Livia Manzella

Pharmacogenetics has transformed oncology by enabling personalized, effective and safer use of anticancer therapies. Among clinically relevant pharmacogenetic biomarkers, polymorphisms in the UGT1A1 gene influence the metabolism of irinotecan and other SN-38-based agents, increasing the risk of treatment-related toxicities. Beyond SN-38-based therapies, UGT1A1 contributes to the metabolism of endogenous compounds and several drugs, and its activity may be affected by concomitant inhibitors or inducers, highlighting its broader clinical relevance. Although substantial evidence supports the association between UGT1A1 genotype and drug safety, implementation of genotype-guided treatment remains inconsistent. This narrative review summarizes the biological basis and clinical relevance of UGT1A1 polymorphisms in patients receiving SN-38-based therapies. Original studies, systematic reviews, meta-analyses, and international and regional pharmacogenetic guidelines were evaluated regarding genotype-guided dosing, clinical outcomes, implementation strategies, and barriers to routine testing. Reduced-function UGT1A1 variants are associated with increased risk of irinotecan-induced neutropenia and diarrhea, particularly at higher doses. However, recommendations from scientific societies differ regarding testing indications, dose adjustment strategies, and patient selection. Uncertainties remain concerning the clinical implications of UGT1A1 variability for newer antibody–drug conjugates carrying an SN-38 payload. Practical barriers, including inconsistent guideline recommendations, limited access to testing, reimbursement issues, and heterogeneous clinical workflows, continue to restrict implementation. UGT1A1 genotyping represents a valuable tool for improving the safety of selected anticancer therapies, yet its incorporation into clinical practice remains incomplete. As precision oncology evolves, pharmacogenetic testing may optimize treatment selection, reduce toxicity, and improve outcomes. Further harmonization of international recommendations, high-quality prospective studies, and wider access to pharmacogenetic testing are needed to support routine implementation.

Background: Acute myeloid leukemia (AML) is genomically heterogeneous, and translating baseline molecular data into individualized prognosis remains difficult. We assessed real-world outcomes and externally validated the Sanger Institute AML multistage prognostic model. Methods: This single-center, retrospective study included 73 AML patients who underwent targeted NGS profiling. In intensively treated patients, the published, validated Sanger AML multistage prognostic model was compared with observed 12- and 36-month clinical outcomes using quadratic-weighted Cohen’s kappa. Results: Median age was 61 years, and median overall survival was 13 months, with the most significant survival differences driven by treatment intensity. TP53 mutations occurred in 7 patients (9.6%) and were linked to primary refractoriness and extremely poor survival. TP53 was the only independent predictor of death (HR 8.07, 95% CI 2.23–29.13; p = 0.0014). Model concordance was moderate at 12 months (29 evaluable cases; weighted κ = 0.52; alive/dead κ = 0.52) and fair-to-moderate at 36 months (23 cases; weighted κ = 0.46). The tool performed best for predicted death without remission, while most discrepancies involved patients expected to remain in first remission who later relapsed and died. Conclusions: TP53 disruption dominates prognosis in real-world AML. The multistage tool supports early high-risk identification but shows limited long-term calibration, motivating the development of dynamic models integrating contemporary therapies and longitudinal min/serial NGS data.

The BCR-ABL1 chimeric oncoprotein plays a pivotal role in the pathogenesis of Chronic Myeloid Leukemia (CML) as its constitutive kinase activity transforms the hematopoietic stem cell, promoting pro-survival signaling. We and others have previously shown that the manipulation of BCR-ABL1 catalytic activity modulates its intracellular localization, thereby transforming the culprit of CML into a pro-apoptotic protein that selectively kills leukemic cells. Here, we investigated the role of the BCR coiled-coil and C2 domains on BCR-ABL1 intracellular localization and leukemogenic potential. We performed a bioinformatic analysis that identified two putative nuclear localization signals (NLSs) in BCR. Using recombinant DNA strategies, we generated multiple BCR and BCR-ABL1 mutants that were ectopically expressed in human cells. The intracellular localization of each construct was analyzed by immunofluorescence, while their biological activity was investigated employing proliferation and transforming assays. We show that BCR displays two nuclear localization signals functionally inactivated by the coiled-coil and C2 domains. The removal of these regions reactivated the nuclear migration of both BCR and BCR-ABL1 mutants. Moreover, BCR-ABL1 constructs devoid of the coiled-coil and C2 domains displayed reduced transforming potential in Ba/F3 cells and in primary human CD34+ progenitors. Finally, we demonstrate that the deletion of the C2 domain compromises TKI efficacy. Our findings identify two nuclear localization signals in the BCR sequence that are functionally suppressed by the coiled-coil and C2 domains. Targeting these regions may provide additional therapeutic strategies to manipulate both BCR-ABL1 intracellular localization and kinase activity.

