Basal and one-month differed neutrophil, lymphocyte and platelet values and their ratios strongly predict the efficacy of checkpoint inhibitors immunotherapy in patients with advanced BRAF wild-type melanoma.

BACKGROUND: To evaluate the capability of basal and one-month differed white blood cells (WBC), neutrophil, lymphocyte and platelet values and their ratios (neutrophils-to-lymphocytes ratio, NLR, and platelets-to-lymphocytes ratio, PLR) in predicting the response to immune checkpoint inhibitors (ICI) in metastatic melanoma (MM). METHODS: We performed a retrospective study of 272 BRAF wild-type MM patients treated with first line ICI. Bivariable analysis was used to correlate patient/tumor characteristics with clinical outcomes. Variations between time 1 and time 0 (Δ) of blood parameters were also calculated and dichotomized using cut-off values assessed by ROC curve. RESULTS: At baseline, higher neutrophils and NLR negatively correlated with PFS, OS and disease control rate (DCR). Higher PLR was also associated with worse OS. In multivariable analysis, neutrophils (p = 0.003), WBC (p = 0.069) and LDH (p = 0.07) maintained their impact on PFS, while OS was affected by LDH (p < 0.001), neutrophils (p < 0.001) and PLR (p = 0.022), while DCR by LDH (p = 0.03) and neutrophils (p = 0.004). In the longitudinal analysis, PFS negatively correlated with higher Δplatelets (p = 0.039), ΔWBC (p < 0.001), and Δneutrophils (p = 0.020), and with lower Δlymphocytes (p < 0.001). Moreover, higher ΔNLR and ΔPLR identified patients with worse PFS, OS and DCR. In the multivariable model, only ΔNLR influenced PFS (p = 0.004), while OS resulted affected by higher ΔWBC (p < 0.001) and lower Δlymphocytes (p = 0.038). Higher ΔWBC also affected the DCR (p = 0.003). When clustering patients in 4 categories using basal LDH and ΔNLR, normal LDH/lower ΔNLR showed a higher PFS than high LDH/higher ΔNLR (20 vs 5 months). Moreover, normal LDH/higher Δlymphocytes had a higher OS than high LDH/lower Δlymphocytes (50 vs. 10 months). CONCLUSIONS: Baseline and early variations of blood cells, together with basal LDH, strongly predict the efficacy of ICI in MM. Our findings propose simple, inexpensive biomarkers for a better selection of patient treatments. Prospective multicenter studies are warranted to confirm these data.

The Tumor Microenvironment in Neuroendocrine Tumors: Biology and Therapeutic Implications.

Neuroendocrine tumors (NETs) include a heterogeneous group of malignancies arising in the diffuse neuroendocrine system and characterized by indolent growth. Complex interactions take place among the cellular components of the microenvironment of these tumors, and the recognition of the molecular mediators of their interplay and cross talk is crucial to discover novel therapeutic targets. NET cells overexpress a plethora of proangiogenic molecules including vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, semaphorins, and angiopoietins that promote both recruitment and proliferation of endothelial cell precursors, thus resulting among the most vascularized cancers with a microvessel density 10-fold higher than epithelial tumors. Also, NETs operate multifaceted interactions with stromal cells, both at local and distant sites, and whether their paracrine secretion of serotonin, connective tissue growth factor, and transforming growth factor ß primarily drives the fibroblast activation to enhance the tumor proliferation, on the other side NET-derived profibrotic factors accelerate the extracellular matrix remodeling and contribute to heart valves and/or mesenteric fibrosis development, namely, major complications of functioning NETs. However, at present, little is known on the immune landscape of NETs, but accumulating evidence shows that tumor-infiltrating neutrophils, mast cells, and/or macrophages concur to promote the neoangiogenic switch of these tumors by either direct or indirect mechanisms. On the other hand, immune checkpoint molecules are heterogeneously expressed in NETs' surrounding cells, and it is unclear whether or not tumor-infiltrating lymphocytes are antitumor armed within the microenvironment, given their low mutational load. Here, we review the current knowledge on both gastroenteropancreatic and pulmonary NETs' microenvironment as well as both established and innovative treatments aimed at targeting the tumor-host interplay.

The Role of Cytotoxic Chemotherapy in Well-Differentiated Gastroenteropancreatic and Lung Neuroendocrine Tumors.

