- Objective: Several strategies for preventing toxicity and resistance to taxane-based chemotherapy have been investigated so far. Lately, findings on the genetic variants associated with neutropenia and neuropathy (N&N) toxicity have been reported. Patients and Methods: A panel assay of single nucleotide polymorphisms (SNP) related to paclitaxel and Docetaxel toxicity on four candidate genes ATP-binding cassette subfamily B member 1 (ABCB1), Beta-tubulin 2A (TUBB2A), Cytochrome P450 3A4* 1B (CYP3A4*1B), Excision-Repair Cross-Complementing group 2 (ERCC3) are validated and discussed. We genotyped 37 cancer patients who received paclitaxel or docetaxel-based therapy. Furthermore, an early outline evaluation of the genotyping costs and benefits was assessed. Results: A total of 37 patients were treated with a taxane of which 17 (45.9%) had adverse N&N events. Pharmacogenomics analysis showed no relation between candidate gene polymorphisms and toxicity, except for the ERCC3 AG+GG allele [OR 2.61 (95% CI: 0.91-7.61)] that showed a significantly weak trend of risk of neurotoxicities vs. the AG allele [OR 1.52 (95% CI: 0.51-4.91)] p=0.03. Conclusions: We propose a useful genotyping panel assay to prevent toxicity in patients undergoing taxane-based therapy at an affordable price, with help from the literature and our experimental results and data. Based on the individual pharmacogenomics profile, clinicians will have additional information to personalize the treatment for the patient to minimize toxicity and maximize benefits, and they can also determine the cost-effectiveness for national healthcare sustainability.

Differentiated thyroid cancer (DTC) includes papillary and follicular carcinomas and is the most common type of thyroid cancer. The incidence of this cancer has increased in the last few years, and even if its prognosis is generally good for a subset of patients that does not respond to radioactive iodine (RAI) therapy, the prognosis is much worse: the median overall survival (OS) from discovery of metastasis is 3–5 years and the 10-year survival rate is only 10%. Several mutations, including RAS or RET, as well as BRAF signaling, are associated with thyroid cancer. Liquid biopsy may be useful in selected patient to identify genomic alterations and thus allowing for a precision medicine approach with target therapy. Sorafenib, an oral multi-kinase inhibitor, can be used in the treatment of DTC. Case presentation: A 77 years old. man with diagnosis of metastatic DTC and evidence of presence of mutation of BRAF K601E on liquid biopsy was treated with sorafenib, showing a good response to the treatment and an improvement in the quality of life (QoL). Currently, this patient is still on treatment with sorafenib, gaining control of a multi-metastatic disease, generally characterized by a very poor prognosis. In conclusion, sorafenib has an active role in the treatment of DTC. It also has been considered the standard of care for patients with advanced unresectable hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). In our case we observe the efficacy of using sorafenib in Papillary thyroid carcinoma (PTC) such as confirming both stable disease (SD) in the CT scan as clinical benefit with an increase in QoL. Therefore, use of sorafenib remains an important treatment option, even in case of BRAF mutation, despite a rapidly evolving treatment landscape. It also seems important to perform liquid biopsies, especially in patients in whom it is not possible to repeat a new tissue biopsy. Ongoing clinical trials continue to evaluate sorafenib in different settings, and in combination with other therapies in DTC and HCC.

Objective: Determination of absolute DNA concentration by Real-Time PCR is mandatory for an accurate molecular diagnosis. The critical step is a precise and reliable calibration curve. Plasmid DNA containing cloned target sequences is widely used as standards in quantitative PCR. Usually, the initial molecular number of plasmid molecules is deduced by theoretical calculation and this can be severely limiting the accuracy of PCR assay. Materials and Methods: We have developed a simple method to generate a precise standard curve made of plasmid DNA by competitive PCR approach. The procedure is the following: i) construction of competitor fragment; ii) generation of the stock standard by cloning the DNA target template in the plasmid vector; iii) determination of the number of molecules in the stock standard by competitive PCR; iv) analytical validation of standard curve, in terms of accuracy, sensitivity and specificity. We have used this procedure to quantify the genetic marker breakpoint, the t(14;18), associated to Follicular Lymphoma (FL) of Non-Hodgkin's Lymphomas (NHL). Results: We found that the assessment of plasmid copy number, obtained by theoretical calculation, is overestimated compared to our competitive PCR approach (error of 22% +/-5). The standard plasmid DNA, used to generate the calibration curve, is stable and produces a high reproducible curve even after a long-term storage compared to genomic DNA curve. Conclusions: The described procedure: i) generates robust standard curves for absolute quantification of any DNA target of interest; ii) is suitable with any molecular diagnostic platform; iii) contributes significantly to improve the accuracy of routine laboratory applications.

