Search

IntroductionInflammatory factors released during severe coronavirus disease-19 (COVID-19) caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are known to influence drug exposure, but data on the effect of mild infection are few. Here we describe for the first time an increase in plasma imatinib and norimatinib concentrations observed in a series of 5 patients treated with imatinib for gastrointestinal stromal tumor (GIST) after mild COVID-19.MethodsThe patients were undergoing routine therapeutic drug monitoring (TDM) and pharmacogenetic (PGx) analyses of polymorphisms in genes involved in imatinib metabolism and transport (CYP3A4, CYP3A5, ABCB1, and ABCG2) when SARS-CoV-2 infection occurred. Imatinib and its active metabolite norimatinib concentrations were determined at Ctrough using a validated LC-MS/MS method. PGx analyses were performed by KASP genotyping assays on a Real-Time PCR system. All patients received imatinib 400 mg/day. Case 1 was prospectively monitored. Cases 2-5 were identified retrospectively.ResultsOn average, imatinib Ctrough increased significantly by 70% during COVID-19, whereas norimatinib showed a 44% increase compared with pre-COVID-19 levels. Elevated plasma imatinib concentrations persisted up to 6 months after infection remission. In 3 cases, this increase reflected the occurrence or worsening of imatinib side effects.ConclusionThis case-series highlights the clinical impact of SARS-CoV-2 infection on the management of patients with GIST treated with imatinib.

Palbociclib, an oral inhibitor of cyclin-dependent kinase 4 and 6, is approved for the treatment of metastatic breast cancer. This study investigated the influence of diverse clinical and biological factors—age, renal function, genetic variations, and concomitant medications (pharmacokinetic covariates)—on palbociclib pharmacokinetics. Employing a validated LC-MS/MS method, we analyzed the minimum plasma concentrations (Ctrough) of palbociclib in 68 women and determined the percentage deviations from the median Ctrough for each dosage group. Variations in a panel of absorption, distribution, metabolism, and excretion (ADME) genes were assessed using end-point allele-specific fluorescence detection and pyrosequencing. Two distinct patient cohorts were defined based on median values of age, creatinine, and eGFR, which exhibited statistically significant differences in percentage deviations (p = 0.0095, p = 0.0288, and p = 0.0005, respectively). Homozygous carriers of the PPARA variants displayed larger positive percentage deviations than the other group (p = 0.0292). Similarly, patients concurrently taking CYP3A and P-glycoprotein inhibitors alongside anticancer therapy exhibited significant variations (p = 0.0285 and p = 0.0334, respectively). Furthermore, exploring the drug–drug–gene interactions between inhibitors of CYP3A and P-glycoprotein with their respective genetic variants revealed two patient groups with statistically different percentage deviations (p = 0.0075, p = 0.0012, and p = 0.0191, respectively). These results could help address cases where pharmacokinetic covariates or subclinical conditions impair palbociclib adherence or response, aiming to offer tailored dosing strategies or monitoring for individual patients.

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure–toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95–106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22–55%),with high recovery rates (81–93%) and minimal matrix effects (ME 0.9–1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Abstract Background Despite a growing number of publications highlighting the potential impact on the therapy outcome, rare genetic variants (minor allele frequency

Objectives: A cost-utility analysis was conducted to evaluate pharmacogenomic (PGx)-guided treatment compared to the standard-of-care intervention among patients diagnosed with colorectal cancer (CRC) in Italy. Methods: Data derived from a prospective, open-label, block randomized clinical trial, as a part of the largest PGx study worldwide (355 patients in both arms) were used. Mortality was used as the primary health outcome to estimate life years (LYs) gained in treatment arms within a survival analysis context. PGx-guided treatment was based on established drug-gene interactions between capecitabine, 5-fluorouracil and irinotecan with DPYD and/or UGT1A1 genomic variants. Utility values for the calculation of Quality Adjusted Life Year (QALY) was based on Visual Analog Scale (VAS) score. Missing data were imputed via the Multiple Imputation method and linear interpolation, when possible, while censored cost data were corrected via the Replace-From-The-Right algorithm. The Incremental Cost-Effectiveness Ratio (ICER) was calculated for QALYs. Raw data were bootstrapped 5000 times in order to produce 95% Confidence Intervals based on non-parametric percentile method and to construct a cost-effectiveness acceptability curve. Cost differences for study groups were investigated via a generalized linear regression model analysis. Total therapy cost per patient reflected all resources expended in the management of any adverse events, including medications, diagnostics tests, devices, surgeries, the utilization of intensive care units, and wards. Results: The total cost of the study arm was estimated at €380 (∼ US$416; 95%CI: 195–596) compared to €565 (∼ US$655; 95%CI: 340–724) of control arm while the mean survival in study arm was estimated at 1.58 (+0.25) LYs vs 1.50 (+0.26) (Log Rank test, X2 = 4.219, df=1, p-value=0.04). No statistically significant difference was found in QALYs. ICER was estimated at €13418 (∼ US$14695) per QALY, while the acceptability curve indicated that when the willingness-to-pay was under €5000 (∼ US$5476), the probability of PGx being cost-effective overcame 70%. The most frequent adverse drug event in both groups was neutropenia of severity grade 3 and 4, accounting for 82.6% of total events in the study arm and 65.0% in the control arm. Apart from study arm, smoking status, Body-Mass-Index and Cumulative Actionability were also significant predictors of total cost. Subgroup analysis conducted in actionable patients (7.9% of total patients) indicated that PGx-guided treatment was a dominant option over its comparator with a probability greater than 92%. In addition, a critical literature review was conducted, and these findings are in line with those reported in other European countries. Conclusion: PGx-guided treatment strategy may represent a cost-saving option compared to the existing conventional therapeutic approach for colorectal cancer patient management in the National Health Service of Italy.

