Germline and Somatic Pharmacogenomics to Refine Rectal Cancer Patients Selection for Neo-Adjuvant Chemoradiotherapy

Neoadjuvant chemoradiotherapy (nCRT) followed by radical surgery is the standard of care for patients with Locally Advanced Rectal Cancer (LARC). Current selection for nCRT is based on clinical criteria regardless of any molecular marker. Pharmacogenomics may be a useful strategy to personalize and optimize nCRT in LARC. This review aims to summarize the most recent and relevant findings about the role of germline and somatic pharmacogenomics in the prediction of nCRT outcome in patients with LARC, discussing the state of the art of their application in the clinical practice. A systematic literature search of the PubMed database was completed to identify relevant English-language papers published up to January 2020. The chemotherapeutic backbone of nCRT is represented by fluoropyrimidines, mainly metabolized by DPD (Dihydro-Pyrimidine Dehydrogenase,DPYD). The clinical impact of testingDPYD*2A, DPYD*13,c.2846A > Tandc.1236G > A-HapB3before a fluoropyrimidines administration to increase treatment safety is widely acknowledged. Other relevant target genes areTYMS(Thymidylate Synthase) andMTHFR(Methylene-Tetrahydro-Folate Reductase), whose polymorphisms were mainly studied as potential markers of treatment efficacy in LARC. A pivotal role of aTYMSpolymorphism in the gene promoter region (rs34743033) was reported and was pioneeringly used to guide nCRT treatment in a phase II study. The pharmacogenomic analysis of other pathways mostly involved in the cellular response to radiation damage, as the DNA repair and the activation of the inflammatory cascade, provided less consistent results. A high rate of somatic mutation in genes belonging to PI3K (Phosphatidyl-Inositol 3-Kinase) and MAPK (Mitogen-Activated Protein Kinase) pathways, asBRAF (V-raf murine sarcoma viral oncogene homolog B1), KRAS(Kirsten Rat Sarcoma viral oncogene homolog), NRAS(Neuroblastoma RAS viral (v-ras) oncogene homolog),PIK3CA(Phosphatidyl-Inositol-4,5-bisphosphate 3-Kinase, Catalytic Subunit Alpha), as well asTP53(Tumor Protein 53) was reported in LARC. Their pharmacogenomic role, already defined in colorectal cancer, is under investigation in LARC with promising results concerning specific somatic mutations inKRASandTP53, as predictors of tumor response and prognosis. The availability of circulating tumor DNA in plasma may also represent an opportunity to monitor somatic mutations in course of therapy

Genetic biomarkers for hepatocellular cancer risk in a caucasian population

AIMTo uncover novel genetic markers that could contribute to predicting hepatocellular carcinoma (HCC) susceptibility in Caucasians.METHODSThe present retrospective case-control study compared genotype frequencies between a cohort of HCC cases and two, independent, HCC-free, age/sex-matched control groups. The HCC cohort comprised 192 homogeneous patients that had undergone orthotopic liver transplantation. The first control group comprised 167 patients that were matched to the HCC cohort for the percentage of hepatitis B (HBV) and/or hepatitis C (HCV) infections. A second control group included 192 virus-free, healthy individuals that were used to evaluate the generalizability of the identified predictive markers. All cases and controls were Caucasian. The three study populations were characterized with a panel of 31 markers derived from 21 genes that encoded key proteins involved in hepatocarcinogenesis-related pathways. The study end-point was to assess the association between genetic variants and HCC onset.RESULTSFive genetic markers were identified as risk factors for HCC in high-risk patients infected with HBV/HCV. According to a dominant model, reduced HCC risk was associated with three polymorphisms: ERCC1 rs3212986 (OR = 0.46, 95% CI: 0.30-0.71, P = 0.0005), GST-P1 rs1138272 (OR = 0.41, 95% CI: 0.21-0.81, P = 0.0097), and CYP17A1 rs743572 (OR = 0.50, 95% CI: 0.31-0.79, P = 0.0032). Conversely, according to a recessive model, increased HCC risk was associated with two polymorphisms: XRCC3 rs1799794 (OR = 3.70, 95% CI: 1.02-13.39, P = 0.0461) and ABCB1 rs1128503 (OR = 2.06, 95% CI: 1.18-3.61, P = 0.0111). These associations remained significant in a subgroup analysis, where patients were stratified according to viral status (HBV-or HCV-positive serology). Two variants exhibited a serology-specific effect: ABCB1 rs1128503 (OR = 4.18, 95% CI: 1.55-11.29, P = 0.0048) showed an effect in the HBV-positive subgroup; and ERCC1 rs3212986 (OR = 0.33, 95% CI: 0.18-0.60, P = 0.0003) showed an effect in the HCV-positive subgroup. Among the five markers identified, ERCC1 rs3212986 (OR = 0.43, P < 0.0001) and CYP17A1 rs743572 (OR = 0.73, P = 0.0310) had a different distribution in patients with HCC compared to healthy individuals. With a recursive partitioning approach, we also demonstrated that significant gene-gene interactions between ERCC1 rs3212986, CYP17A1 rs743572, GST-P1 rs1138272, and the previously described UGT1A7*3 predictive marker, played a role in the complex trait of HCC susceptibility.CONCLUSIONWe identified five polymorphisms and interactions that contributed crucially to predicting HCC risk. These findings represented an important step towards improving HCC diagnosis and management