Introduction: The renin-angiotensin-aldosterone system (RAAS), a metabolic cascade regulating pressure and circulating blood volume, has been considered the main system involved in the pathogenesis of severe lung injury and organs decline in COVID-19 patients. The angio-tensin I-converting enzyme (ACE1), angiotensin-converting enzyme 2 (ACE2), angiotensinogen (AGT) and receptors angiotensin II receptor type 1 (AGTR1) are key factors for SARS-CoV-2 entering in the cells, sodium and water retention with an increase blood pressure, promotion of fibrotic and inflammatory phenomena resulting in a cytokine storm. Methods: In this pilot study, the frequencies of six polymorphisms in the ACE1, ACE2, AGT and AGTR1 genes were analysed in symptomatic patients affected by COVID-19 and compared with the results obtained from asymptomatic subjects. Results: Thus, we have identified that rs2074192 (ACE2), rs1799752 (ACE1) and rs699 (AGT) SNPs could potentially be a valuable tool for predicting the clinical outcome of SARS-CoV-2 infected patients. A genetic predisposition may be prospected for severe internal organ damages and poor prognosis in patients with COVID-19 disease, as observed in symptomatic vs asymptomatic. Conclusion: This study provides evidence that analysis of RAAS polymorphisms could be considered the key point in understanding and predicting the SARS-CoV-2 course infection.

Objective: Prostate Specific Antigen (PSA) is a commonly used marker for the diagnosis and follow-up of Prostate Cancer (PC) and Prostatic Intraepithelial Neoplasia (PIN). Furthermore, in order to ensure early detection of the patients at risk of PC and PIN, there is a growing need for new tools able to early identify this subject. Molecular analysis of neoplastic prostate tissues showed the inactivation of the Glutathione-S-Transferase gene (GSTP1) due to the hyper-methylation. The aim of this study is the validation of the specific and sensitive detection of the methylation status of the GSTP1 gene for potential biomarker to assess early detection of PIN. Patients and Methods: The methylation status of 5' promoter region of the GSTP1 gene was obtained by Methylation Sensitivity-PCR (MS-PCR). The test was optimized in terms of the specificity and sensitivity. The cost-efficacy of the test was tested on the DNA from 20 donors healthy subject, 57 benign prostatic hypertrophy (BPH), and 57 PC patients. Results: GSTP1 promoter gene methylation was detected in 0% of healthy subjects (20/20, median age 32,7 years), in 43,9% of patients with BPH (25/57 mean age 60,5 years) and in 57,6% of patients with PC (34/57 mean age 67,8 years). Significantly, the 81,8% of patients with PC, age >65 years and total PSA <= 4 ng/ml were positive for the methylation status of GSTP1 gene. Conclusions: In this way, specific evaluation of the methylation status of the GSTP1 gene may be a useful tool for the prediction of patients at risk of PC. In addition, the test is cost-effectiveness and could be used extensively for cancer prevention.

OBJECTIVE:Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease that leads to local and systemic arthritis and bone loss. Exploring genetic markers of candidate genes in osteoporosis and inflammatory cytokine genes could be a useful tool for the early identification of bone loss and fracture risk in RA patients. The target of this study is the evaluation and correlation between of Single Nucleotide Polymorphisms (SNPs) of Vitamin D Receptor (VDR) and possible effects on bone loss in RA.PATIENTS AND METHODS:40 Caucasian patients with RA (26 of them with a severe bone loss) and 40 healthy donors as control samples were genotyped for the VDR SNPs (called BsmI, ApaI, TaqI and FokI). The detection method is based on Restriction Fragment Length Polymorphism (RFLP).RESULTS:Genotyping profile shown no difference between RA patients and controls. Only VDR-TaqI genotype (TT vs. tt) seem to influence the bone density in females, but not in males. The mean differences of Bone Mass Density (BMD) at the lumbar spine in RA women with the tt allele were 4.7% compared to 0.1% in women with the TT allele (p < 0.05).CONCLUSIONS:The results of these studies support an association between specific VDR alleles and bone loss in RA. The TaqI t and BsmI B alleles were associated with an accelerated bone loss in RA, but not with a focal bone loss. These effects of VDR genotypes and vitamin D supplementation are not unexpected, given that the central pathological feature in RA is bone and joint destruction. The VDR SNPs genotyping should be a useful tool to screen early women RA patients with the bone loss.

It is well-known that 75% of risk factors of chronic liver disease (CLD) are related to nutrition. These circumstances potentially progress towards liver steatosis, fibrosis and hepatocellular carcinoma (HCC). It still represents an enormous problem for the economy of public health worldwide. Furthermore, validated prevention programs could be the solution. Recent knowledge in understanding molecular determinants of energy liver metabolism and new genetic markers offers new insights into the pathogenesis of CLD and HCC. The main rationale of the present issue is to provide a summary of recent insights into the inherited variants regulating lipid metabolism (steatohepatitis) and acquired mutation for early diagnosis of HCC, specifically focusing on the significance of antioxidant agents and genotyping tests as a cost-effectiveness tool for the prevention of liver disease. Several national healthy programs worldwide promote the daily use of antioxidant nutrients either for the prevention and/or as complementary and alternative medicines (CAM). This review could be advising for the planning of a large-scale clinical trial including a combination strategy of antioxidant agents and genotyping tests in patients with high risk of CLD.