A wide interindividual variability in therapeutic response to cyclin-dependent kinases 4 and 6 inhibitors (CDKis) palbociclib, ribociclib and abemaciclib, among patients with HR+/HER2- metastatic breast cancer has been reported. This study explored the impact of genetic polymorphisms in ADME genes (responsible for drug absorption, distribution, metabolism, and elimination) on CDKis safety profiles in 230 patients. Selected endpoints include grade 3/4 neutropenia at day 14 of the first treatment cycle, early dose-limiting toxicities (DLTs), and dose reductions within the initial three cycles. Our analysis revealed associations between these endpoints and polymorphisms in CYP3A4, CYP3A5, ABCB1, and ABCG2 genes. Their impact on CDKis plasma concentrations (Ctrough) was also examined. Specifically, ABCB1 c.1236C>T and c.2677C>T polymorphisms correlated significantly with grade 3/4 neutropenia at day 14 (OR 3.94, 95% CI 1.32–11.75; p = 0.014 and OR 3.32, 95% CI 1.12-9.85; p = 0.030). Additionally, ABCB1 c.3435C>T was associated with an elevated risk of early DLTs and dose reductions (OR 3.28, 95% CI 1.22–8.84, p = 0.019; OR 2.60, 95% CI 1.20–5.60, p = 0.015). Carriers of the CYP3A4*22 allele also demonstrated in univariate a higher risk of early DLTs (OR 3.10, 95% CI 1.01–9.56, p = 0.049). Furthermore, individuals with the ABCB1 1236T–3435T–2677T(A) variant haplotype exhibited significant associations with grade 3/4 neutropenia at day 14 (OR 3.36, 95% CI 1.20–9.41; p = 0.021) and early DLTs in univariate (OR 3.08, 95% CI 1.19–7.95; p = 0.020). Homozygous carriers of the ABCB1 T-T-T(A) haplotype tended to have a higher mean ribociclib Ctrough (934.0 ng/mL vs. 752.0 ng/mL and 668.0 ng/mL). Regardless preliminary, these findings offer promising insights into the role of pharmacogenetic markers in CDKis safety profiles, potentially contributing to address the interindividual variability in CDKis responses.

The impact of body mass index (BMI) on treatment outcomes in patients with cancer is gaining increasing attention given the limited data available. The aim of this study was to investigate the contribution of BMI on the safety and efficacy profile of palbociclib in 134 patients with metastatic luminal-like breast cancer treated with palbociclib and endocrine therapy (ET). Normal-weight and underweight patients (BMI

Abstract 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.

Background: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). Aims: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. Methods: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients’ erythrocytes by HPLC and associated with patients’ age group and TPMT DNA methylation. Results: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10−5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. Conclusion: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

Preemptive targeted pharmacogenetic testing of candidate variations in DPYD is currently being used to limit toxicity associated with fluoropyrimidines. The use of innovative next generation sequencing (NGS) approaches could unveil additional rare (minor allele frequency

A wide inter-individual variability in the therapeutic response to cyclin-dependent kinases 4 and 6 inhibitors (CDKis) has been reported. We herein present a case series of five patients treated with either palbociclib or ribociclib referred to our clinical pharmacological counselling, including therapeutic drug monitoring (TDM), pharmacogenetics, and drug–drug interaction analysis to support clinicians in the management of CDKis treatment for metastatic breast cancer. Patients’ plasma samples for TDM analysis were collected at steady state and analyzed by an LC-MS/MS method for minimum plasma concentration (Cmin) evaluation. Under and overexposure to the drug were defined based on the mean Cmin values observed in population pharmacokinetic studies. Polymorphisms in selected genes encoding for proteins involved in drug absorption, distribution, metabolism, and elimination were analyzed (CYP3A4, CYP3A5, ABCB1, SLCO1B1, and ABCG2). Three of the five reported cases presented a CDKi plasma level above the population mean value and were referred for toxicity. One of them presented a low function ABCB1 haplotype (ABCB1-rs1128503, rs1045642, and rs2032582), possibly causative of both increased drug oral absorption and plasmatic concentration. Two patients showed underexposure to CDKis, and one of them was referred for early progression. In one patient, a CYP3A5*1/*3 genotype was found to be potentially responsible for more efficient drug metabolism and lower drug plasma concentration. This intensified pharmacological approach in clinical practice has been shown to be potentially effective in supporting prescribing oncologists with dose and drug selection and could be ultimately useful for increasing both the safety and efficacy profiles of CDKi treatment.