Inositol and Non-Alcoholic Fatty Liver Disease: A Systematic Review on Deficiencies and Supplementation

Liver lipid accumulation is a hallmark of non-alcoholic fatty liver disease (NAFLD), broadly associated with insulin resistance. Inositols (INS) are ubiquitous polyols implied in many physiological functions. They are produced endogenously, are present in many foods and in dietary supplements. Alterations in INS metabolism seems to play a role in diseases involving insulin resistance such as diabetes and polycystic ovary syndrome. Given its role in other metabolic syndromes, the hypothesis of an INS role as a supplement in NAFLD is intriguing. We performed a systematic review of the literature to find preclinical and clinical evidence of INS supplementation efficacy in NAFLD patients. We retrieved 10 studies on animal models assessing Myoinosiol or Pinitol deficiency or supplementation and one human randomized controlled trial (RCT). Overall, INS deficiency was associated with increased fatty liver in animals. Conversely, INS supplementation in animal models of fatty liver reduced hepatic triglycerides and cholesterol accumulation and maintained a normal ultrastructural liver histopathology. In the one included RCT, Pinitol supplementation obtained similar results. Pinitol significantly reduced liver fat, post-prandial triglycerides, AST levels, lipid peroxidation increasing glutathione peroxidase activity. These results, despite being limited, indicate the need for further evaluation of INS in NAFLD in larger clinical trials

Impact of ABCG2 and ABCB1 Polymorphisms on Imatinib Plasmatic Exposure: An Original Work and Meta-Analysis

Adequate imatinib plasma levels are necessary to guarantee an efficacious and safe treatment in gastrointestinal stromal tumor (GIST) and chronic myeloid leukemia (CML) patients. Imatinib is a substrate of the drug transporters ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) that can affect its plasma concentration. In the present study, the association between three genetic polymorphisms in ABCB1 (rs1045642, rs2032582, rs1128503) and one in ABCG2 (rs2231142) and the imatinib plasma trough concentration (Ctrough) was investigated in 33 GIST patients enrolled in a prospective clinical trial. The results of the study were meta-analyzed with those of other seven studies (including a total of 649 patients) selected from the literature through a systematic review process. The ABCG2 c.421C>A genotype demonstrated, in our cohort of patients, a borderline association with imatinib plasma trough levels that became significant in the meta-analysis. Specifically, homozygous carriers of the ABCG2 c.421 A allele showed higher imatinib plasma Ctrough with respect to the CC/CA carriers (Ctrough, 1463.2 ng/mL AA, vs. 1196.6 ng/mL CC + AC, p = 0.04) in 293 patients eligible for the evaluation of this polymorphism in the meta-analysis. The results remained significant under the additive model. No significant association could be described between ABCB1 polymorphisms and imatinib Ctrough, neither in our cohort nor in the meta-analysis. In conclusion, our results and the available literature studies sustain an association between ABCG2 c.421C>A and imatinib plasma Ctrough in GIST and CML patients

IL15RA and SMAD3 Genetic Variants Predict Overall Survival in Metastatic Colorectal Cancer Patients Treated with FOLFIRI Therapy: A New Paradigm