Hydroxyurea (HU) cytoreduction is usually administered to patients with chronic myeloid leukemia before starting any tyrosine kinase inhibitors (TKIs) therapy. However, up to date, there is no evidence of any benefit of hydroxyurea pre-treatment. Conversely, evidence has been provided on both the prognostic significance of the quantitative assessment of BCR::ABL1 expression at diagnosis and the individual decline of the BCR::ABL1 slope. In this view, we assumed that any kind of treatment administered before a confirmed diagnosis of chronic myeloid leukemia might change the amount of BCR::ABL1 transcript levels. To this purpose, we evaluated leukocyte counts and BCR::ABL1 quantitative expression either at diagnosis (baseline and no therapy) and on day 7 and day 14 of treatment in a cohort of 45 unselected patients with newly diagnosed chronic myeloid leukemia in the chronic phase. After informed consent, 21 of them received HU cytoreduction for 14 days before starting TKI treatment (HU group), whereas the other 24 patients received frontline TKI therapy without HU pre-treatment (TKI group). Our findings showed that: (i) there is no benefit from HU cytoreduction in patients affected with chronic myeloid leukemia before starting treatment with TKIs; (ii) any kind of therapy administered before a confirmed diagnosis of chronic myeloid leukemia might change the amount of BCR::ABL1 expression levels.

Background: Despite advances in uveal melanoma (UM) diagnosis and treatment, about 50% of patients develop distant metastases, thereby displaying poor overall survival. Molecular profiling has identified several genetic alterations that can stratify patients with UM into different risk categories. However, these genetic alterations are currently dispersed over multiple studies and several methodologies, emphasizing the need for a defined workflow that will allow standardized and reproducible molecular analyses. Methods: Following the findings published by “The Cancer Genome Atlas–UM” (TCGA-UM) study, we developed an NGS-based gene panel (called the UMpanel) that classifies mutation sets in four categories: initiating alterations (CYSLTR2, GNA11, GNAQ and PLCB4), prognostic alterations (BAP1, EIF1AX, SF3B1 and SRSF2), emergent biomarkers (CDKN2A, CENPE, FOXO1, HIF1A, RPL5 and TP53) and chromosomal abnormalities (imbalances in chromosomes 1, 3 and 8). Results: Employing commercial gene panels, reference mutated DNAs and Sanger sequencing, we performed a comparative analysis and found that our methodological approach successfully predicted survival with great specificity and sensitivity compared to the TCGA-UM cohort that was used as a validation group. Conclusions: Our results demonstrate that a reproducible NGS-based workflow translates into a reliable tool for the clinical stratification of patients with UM.

Germline BRCA1/2 alteration has been linked to an increased risk of hereditary breast and ovarian cancer syndromes. As a result, genetic testing, based on NGS, allows us to identify a high number of variants of uncertain significance (VUS) or conflicting interpretation of pathogenicity (CIP) variants. The identification of CIP/VUS is often considered inconclusive and clinically not actionable for the patients’ and unaffected carriers’ management. In this context, their assessment and classification remain a significant challenge. The aim of the study was to investigate whether the in silico prediction tools (PolyPhen-2, SIFT, Mutation Taster and PROVEAN) could predict the potential clinical impact and significance of BRCA1/2 CIP/VUS alterations, eventually impacting the clinical management of Breast Cancer subjects. In a cohort of 860 BC patients, 10.6% harbored BRCA1 or BRCA2 CIP/VUS alterations, mostly observed in BRCA2 sequences (85%). Among them, forty-two out of fifty-five alterations were predicted as damaging, with at least one in silico that used tools. Prediction agreement of the four tools was achieved in 45.5% of patients. Moreover, the highest consensus was obtained in twelve out of forty-two (28.6%) mutations by considering three out of four in silico algorithms. The use of prediction tools may help to identify variants with a potentially damaging effect. The lack of substantial agreement between the different algorithms suggests that the bioinformatic approaches should be combined with the personal and family history of the cancer patients.