Background The clinical implementation of pharmacogenomics (PGx) could be one of the first milestones towards realizing personalized medicine in routine care. However, its widespread adoption requires the availability of suitable clinical decision support (CDS) systems, which is often impeded by the fragmentation or absence of adequate health IT infrastructures. We report results of CDS implementation in the large-scale European research project Ubiquitous Pharmacogenomics (U-PGx), in which PGx CDS was rolled out and evaluated across more than 15 clinical sites in the Netherlands, Spain, Slovenia, Italy, Greece, United Kingdom and Austria, covering a wide variety of healthcare settings. Methods We evaluated the CDS implementation process through qualitative and quantitative process indicators. Quantitative indicators included statistics on generated PGx reports, median time from sampled upload until report delivery and statistics on report retrievals via the mobile-based CDS tool. Adoption of different CDS tools, uptake and usability were further investigated through a user survey among healthcare providers. Results of a risk assessment conducted prior to the implementation process were retrospectively analyzed and compared to actual encountered difficulties and their impact. Results As of March 2021, personalized PGx reports were produced from 6884 genotyped samples with a median delivery time of twenty minutes. Out of 131 invited healthcare providers, 65 completed the questionnaire (response rate: 49.6%). Overall satisfaction rates with the different CDS tools varied between 63.6% and 85.2% per tool. Delays in implementation were caused by challenges including institutional factors and complexities in the development of required tools and reference data resources, such as genotype-phenotype mappings. Conclusions We demonstrated the feasibility of implementing a standardized PGx decision support solution in a multinational, multi-language and multi-center setting. Remaining challenges for future wide-scale roll-out include the harmonization of existing PGx information in guidelines and drug labels, the need for strategies to lower the barrier of PGx CDS adoption for healthcare institutions and providers, and easier compliance with regulatory and legal frameworks.

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.

Identifying patients at risk of poor response to neoadjuvant chemoradiotherapy (nCRT) is an emerging clinical need in locally advanced rectal cancer (LARC). SMAD3 is a key player in the chemoradio-resistance phenotype and its expression is both constitutive and locally induced. The aim was to investigate both host (genetic polymorphisms) and tumor SMAD3 profiling to predict response to nCRT. In a group of 76 LARC patients, SMAD3 and phosphorylated-SMAD3 expression was assessed by immunohistochemistry in preoperative tumor tissue. In an expanded study group (n = 378), a set of SMAD3 polymorphisms (rs35874463, rs1065080, rs1061427, rs17228212, rs744910, and rs745103) was analyzed. Association with tumor regression grade (TRG) and patient prognosis (progression-free survival [PFS] and overall survival [OS]) was assessed. Patients with high tumor expression of SMAD3 had a significantly increased risk of poor response (TRG≥2) [cellularity >55% (OR:10.36, p = 0.0004), or moderate/high intensity (OR:5.20, p = 0.0038), or an H-score≥1 (OR:9.84, p = 0.0004)]. Patients carrying the variant SMAD3 rs745103-G allele had a poorer response (OR:0.48, p = 0.0093), a longer OS (HR:0.65, p = 0.0307), and a trend for longer PFS (HR:0.75, p = 0.0944). Patients who carried both high SMAD3 tumor expression and the wild-type rs745103-A allele had an extremely high risk of not achieving a complete response (OR:13.45, p = 0.0005). Host and tumor SMAD3 status might be considered to improve risk stratification of LARC patients to facilitate selection for alternative personalized neoadjuvant strategies including intensified regimens.

Microenvironmental factors such as non-classical human leukocyte antigen-G (HLA-G) have been associated with cancer invasiveness and metastatic progression. HLA-G expression has been associated with specific single-nucleotide polymorphisms (SNP) in HLA-G 3′untranslated region (UTR) in several diseases. The primary aim was to investigate the predictive role of HLA-G polymorphisms on treatment efficacy in metastatic colorectal cancer (mCRC) patients homogeneously treated with first-line FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin) and their association with soluble HLA-G (sHLA-G) plasma concentration. HLA-G 3′UTR was sequenced in 248 patients. A set of eight polymorphisms and related haplotypes were analyzed for their association with best tumor response, overall survival (OS), and progression-free survival (PFS). sHLA-G was measured by immunoassay in 35 available plasma samples and correlated with HLA-G 3′UTR polymorphisms/haplotypes. Our results showed that carriers of rs371194629 (+2960)-Ins allele were at risk for lack of complete response (hazard ratio (HR):0.29, pBH = 0.0336), while carriers of rs1710 (+3010)-G allele (rs1063320 (+3142)-C allele in linkage-disequilibrium), and rs9380142 (+3187)-G allele had a higher chance of complete response according to additive models (HR:4.58, pBH = 0.0245; HR:3.18, pBH = 0.0336, respectively). The combination of rs371194629-Del, rs1710-G, and rs9380142-G alleles forms the UTR1 haplotype. Patients who were carriers of UTR1/UTR-1 diplotype had a greater chance of complete response to therapy (HR:10.59, pBH = 0.0294). The same three beneficial alleles showed a trend toward higher pre-treatment sHLA-G plasma levels, supporting a functional role for polymorphisms in protein secretion. In conclusion, genetic variants of HLA-G are associated with treatment efficacy in mCRC patients treated with first-line FOLFIRI. This finding shed light on the combined effect of this immune system factor and chemotherapy in cancer patients.