Simple SummaryThere is an increasing scientific interest in the study of the interaction between the immune system and drugs in cancer that can affect the efficacy of an anti-cancer treatment. This study was undertaken to better understand if the genetic characteristic of a cancer patient's immune system can predict the tumor response to the treatment and the duration of survival. The topic was studied on 335 metastatic colorectal cancer patients treated with a first-line chemotherapy (FOLFIRI regimen, irinotecan-5-fluorouracil-leucovorin). The research highlighted two markers, IL15RA-rs7910212 and SMAD3-rs7179840, significantly associated with the patient's survival. When considering IL15RA-rs7910212 and SMAD3-rs7179840 in combination with other two genetic markers previously investigated (NR1I2-rs1054190, VDR-rs7299460), we built up a highly predictive genetic score of survival. The herein identified markers must be further validated, but still represent good candidates to understand how much a patient with a metastatic colorectal cancer can benefit from a chemotherapy with FOLFIRI regimen.A new paradigm in cancer chemotherapy derives from the interaction between chemotherapeutics, including irinotecan and 5-fluorouracil (5-FU), and the immune system. The patient's immune response can modulate chemotherapy effectiveness, and, on the other hand, chemotherapeutic agents can foster tumor cell immunogenicity. On these grounds, the analysis of the cancer patients' immunogenetic characteristics and their effect on survival after chemotherapy represent a new frontier. This study aims to identify genetic determinants in the immuno-related pathways predictive of overall survival (OS) after FOLFIRI (irinotecan, 5-FU, leucovorin) therapy. Two independent cohorts comprising a total of 335 patients with metastatic colorectal cancer (mCRC) homogeneously treated with first-line FOLFIRI were included in the study. The prognostic effect of 192 tagging genetic polymorphisms in 34 immune-related genes was evaluated using the bead array technology. The IL15RA rs7910212-C allele was associated with worse OS in both discovery (HR: 1.57, p = 0.0327, Bootstrap p-value = 0.0280) and replication (HR: 1.71, p = 0.0411) cohorts. Conversely, SMAD3 rs7179840-C allele was associated with better OS in both discovery (HR: 0.65, p = 0.0202, Bootstrap p-value = 0.0203) and replication (HR: 0.61, p = 0.0216) cohorts. A genetic prognostic score was generated integrating IL15RA-rs7910212 and SMAD3-rs7179840 markers with inflammation-related prognostic polymorphisms we previously identified in the same study population (i.e., PXR [NR1I2]-rs1054190, VDR-rs7299460). The calculated genetic score successfully discriminated patients with different survival probabilities (p < 0.0001 log-rank test). These findings provide new insight on the prognostic value of genetic determinants, such as IL15RA and SMAD3 markers, and could offer a new decision tool to improve the clinical management of patients with mCRC receiving FOLFIRI

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma

Innovative strategies for tailoring therapy in cancer patient pharmacogenetics and therapy personalization in metastatic colorectal cancer patients treated with irinotecan

