to tissue damage and surgical complication.

Unrestrained activation of the proteolytic systems in anastomotic tissue during repair has been implicated in the pathogenesis of anastomotic leakage. We hypothesized that this mechanism may promote an up‐regulation of the urokinase‐type plasminogen activator system and a spillover of soluble urokinase‐type plasminogen activator receptor (suPAR) into blood. In this retrospective analysis patients with anastomotic leakage were compared with a group of matched uncomplicated patients. Anastomotic leakage complicated patients had significantly higher suPAR (p = 0.04) levels until day 3 after surgery. The area under the receiver‐operating characteristic (ROC) for suPAR was higher than that CRP (0.874 vs. 0.836). Their analysis suggests the possible use of suPAR as serum marker to characterize the persistent inflammatory response that lead to tissue damage and surgical complication.

Body mass index and clinical benefit of fulvestrant in postmenopausal women with advanced breast cancer.

Purpose Obesity is a known risk factor for breast cancer and has been linked to increased risk of recurrence and death in breast cancer patients. Little is known about the predictive value of obesity. As endocrine therapy is widely used for breast cancer treatment worldwide, we aimed at correlating baseline body mass index (BMI) with clinical benefit derived from fulvestrant in postmenopausal women with advanced breast cancer. Methods We analyzed consecutive patients treated with fulvestrant in our center between January 2009 and March 2015. Patients were categorized as normal (BMI 18.5-24.9 kg/m2), overweight (BMI 25-29 kg/m2) and obese (BMI >30 kg/m2). The antitumor activity of fulvestrant was evaluated in terms of the clinical benefit rate (CBR). Results Seventy-five consecutive patients matched the eligibility criteria for analysis. Fulvestrant was administered as first-line therapy in 4 (5%) cases, as second line in 27 (36%) and as third line and beyond in 44 (59%) cases. According to BMI, 44 (59%) patients were classified as normal weight, 19 (25%) as overweight, and 12 (16%) as obese. No difference in estrogen receptor expression was found in relation to BMI. CBR was 53% overall, but rose to 70.5% in normal-weight patients and dropped to 31.6% and 25% in overweight and obese patients, respectively (p<0.001). Conclusions Increased BMI has a negative influence on treatment outcome. Even with the limitation of the relatively small sample size, it appears that patients of normal weight are 2.5-fold more likely to benefit from fulvestrant as overweight and obese patients.

532PPROSPECTIVE OBSERVATIONAL STUDY FOR DPYD AND UGT1A1 DEFICIENCY-ASSOCIATED TOXICITY IN PATIENTS WITH METASTATIC COLORECTAL CANCER (MCRC) RECEIVING TRIPLET CHEMOTHERAPY WITH CAPECITABINE, IRINOTECAN AND OXALIPLATIN (COI)

ABSTRACT Aim: Triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan is a standard treatment of mCRC, even if toxicity is significantly increased over doublets. Genetic variations in DPYD and UGT1A1 genes influence fluoropyrimdines and irinotecan adverse events (AEs), respectively. Low-frequency variants - such as DPYD c.1905 + 1G > A, c.1679T > G, c.2846A > T and homozygous UGT1A1*28 - are validated. More common DPYD variants– such as c.496A > G and the deep intronic variant c.1129-5923C > G–are controversial and not routinely used. Their assessment, particularly in association with altered UGT1A1 metabolism, may be valuable for patients receiving intensive triplet combinations. Methods: From 2008 to 2013, 64 pts enrolled in two phase II trials of COI plus bevacizumab [EudraCT No. 2008-008749-39] or cetuximab [EudraCT No. 2008-001062-93] - gave written consent at Fondazione IRCCS Istituto Nazionale dei Tumori. All genotypes were determined by Real-Time PCR, using LightSNiP (Roche). Inclusion criteria: absence of c.1905 + 1G > A, c.1679T > G, c.2846A > T. We aimed at genotyping c.496A > G and c.1129-5923C > G and UGT1A1*28 to assess associations with grade 3-4 chemotherapy-induced AEs (cAEs). Results: We found heterozygous DPYD 1129-5923C > G and 496A > G variants in 4 (6,3%) and 12 (18,8%) pts (concomitantly only in 1); 32 (50%) pts heterozygous and 5 (7,8%) homozygous for UGT1A1*28; concomitant heterozygosis of DPYD 496A > G and UGT1A1*28 in 7 (11%). Grade 3-4 cAE observed in 22 pts (35%; diarrhea 28%, neutropenia 8%, asthenia 3%). Probably due to low frequency, DPYD 1129-5923C > G was not significantly associated with severe toxicity. Grade 3-4 cAEs were observed in 58% pts with 496A > G polymorphism vs. 29% others (p = 0.053). As expected, toxicity was increased in pts with UGT1A1*28/*28 (p = 0.038). Pts with concomitant heterozygosis of DPYD 496A > G and UGT1A1*28 had higher incidence of cAEs as compared to all others (71% vs. 30%; p = 0.029). Conclusions: Concomitant DPYD 496A > G and UGT1A1*28 assessment is promising to predict severe toxicity following triplet chemotherapy with COI regimen. A prospective trial of dose modulation according to our pharmacogenetic panel is recruiting at our Institution. Disclosure: All authors have declared no conflicts of interest.