Pharmacogenetics plays a key role in personalized cancer treatment. Currently, the clinically available pharmacogenetic markers for metastatic colorectal cancer (mCRC) are in genes related to drug metabolism, such as DPYD for fluoropyrimidines and UGT1A1 for irinotecan. Recently, the impact of host variability in inflammatory and immune-response genes on treatment response has gained considerable attention, opening innovative perspectives for optimizing tailored mCRC therapy. A literature review was performed on the predictive role of immune-related germline genetic biomarkers on pharmacological outcomes in patients with mCRC. Particularly, that for efficacy and toxicity was reported and the potential role for clinical management of patients was discussed. Most of the available data regard therapy effectiveness, while the impact on toxicity remains limited. Several studies focused on the effects of polymorphisms in genes related to antibody-dependent cellular cytotoxicity (FCGR2A, FCGR3A) and yielded promising but inconclusive results on cetuximab efficacy. The remaining published data are sparse and mainly hypothesis-generating but suggest potentially interesting topics for future pharmacogenetic studies, including innovative gene–drug interactions in a clinical context. Besides the tumor immune escape pathway, genetic markers belonging to cytokines/interleukins (IL-8 and its receptors) and angiogenic mediators (IGF1) seem to be the best investigated and hopefully most promising to be translated into clinical practice after validation.

Background: Targeted therapies (TKI) have improved the prognosis of ALK-rearranged lung cancer (ALK+ NSCLC), but clinical courses vary widely. Early identification and molecular characterisation of treatment failure have key importance for subsequent therapies. We performed copy number variation (CNV) profiling and targeted panel sequencing from cell-free DNA (cfDNA) to monitor ALK+ NSCLC. Methods: 271 longitudinal plasma DNA samples from 73 patients with TKI-treated metastatic ALK+ NSCLC were analysed by capture-based targeted (average coverage 4,100x), and shallow whole genome sequencing (sWGS, 0.5x). Mutations were called using standard algorithms. CNVs were quantified using the trimmed median absolute deviation from copy number neutrality (t-MAD). Findings: cfDNA mutations were identified in 58% of patients. They included several potentially actionable alterations, e.g. in the genes BRAF, ERBB2, and KIT. sWGS detected CNVs in 18% of samples, compared to 6% using targeted sequencing. Several of the CNVs included potentially druggable targets, such as regions harboring EGFR, ERBB2, and MET. Circulating tumour DNA (ctDNA) mutations and t-MAD scores increased during treatment, correlated with markers of higher molecular risk, such as the EML4-ALK variant 3 and/or TP53 mutations, and were associated with shorter patient survival. Importantly, t-MAD scores reflected the tumour remission status in serial samples similar to mutant ctDNA allele frequencies, and increased with disease progression in 79% (34/43) of cases, including those without detectable single nucleotide variant (SNV). Interpretation: Combined copy number and targeted mutation profiling could improve monitoring of ALK+ NSCLC. Potential advantages include the identification of treatment failure, in particular for patients without detectable mutations, and broader detection of genomic changes acquired during therapy, especially in later treatment lines and in high-risk patients. Funding: This work was supported by the German Center for Lung Research (DZL), by the German Cancer Consortium (DKTK), by the Heidelberg Center for Personalized Oncology at the German Cancer Research Center (DKFZ-HIPO), and by Roche Sequencing Solutions (Pleasanton, CA, USA).

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 testing DPYD*2A, DPYD*13, c.2846A > T and c.1236G > A-HapB3 before a fluoropyrimidines administration to increase treatment safety is widely acknowledged. Other relevant target genes are TYMS (Thymidylate Synthase) and MTHFR (Methylene-Tetrahydro-Folate Reductase), whose polymorphisms were mainly studied as potential markers of treatment efficacy in LARC. A pivotal role of a TYMS polymorphism 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, as BRAF (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 as TP53 (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 in KRAS and TP53, 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.

The standard of care for the first-line treatment of advanced gastrointestinal stromal tumor (GIST) is represented by imatinib, which is given daily at a standard dosage until tumor progression. Resistance to imatinib commonly occurs through the clonal selection of genetic mutations in the tumor DNA, and an increase in imatinib dosage was demonstrated to be efficacious to overcome imatinib resistance. Wild-type GISTs, which do not display KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations, are usually primarily insensitive to imatinib and tend to rapidly relapse in course of treatment. Here we report the case of a 53-year-old male patient with gastric GIST who primarily did not respond to imatinib and that, despite the administration of an increased imatinib dose, led to patient death. By using a deep next-generation sequencing barcode-aware approach, we analyzed a panel of actionable cancer-related genes in the patient cfDNA to investigate somatic changes responsible for imatinib resistance. We identified, in two serial circulating tumor DNA (ctDNA) samples, a sharp increase in the allele frequency of a never described TP53 mutation (c.560-7_560-2delCTCTTAinsT) located in a splice acceptor site and responsible for a protein loss of function. The same TP53 mutation was retrospectively identified in the primary tumor by digital droplet PCR at a subclonal frequency (0.1%). The mutation was detected at a very high allelic frequency (99%) in the metastatic hepatic lesion, suggesting a rapid clonal selection of the mutation during tumor progression. Imatinib plasma concentration at steady state was above the threshold of 760 ng/ml reported in the literature for the minimum efficacious concentration. The de novo TP53 (c.560-7_560-2delCTCTTAinsT) mutation was in silico predicted to be associated with an aberrant RNA splicing and with an aggressive phenotype which might have contributed to a rapid disease spread despite the administration of an increased imatinib dosage. This result underlies the need of a better investigation upon the role of TP53 in the pathogenesis of GISTs and sustains the use of next-generation sequencing (NGS) in cfDNA for the identification of novel genetic markers in wild-type GISTs.