In the field of breast cancer care, a significant breakthrough has occurred with the recognition of HER2-low expression as a target for novel anti-HER2 antibody–drug conjugates (ADC). This discovery is reshaping the treatment landscape, challenging previous perceptions that considered HER2-low as clinically insignificant. The ability to target HER2-low expression is expected to have substantial clinical implications, irrespective of gender, including in cases of male breast cancer (MBC). However, an estimate of the prevalence of the HER2-low subtype in MBC is missing. This retrospective, observational, multicenter study was aimed at characterizing the HER2-low subtype in MBC. For the purpose of this study, the three-tiered categorization of HER2 (HER2-0, HER2-low, and HER2-positive) was used to reclassify the HER2-negative group into HER-0 or HER2-low subtypes. In the whole series of 144 invasive MBCs, 79 (54.9%) were HER2-0 (IHC scores of 0), 39 (27.1%) HER2-low (IHC scores of 1+/2+ with negative ISH), and 26 (18.0%) HER2-positive (IHC scores of 3+/2+ with positive ISH). Specifically, among hormone receptor-positive (HR+) HER2-negative invasive MBCs, 34.8% were HER2-low and 65.2% HER2-0. Compared with HER2-0, HER2-low subtype was associated with a positive lymph node involvement (p = 0.01). Other pathologic characteristics including histology, staging, and grading did not show notable variations between the two subtypes. The presence of germline BRCA1/2 pathogenic variants (PVs) did not significantly differ between HER2-0 and HER2-low MBCs. However, about 13% of HER2-low MBCs had germline PVs in BRCA1/2 genes, mainly BRCA2, a clinically relevant observation in the context of combined target therapy. Overall, our data, which focused on the largest gender-specific breast cancer series, to our knowledge, confirm that the emerging three-tiered categorization of HER2 (HER2-0, HER2-low, and HER2-positive) can also be considered in MBC, to mitigate both the gender gap and the underrepresentation of males in clinical trials.

Cancer molecular profiling obtained with conventional bulk sequencing describes average alterations obtained from the entire cellular population analyzed. In the era of precision medicine, this approach is unable to track tumor heterogeneity and cannot be exploited to unravel the biological processes behind clonal evolution. In the last few years, functional single-cell omics has improved our understanding of cancer heterogeneity. This approach requires isolation and identification of single cells starting from an entire population. A cell suspension obtained by tumor tissue dissociation or hematological material can be manipulated using different techniques to separate individual cells, employed for single-cell downstream analysis. Single-cell data can then be used to analyze cell–cell diversity, thus mapping evolving cancer biological processes. Despite its unquestionable advantages, single-cell analysis produces massive amounts of data with several potential biases, stemming from cell manipulation and pre-amplification steps. To overcome these limitations, several bioinformatic approaches have been developed and explored. In this work, we provide an overview of this entire process while discussing the most recent advances in the field of functional omics at single-cell resolution.

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are two of the main BCR-ABL1-negative chronic myeloproliferative neoplasms (MPNs) characterized by abnormal megakaryocytic proliferation. Janus kinase 2 (JAK2) mutations are detected in 50–60% of ET and PMF, while myeloproliferative leukemia (MPL) virus oncogene mutations are present in 3–5% of cases. While Sanger sequencing is a valuable diagnostic tool to discriminate the most common MPN mutations, next-generation sequencing (NGS) is a more sensitive technology that also identifies concurrent genetic alterations. In this report, we describe two MPN patients with simultaneous double MPL mutations: a woman with ET presenting both MPL^V501A-W515R and JAK2^V617F mutations and a man with PMF displaying an uncommon double MPL^V501A-W515L. Using colony-forming assays and NGS analyses, we define the origin and mutational landscape of these two unusual malignancies and uncover further gene alterations that may contribute to the pathogenesis of ET and PMF.