Pharmacogenetics focuses on inter-subject variation in drug therapeutic effects and toxicity depending on genetic polymorphisms. Irinotecan and fluoropyrimidine, currently used in cancer chemotherapy, are characterized by a sometimes unpredictably severe toxicity. Pharmacogenomics was largely applied in the last years to the irinotecan-based colorectal cancer (CRC) treatment personalization with limited data regarding validated marker of severe toxicity. In the first part of my thesis, I have been focusing on the investigation of innovative pharmacogenetic markers of neutropenia or gastrointestinal toxicity irinotecan-related using the “tagging polymorphisms (SNPs)” (TagSNPs) approach. Since therapeutic implications of cancer-related inflammation have gained great attention in recent years, innovative prospects for the optimization of tailored therapy arose. Two hundred and fifty metastatic CRC patients, homogeneously treated with an irinotecan-including regimen (FOLFIRI), have been collected retrospectively for this study. Clinical parameters of toxicity (by NCI-CTC scale) and response to the therapy (by WHO criteria) were monitored all along the study. They were genotyped for 246 htSNPs characterizing 22 transcriptional regulators and cytokines inflammation-related genes; positive findings were replicated in a cohort of 167 metastatic CRC patients receiving FOLFIRI-based therapy. One polymorphism (rs1053004) in STAT-3 gene resulted predictive of severe GI toxicity in both discovery and replication cohort with a protective effect toward the risk of developing grade 3-4 events (OR=0.51 CI=0.27-0.99 p=0.045; OR=0.38 CI=0.15-0.95 p=0.038, respectively). Additional variants in NRs genes, especially HNF4α and VDR, although not validated, were suggested to contribute to determining the risk of developing neutropenia and GI toxicity. Preliminary pharmacokinetic data supported the observed genotype/phenotype clinical associations. A validated contribution of STAT-3 rs1053004 in determining GI toxicity risk after FOLFIRI therapy was pointed out. Further potential predictive markers of irinotecan-related toxicity were suggested. These findings could represent a further step towards personalized FOLFIRI therapy. UGT1A1*28 polymorphism has been demonstrated in the last years to have an impact on irinotecan pharmacokinetics and toxicity to the treatment. Although, the adoption of a pre-emptive UGT1A1*28 genotyping to increase irinotecan safety and to better characterize patient “Diagnosis Related Groups”, for therapy reimbursement purposes in clinical practice, is still limited. The second part of my thesis aimed to estimate the effect of UGT1A1*28 on the costs associated with irinotecan-related toxicity. A retrospective analysis of the costs of toxicity management was conducted on a subset of the aforementioned population of 250 mCRC patients. 243 mCRC patients treated with FOLFIRI have been genotyped for UGT1A1*28. The mean predicted cost per patient was higher for *1/*28 (1,119€) and *28/*28 (4,886€), as compared to *1/*1 (812€) (P<0.001). This is consistent with a different grade 4 toxicity profile among the three groups of patients, and a higher frequency of costly interventions like hospitalization among patients with the *28 allele. The aim of the third part of my thesis consisted of evaluating the implementation of the routine application of prospective DPYD risk variants and UGT1A1*28 screening at the National Cancer Center CRO of Aviano. A Pharmacogenetic implementation infrastructure has been set-up starting from January 2014 for the prevention of irinotecan (UGT1A1*28 rs8175347) and/or fluoropyrimidine (DPYD rs3918290, rs55886062, rs67376798)-associated toxicity in the clinical routine of the National Cancer Center CRO of Aviano. Genotyping was performed by PCR-based methods, such as pyrosequencing, Sanger sequencing, and fragment analysis. A digital Pharmacogenomic report including the dose-adjustment recommended according to the published pharmacogenetics guidelines will finally be embedded in patients’ clinical record and ultimately made available to the medical personnel. From September 2011 to September 2016, a total of 393 patients were genotyped for such variants at CRO-Aviano. Three hundred and eighty-six out of 393 patients were screened for at least one DPYD variants and 40 for UGT1A1*28. Of these patients, 9 patients (2.58%) were found to carry at least one DPYD variants, and two patients (5.00%) were found to carry two *28 risk alleles for UGT1A1. Moreover, twenty-three patients out of 393 (5.85%) were referred for toxicity from the CRO-Aviano oncologists. In conclusion, in this work of thesis, interesting molecular markers with a predictive value on pharmacokinetics and pharmacodynamics of irinotecan were described. A possible application of these parameters in the clinical practice will be useful to design a tailored irinotecan dosing based on peculiar characteristics of each patient. In addition to the prevention of severe toxicity, pre-treatment UGT1A1*28 genotyping should be considered to save economic resources related to the management of irinotecan-related toxicities and for innovative reimbursement strategies. Plus, the implementation of pre-emptive pharmacogenetics tests is now part of a European Project (U-PGx) with the aim of providing the final proof of pharmacogenetics efficacy in increasing drug safety when fully integrated into the clinical practice

Lipid rafts as viral entry routes and immune platforms: A double-edged sword in SARS-CoV-2 infection?

Lipid rafts are nanoscopic compartments of cell membranes that serve a variety of biological functions. They play a crucial role in viral infections, as enveloped viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can exploit rafts to enter or quit target cells. On the other hand, lipid rafts contribute to the formation of immune synapses and their proper functioning is a prerequisite for adequate immune response and viral clearance. In this narrative review we dissect the panorama focusing on this singular aspect of cell biology in the context of SARS-CoV-2 infection and therapy. A lipid raft-mediated mechanism can be hypothesized for many drugs recommended or considered for the treatment of SARS-CoV-2 infection, such as glucocorticoids, antimalarials, immunosuppressants and antiviral agents. Furthermore, the additional use of lipid-lowering agents, like statins, may affect the lipid composition of membrane rafts and thus influence the processes occurring in these compartments. The combination of drugs acting on lipid rafts may be successful in the treatment of more severe forms of the disease and should be reserved for further investigation