Sex differences in adaptive and innate immune responses have been shown to occur and anecdotal reports suggest that vaccine efficacy and safety may be sex-dependent. We investigated the influence of sex on the efficacy of COVID-19 vaccines through a systematic review and meta-analysis of clinical trials on COVID-19 vaccines. The safety profile of COVID-19 vaccines was also investigated. A systematic review included eligible articles published in three databases and three websites. A meta-analysis of available data, stratified by sex, was conducted. Statistical analysis was performed using the Hartung–Knapp–Sidik–Jonkman method, as well as influence and heterogeneity analysis. Pooled analysis showed significantly higher efficacy, measured as the rate of new COVID-19 cases, in men compared to women in the vaccine group (OR = 0.67, 95% CI 0.48–0.94). No sex differences were found in the rate of new cases in the control group (OR = 0.92, 95% CI 0.78–1.09). Safety profiles derived from pharmacovigilance reports appear to indicate increased toxicity in women. In conclusion, evidence of a potential role of sex in COVID-19 vaccine efficacy was described. It strengthens the need to include sex as a core variable in the clinical trial design of COVID-19 vaccines.

Nuclear receptors act as mediators of cancer-related inflammation and gene expression. They have a regulatory effect on genes encoding proteins related to drug adsorption, distribution, metabolism, and excretion. The aim of the present study was to highlight novel prognostic markers among polymorphisms in genes encoding for nuclear receptor proteins and inflammation-related cytokines in patients treated with a FOLFIRI regimen. This study included two independent cohorts comprising a total of 337 mCRC patients homogeneously treated with first-line FOLFIRI. Genotyping of 246 haplotype-tagging polymorphisms in 22 genes was performed using bead array technology. The NR1I2 (PXR)-rs1054190 and VDR-rs7299460 polymorphisms were significantly associated with patient overall survival (OS). A detrimental effect of the NR1I2 rs1054190-TT genotype on OS was observed in both the discovery and replication cohorts (HR = 6.84, P = 0.0021, q-value = 0.1278 and HR = 3.56, P = 0.0414, respectively). Patients harboring the NR1I2 rs1054190-TT genotype had a median OS of 9 months vs. 21 months in patients with C-allele (P < 0.0001 log-rank test). VDR rs7299460-T was consistently associated with a longer OS in both cohorts (discovery: HR = 0.61, P = 0.0075, q-value = 0.1535; replication: HR = 0.57, P = 0.0477). Patients with the VDR rs7299460-T allele had a median OS of 23 months compared to 18 months in those with the CC genotype (P = 0.0489, log-rank test). The NR1I2-rs1054190 polymorphism also had an effect on the duration of progression-free survival, consistent with the effect observed on OS. Two novel prognostic markers for mCRC treated with FOLFIRI were described and, if validated by prospective trials, have a potential application in the management of these patients.

Palbociclib, ribociclib, and abemaciclib belong to the third generation of cyclin-dependent kinases inhibitors (CDKis), an established therapeutic class for advanced and metastatic breast cancer. Interindividual variability in the therapeutic response of CDKis has been reported and some individuals may experience increased and unexpected toxicity. This narrative review aims at identifying the factors potentially concurring at this variability for driving the most appropriate and tailored use of CDKis in the clinic. Specifically, concomitant medications, pharmacogenetic profile, and pathophysiological conditions could influence absorption, distribution, metabolism, and elimination pharmacokinetics. A personalized therapeutic approach taking into consideration all factors potentially contributing to an altered pharmacokinetic/pharmacodynamic profile could better drive safe and effective clinical use.

There are clinical challenges related to adjuvant treatment in colorectal cancer (CRC) and novel molecular markers are needed for better risk stratification of patients. Our aim was to integrate our previously reported clinical-genetic prognostic score with new immunogenetic markers of 5-year disease-free survival (DFS) to evaluate the recurrence risk stratification before fluoropyrimidine (FL)-based adjuvant therapy. The study population included a total of 270 stage II-III CRC patients treated with adjuvant FL with (FL + OXA, n = 119) or without oxaliplatin (FL, n = 151). Patients were genotyped for a panel of 192 tagging polymorphisms in 34 immune-related genes. The IFNG-rs1861494 polymorphism was associated with worse DFS in the FL + OXA (HR = 2.14, 95%CI 1.13–4.08; P = 0.020, q-value = 0.249) and FL (HR = 1.97, 95%CI 1.00–3.86; P = 0.049) cohorts, according to a dominant model. The integration of IFNG-rs1861494 in our previous clinical genetic multiparametric score of DFS improved the patients’ risk stratification (Log-rank P = 0.0026 in the pooled population). These findings could improve the discrimination of patients who would benefit from adjuvant treatment. In addition, the results may help better elucidate the interplay between the immune system and chemotherapeutics and help determine the efficacy of anti-tumor strategies.