OBJECTIVE: Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma (NHL) featured by participation of the lymph nodes, spleen, blood and bone marrow with a short remission period to standard therapies and a median overall survival of 4-5 years.PATIENTS AND METHODS: In this study, we compare the levels of bcl-1/JH fusion products detected by q-PCR in the concurrent peripheral blood (PB) and bone marrow (BM) aspirate samples from 7 patients with MCL.RESULTS: In patients with moderate to high levels of bcl-1/JH copies, the results of q-PCR analysis of PB and BM aspirate samples correlate well. In patients with high levels of bcl-1/JH copies, instead, PB levels are a good indication of tumor burden. Finally, in patients with low levels of bcl-1/JH copies, the t(11;14) may be detected by identification of neoplastic cells.CONCLUSIONS: Our data suggest that PB can be reliably used in place of BM aspirate both for detection of translocation status during minimal residual disease monitoring and for a possible molecular relapse, especially in those patients who have moderate to high levels of bcl-1/JH copies. If these results will be confirmed on a wider number of MCL patients, future study will be required to address the issue.

Background: Breast cancer is a heterogeneous disease, presents various pathological signs such as axillary lymph node metastasis which is associated with a high risk of recurrence and considered as an important prognosis factor in the early stages of the disease. Invasion and metastasis are two important hallmarks of malignant tumors caused by complex genetic and epigenetic alterations. The present study investigated the contribution of aberrant methylation profiles of cancer related genes, BRCA1, GSTP1, and, p16 (CDKN2A), in the sporadic Breast Cancer biopsy. Patients andMethods: A total of 35 subjects, (7 healthy women with a family hystory of breast cancer and 26 with monolateral breast cancer) have been studied in order to detect the hypermethylation status of the promoter region of the following genes: p16, GSTP1 and BRCA1.Results: The DNA methylation analysis of the candidate genes showed higher methylation proportion in the primary tumor tissue than that of the matched normal tissue and the differences were significant for the, BRCA1, and p16, promoter regions (p<0.05). Among those candidate methylated genes, BRCA1 and p16 displayed higher methylation proportion in the matched lymph node metastasis than that found in the normal tissue (p<0.05).Conclusions: The results of the present study showed methylation heterogeneity between primary tumors and metastatic lesion. The pathway analysis revealed that, BRCA1 and p16 have a role in prevention of neoplasm metastasis. The contribution of aberrant methylation alterations of, BRCA1 and p16 genes in lymph node metastasis might provide a further sign to establish useful biomarkers for screening metastasis. In summary, our DNA methylated assay performed from blood cells from BRCA1 carriers is able to predict breast cancer risk years in advance of diagnosis.

Backbone: Gemcitabine is an anticancer drug routinely used to treat diverse cancers disease. Discontinuation of Gemcitabine based treatments is mostly caused either by toxicity or more often for tumor progression, potentially compromising patient benefit. Several strategies to prevent toxicity/resistance have been so far investigated.Content: In order to overreach this life-conditioning side effect, we describe in detail recent findings about the underlying mechanism of genetic variants associated with toxicity and resistance to Gemcitabine-based chemotherapy. A comprehensive panel of 6 polymorphisms detected on 4 genes, previously validated as significant markers related to toxicity/resistance, arer proposed and finely described. In addition, an early outline evaluation of the genotyping costs and methods are taken in consideration.Summary: Based on the individual pharmacogenomics profile, the oncologists will have new means to make treatment decisions for their patients in order to maximize benefit and minimize toxicity. Based on this purpose, the clinician and lab manager should cooperate to evaluate advantages and limitations, in terms of costs and applicability, of the most appropriate pharmacogenomics tests to incorporate routinely into clinical practice.

Background: Currently, a widely used marker for the diagnosis and follow-up of Prostate cancer ( PC) is the Prostate Specific Antigen (PSA). Furthermore, in order to ensure an efficient monitoring of the patients at risk of PC, there is a growing need of new tools able to early identify these subject. Molecular analysis of neoplastic prostate tissues shown the inactivation of the Glutatione-S-Transferase gene (GSTP1), due to the hypermethylation. This features could be a potential biomarker for PC. The aim of this study is the specific and sensitive detection of the methylation status of GSTP1 gene in plasma.Methods: The methylation status of 5' promoter region of GSTP1 gene was obtained by methylation Sensitivity-PCR (MS-PCR). The test was optimaized in terms of the specificity, sensitivity. The diagnostic efficacy of the test was tested on the DNA from 20 healthy donors, 57 benign prostatic hypertrophy (BPH), and 57 PC patients.Results: GSTP1 promoter gene hypermethylation was detected in 0% of healthy subjects (20/20, median age 32.7 years), in 43.9% of patients with BPH (25/57 mean age 60.5 years) and in 57.6% of patients with PC (34/57 mean age 67.8 years). Significantly, the 81.8% of patients with PC, age > 65 years and total PSA <= 4 ng/ml were positive for the hyper-methylation status of GSTP1 gene.Conclusions: By this means, specific evaluation of methylation status of GSTP1 gene may be an useful tool for the prediction of patients at risk of PC. In addition the test is cost-effectiveness and could be used extensively for cancer prevention.