Pharmacogenomics has largely been applied to the personalization of irinotecan-based treatment, focusing mainly on the study of genetic variants in adsorption, distribution, metabolism, and excretion (ADME) genes. The transcriptional control of ADME gene expression is mediated by a set of nuclear factors responding to cancer-related inflammation, which could have pharmacological implications. The aim of the present study was to uncover novel genetic predictors of neutropenia and gastrointestinal toxicity risk among 246 haplotype-tagging polymorphisms in 22 genes encoding inflammation-related cytokines and transcriptional regulators of ADME genes. The study comprised overall more than 400 metastatic colorectal cancer (mCRC) patients treated with first-line FOLFIRI, grouped in a discovery and a replication cohorts. A concordant protective effect of STAT-3 rs1053004 polymorphism against the risk of grade 3–4 gastrointestinal toxicity was observed in both the cohorts of patients (OR = 0.51, p = 0.045, q = 0.521 and OR = 0.39, p = 0.043, respectively). VDR rs11574077 polymorphism was demonstrated to affect both irinotecan biliary index (BI) and glucuronidation ratio (GR) by a pharmacokinetic analysis. This effect was consistent with an increased risk of grade 3–4 gastrointestinal toxicity in the discovery cohort (OR = 4.46, p = 0.010, q = 0.305). The association was not significant in the replication cohort (OR = 1.44, p = 0.601). These findings suggest an effect of STAT-3 and VDR polymorphisms on FOLFIRI-related gastrointestinal toxicity. If prospectively validated as predictive markers, they could be used to improve the clinical management of mCRC.

Hepatocyte nuclear factor 1-alpha (HNF1A) is a liver-enriched transcription factor that plays a key role in many aspects of hepatic functions including detoxification processes. We examined whether HNF1A polymorphisms are associated with clinical outcomes in two independent cohorts combining 417 European ancestry patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based chemotherapy. The intronic rs2244608A>G marker was predictive of an improved progression-free survival with a trend in the Canadian cohort and reaching significance in the Italian cohort, with hazard ratios (HR) of 0.74 and 0.72, P = 0.076 and 0.038, respectively. A strong association between rs2244608A>G and improved PFS was found in the combined analysis of both cohorts (HR = 0.72; P = 0.002). Consistent with an altered HNF1A function, mCRC carriers of the rs2244608G minor allele displayed enhanced drug exposure by 45% (P = 0.032) compared to non-carriers. In Caucasians, rs2244608A>G is in strong linkage with the coding variant rs1169288c.79A>C (HNF1A p.I27L). In healthy donors, we observed an altered hepatic (ABCC1, P = 0.009, ABCC2, P = 0.048 and CYP3A5, P = 0.001; n = 89) and intestinal (TOP1, P = 0.004; n = 75) gene expression associated with the rs1169288C allele. In addition, the rs1169288C polymorphism could significantly increase the ABCC1 promoter activity by 27% (P = 0.008) and 15% (P = 0.041) in the human kidney HEK293 and the human liver HepG2 cell lines, respectively. Our findings suggest that the HNF1A rs2244608, or the tightly linked functional coding variant p.I27L, might be a potential prognostic marker with irinotecan-based regimens.

Introduction: Long-term adverse symptoms of men who used oral finasteride against androgenic alopecia have been recently described as post-finasteride syndrome (PFS). Aim: To determine whether (CAG)n-rs4045402 and (GGN)n-rs3138869 polymorphisms in the androgen receptor (AR) gene are implicated in PFS. Methods: AR polymorphisms were studied according to PFS symptoms in 66 white participants (31.8% Italian, 28.8% American, and 39.4% other). Main Outcome Measures: Symptoms were investigated by an ad hoc 100-item questionnaire and the Arizona Sexual Experience Scale and Aging Male Symptom Scale (AMS). (CAG)n and (GGN)n repeats were categorized as short ([CAG]9–19, [GGN]23). Results: Median age was 32 years, duration of finasteride use was 360 days, and time from finasteride discontinuation was 1,053 days. We observed several frequency differences in symptoms according to (CAG)n and (GGN)n repeat numbers. Three AMS items were worse for medium (GGN)23 than for long (GGN)>23 carriers and one item was worse for short (GGN)2 kg), increased skin dryness, and onset of symptoms after finasteride use. Conclusion: This study showed that short and/or long (CAG)n and (GGN)n repeats had different frequencies according to symptoms reported by patients with PFS, likely reflecting the vast array of genes modulated by the AR. This study showed a U-curvilinear profile of (CAG)n repeats for skin dryness symptoms, where the two extremes exhibited a worse condition than medium repeats. Further studies are necessary to investigate the PFS pathophysiology using a precision medicine approach.

Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide, representing the third leading cause of cancer-related deaths. HCC genetic characterization at the tumor level has been recently completed, highlighting how a number of genes are frequently mutated in this pathology. Actionable somatic mutations found in a HCC tumor may represent targets for innovative drugs as well as prognostic/predictive markers. Nonetheless, surgical or bioptic tissue is hardly accessible in HCC and a single tumor sample is poorly representative of the tumor genetic heterogeneity. In this context, analyzing the circulating cell-free DNA (ccfDNA) and its tumor-derived fraction (ctDNA) could represent a promising strategy of liquid biopsy. Recent data suggested that the fluctuation of the ccfDNA quantity in the plasma of HCC patients could anticipate the detection of tumor progression. The presence of somatic mutations in p53 signaling, Wnt/β-catenin, chromatin remodeling, response to oxidative stress and telomerase maintenance pathways can also be studied in ccfDNA bypassing the need to perform a tumor biopsy. The profiling of ccfDNA fragmentation and the methylation pattern could further improve the clinical management of HCC patients. Performing a dynamic monitoring in the course of systemic treatment with sorafenib or regorafenib is a possible way to provide insights into the resistance mechanism, and to identify predictive and prognostic genetic alterations, helping the clinicians in terms of treatment decision making. This review will discuss the most recent literature data about the use of ccfDNA to monitor and improve the treatment of HCC.

Carboplatin/paclitaxel is the reference regimen in the treatment of advanced high-grade serous ovarian cancer (HGSOC) in neo-adjuvant chemotherapy (NACT) before interval debulking surgery (IDS). To identify new genetic markers of platinum-resistance, next-generation sequencing (NGS) analysis of 26 cancer-genes was performed on paired matched pre- and post-NACT tumor and blood samples in a patient with stage IV HGSOC treated with NACT-IDS, showing platinum-refractory/resistance and poor prognosis. Only the TP53 c.375+1G>A somatic mutation was identified in both tumor samples. This variant, associated with aberrant splicing, was in trans configuration with the 72Arg allele of the known germline polymorphism TP53 c.215C>G (p. Pro72Arg). In the post-NACT tumor sample we observed the complete expansion of the TP53 c.375+1G>A driver mutant clone with somatic loss of the treatment-sensitive 72Arg allele. NGS results were confirmed with Sanger method and immunostaining for p53, BRCA1, p16, WT1, and Ki-67 markers were evaluated. This study showed that (i) the splice mutation in TP53 was present as an early driver mutation at diagnosis; (ii) the mutational profile was shared in pre- and post-NACT tumor samples; (iii) the complete expansion of a single dominant mutant clone through loss of heterozygosity (LOH) had occurred, suggesting a possible mechanism of platinum-resistance in HGSOC under the pressure of NACT.

Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III−IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor.

Up to 30−50% of patients with locally advanced prostate cancer (PCa) undergoing radiotherapy (RT) experience biochemical recurrence (BCR). The immune system affects the RT response. Immunogenetics could define new biomarkers for personalization of PCa patients’ treatment. The aim of this study is to define the immunogenetic biomarkers of 10 year BCR (primary aim), 10 year overall survival (OS) and 5 year BCR (secondary aims). In this mono-institutional retrospective study, 549 Caucasian patients (a discovery set n = 418; a replication set n = 131) were affected by locally advanced PCa and homogeneously treated with RT. In the training set, associations were made between 447 SNPs in 77 genes of the immune system; and 10 year BCR and 10 year OS were tested through a multivariate Cox proportional hazard model. Significant SNPs (p-value < 0.05, q-value < 0.15) were analyzed in the replication set. Replicated SNPs were tested for 5 year BCR in both sets of patients. A polymorphism in the PDL1 gene (rs4143815) was the unique potential genetic variant of 10 year BCR (training set: p = 0.003, HR (95% CI) = 0.58 (0.41−0.83); replication set: p = 0.063, HR (95% CI) = 0.52 (0.26−1.04)) that was significantly associated with 5 year BCR (training set: p = 0.009, HR (95% CI) = 0.59 (0.40−0.88); replication set: p = 0.036, HR (95% CI) = 0.39 (0.16−0.94)). No biomarkers of OS were replicated. rs4143815-PDL1 arose as a new immunogenetic biomarker of BCR in PCa, giving new insights into the RT/immune system interaction, which could be potentially useful in new approaches using anti-PDL1 therapies for PCa.

An important hallmark of CRC is the evasion of immune surveillance. HLA-G is a negative regulator of host's immune response. Overexpression of HLA-G protein in primary tumour CRC tissues has already been associated to worse prognosis; however a definition of the role of immunogenetic host background is still lacking. Germline polymorphisms in the 3'UTR region of HLA-G influence the magnitude of the protein by modulating HLA-G mRNA stability. Soluble HLA-G has been associated to 3'UTR +2960 Ins/Ins and +3035 C/T (lower levels) and +3187 G/G (high levels) genotypes. HLA-G 3'UTR SNPs have never been explored in CRC outcome. The purpose of this study was to investigate if common HLA-G 3'UTR polymorphisms have an impact on DFS and OS of 253 stage II-III CRC patients, after primary surgery and ADJ-CT based on FL. The 3'UTR was sequenced and SNPs were analyzed for their association with survival by Kaplan-Meier and multivariate Cox models; results underwent internal validation using a resampling method (bootstrap analysis). In a multivariate analysis, we estimated an association with improved DFS in Ins allele (Ins/Del +Ins/Ins) carriers (HR 0.60, 95% CI 0.38-0.93, P = 0.023) and in patients with +3035 C/T genotype (HR 0.51, 95% CI 0.26-0.99, P = 0.045). The +3187 G/G mutated carriers (G/G vs A/A+A/G) were associated to a worst prognosis in both DFS (HR 2.46, 95% CI 1.19-5.05, P = 0.015) and OS (HR 2.71, 95% CI 1.16-6.63, P = 0.022). Our study shows a prognostic and independent role of 3 HLA-G 3'UTR SNPs, +2960 14-bp INDEL, +3035 C>T, and +3187 A>G.

The use of targeted agents in the treatment of metastatic colorectal cancer (CRC) has improved patient outcomes. Anti-epidermal growth factor receptor (anti-EGFR) agents (cetuximab and panitumumab) and antiangiogenic molecules (bevacizumab, regorafeninb, ramucirumab, and aflibercept) have been successfully integrated into clinical practice. Other drugs have been designed to target additional deregulated pathways in CRC, such as MAPK (mitogen-activated protein kinase)/PI3K-AKT (phosphatidylinositol-3-kinase-AKT serine/threonine kinase)/mTOR (mammalian target of rapamycin), HER-2 and 3 ( human epidermal growth factor receptor-2 and -3), and BRAF. A major issue with targeted treatment is early identification of patients with primary or secondary drug resistance. Pharmacogenomic research has demonstrated its value in this field, highlighting some tumor mutations that could discriminate responders from non-responders. The tumor genetic profile of the RAS/RAF pathway is needed before treatment with anti-EGFR agents; mutations in EGFR pathway genes have also been explored in relation to antiangiogenic molecules although further data are required prior to their integration into clinical practice. The introduction of immunotherapy has paved the way for a new generation of predictive markers, including genome-wide assessment of the tumor landscape. Furthermore, the development of next generation sequencing technology and non-invasive approaches to analyze circulating tumor DNA will make real-time monitoring of the tumor pharmacogenomic markers possible in the clinical routine, rendering precision medicine available to every patient.

Polymorphisms in drug-metabolizing enzymes might not completely explain inter-individual differences in toxicity profiles of patients with colorectal cancer (CRC) that receive folinic acid/5-fluorouracil/oxaliplatin (FOLFOX4). Recent data indicate that the immune system could contribute to FOLFOX4 outcomes. In light of the immune inhibitory nature of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex (MHC) class I molecule, we aimed to identify novel genomic markers of grades 3 and 4 (G3-4) toxicity related to FOLFOX4 therapy in patients with CRC. We retrospectively analyzed data for 144 patients with stages II-III CRC to identify HLA-G 3′ untranslated region (3′UTR) polymorphisms and related haplotypes and evaluate their impact on the risk of developing G3-4 toxicities (i.e., neutropenia, hematological/non-hematological toxicity, neurotoxicity) with logistic regression. The rs1610696-G/G polymorphism was associated with increased risk of G3-4 neutropenia (OR = 3.76, p = 0.015) and neurotoxicity (OR = 8.78, p = 0.016); rs371194629-Ins/Ins was associated with increased risk of neurotoxicity (OR = 5.49, p = 0.027). HLA-G 3′UTR-2, which contains rs1610696-G/G and rs371194629-Ins/Ins polymorphisms, was associated with increased risk of G3-4 neutropenia (OR = 3.92, p = 0.017) and neurotoxicity (OR = 11.29, p = 0.009). A bootstrap analysis confirmed the predictive value of rs1610696 and rs371194629, but the UTR-2 haplotype was validated only for neurotoxicity. This exploratory study identified new HLA-G 3′UTR polymorphisms/haplotypes as potential predictive markers of G3-4 toxicities in CRC.

To explore genes of the killer-cell immunoglobulin-like receptor (KIR) and of the HLA ligand and their relationship with the outcome of metastatic colorectal cancer (mCRC) patients treated with first-line 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI).A total of 224 mCRC patients were screened for KIR/HLA typing. The determination of the KIR/HLA combinations was based upon the gene content and variants. Genetic associations with complete response (CR), time to progression (TTP) and overall survival (OS) were evaluated by calculating odds and hazard ratios. Multivariate modeling with prognostic covariates was also performed.For CR, the presence of KIR2DL5A, 2DS5, 2DS1, 3DS1, and KIR3DS1/HLA-Bw4-I80 was associated with increased CR rates, with median ORs ranging from 2.1 to 4.3, while the absence of KIR2DS4 and 3DL1 was associated with increased CR rates (OR 3.1). After univariate analysis, patients that underwent resective surgery of tumor, absence of KIR2DS5, and presence of KIR3DL1/HLA-Bw4-I80 showed a significant better OS (HR 1.5 to 2.8). Multivariate analysis identified as parameters independently related to OS the type of treatment (surgery; HR 2.0) and KIR3DL1/HLA-Bw4-I80 genotype (HR for T-I80 2.7 and for no functional KIR/HLA interaction 1.8). For TTP, no association with KIR/HLA genes was observed.This study, for the first time, evidences that the genotyping for KIR-HLA pairs are found predictive markers associated with complete response and improves overall survival prediction of FOLFIRI treatment response in metastatic colorectal cancer. These results suggest a role of the KIR/HLA system in patient outcome, and guide new research on the immunogenetics of mCRC through mechanistic studies and clinical validation.

Background: Pathological complete response (pCR) to neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC) is still ascribed to a minority of patients. A pathway based-approach could highlight the predictive role of germline single nucleotide polymorphisms (SNPs). The primary aim of this study was to define new predictive biomarkers considering treatment specificities. Secondary aim was to determine new potential predictive biomarkers independent from radiotherapy (RT) dosage and cotreatment with oxaliplatin. Methods: Thirty germ-line SNPs in twenty-one genes were selected according to a pathway-based approach. Genetic analyses were performed on 280 LARC patients who underwent fluoropyrimidine-based CRT. The potential predictive role of these SNPs in determining pathological tumor response was tested in Group 1 (94 patients undergoing also oxaliplatin), Group 2 (73 patients treated with high RT dosage), Group 3 (113 patients treated with standard RT dosage), and in the pooled population (280 patients). Results: Nine new predictive biomarkers were identified in the three groups. The most promising one was rs3136228-MSH6 (p = 0.004) arising from Group 3. In the pooled population, rs1801133-MTHFR showed only a trend (p = 0.073). Conclusion: This exploratory study highlighted new potential predictive biomarkers of neoadjuvant CRT and underlined the importance to strictly define treatment peculiarities in pharmacogenetic analyses.