OPINION STATEMENT: The treatment landscape of well-differentiated neuroendocrine tumors (NETs) has considerably expanded in recent years, and both somatostatin analogs, radiolabeled somatostatin analogs, everolimus, and sunitinib have been incorporated within the therapeutic armamentarium against these malignancies. Even in the context of multiple treatment options available, cytotoxic chemotherapy plays a pivotal role in the management of pancreatic NETs (panNETs), while its activity in midgut carcinoids and lung NETs is still debated. High response rates, ranging from 30 to 70%, have been consistently reported in studies of panNETs investigating streptozotocin-, temozolomide-, or platinum-based regimens, and an unprecedented prolongation of progression-free survival has been recently demonstrated in a prospective, randomized trial of capecitabine and temozolomide in patients with progressive panNETs. As a general principle, cytotoxic chemotherapy appears particularly appropriate in patients with bulky, symptomatic, or rapidly progressing tumors, especially of pancreatic origin, or in the salvage setting of NET patients who have failed alternative therapeutic options. Emerging evidence has also shown the potential efficacy of induction chemotherapy in patients with locally advanced or oligometastatic panNET, but prospective validation is needed before implementation of this approach in routine clinical practice. At present, there is no consensus on adjuvant therapy in pulmonary NETs, and differences between guideline recommendations at this regard mainly stem from the lack of high-level evidence. In the future, the identification of molecular biomarkers of response to chemotherapy might allow better patient preselection, thus leading to improved outcomes.

Double Heterozygosity for BRCA1 Pathogenic Variant and BRCA2 Polymorphic Stop Codon K3326X: A Case Report in a Southern Italian Family.

Here, we describe a patient with bilateral breast cancer and melanoma, and with a concomitant double variant, namely p.Gln563Ter in BRCA1 and p.Lys3326Ter in BRCA2. The BRCA2 p.Lys3326Ter (K3326X) (rs11571833) mutation identified in our patient is a debated substitution of thymidine for adenine which is currently regarded as benign polymorphism in main gene databases. Recent studies, however, describe this variant as associated with breast and ovarian tumors. Based on the observation of the cancer's earliest age of onset in this subject, our purpose was to reevaluate this variant according to recent papers indicating a role of powerful modifier of the genetic penetrance. Genetic testing was performed in all consenting patient's relatives, and in the collection of the clinical data particular attention was paid to the age of onset of the neoplasia. Following our observation that the our patient with double heterozygosis had an early age of onset for cancer similar to a few rare cases of double mutation for BRCA1 and BRCA2, we also performed an extensive review of the literature relative to patients carrying a double heterozygosity for both genes. In line with previous studies relative to the rare double heterozygosity in both BRCA1/2 genes, we found the earlier onset of breast cancer in our patient with both BRCA1/2 mutations with respect to other relatives carrying the single BRCA1 mutation. The presence of the second K3326X variant in our case induces a phenotype characterized by early onset of the neoplasia in a manner similar to the other cases of double heterozygosity previously described. Therefore, we suggest that during the genetic counseling, it should be recommendable to evaluate the presence of the K3326X variant in association with other pathogenic mutations.

Rare histotypes of epithelial biliary tract tumors: A literature review.

Adenocarcinoma represents the most frequent biliary tract cancer. However, other rare histotypes can be found in the biliary tract, such as cholangiolocellular carcinoma, cholangiocarcinoma with ductal plate malformation pattern, adenosquamous carcinoma, mucinous carcinoma, signet ring cell carcinoma, clear cell carcinoma, mucoepidermoid carcinoma, lymphoepithelioma-like carcinoma, and sarcomatous cholangiocarcinoma. These cancer types account for less than 10 % of all the already rare biliary tract tumors. Yet, they represent a relevant issue in everyday clinical practice, given the lack of therapeutic recommendations and the overall scarcity of data, mainly deriving from isolated small center-specific cohorts of patients.The shifts of such histotypes from the most common ones reflect genetic and molecular differences, determine changes in clinical aggressiveness, and suggest a possible variability in sensitivity to the standard treatments of biliary adenocarcinomas. The consistency and degree of these variables are still to be solidly demonstrated and investigated. Therefore, this paper aims to review the current literature concerning very infrequent and rare epithelial biliary tract cancers, focusing our attention on the clinical, molecular, and immunohistochemical features of these tumors.

An Invasive Disease Event-Free Survival Analysis to Investigate Ki67 Role with Respect to Breast Cancer Patients' Age: A Retrospective Cohort Study.

Characterization of breast cancer into intrinsic molecular profiles has allowed women to live longer, undergoing personalized treatments. With the aim of investigating the relation between different values of ki67 and the predisposition to develop a breast cancer-related IDE at different ages, we enrolled 900 patients with a first diagnosis of invasive breast cancer, and we partitioned the dataset into two sub-samples with respect to an age value equal to 50 years. For each sample, we performed a Kaplan−Meier analysis to compare the IDE-free survival curves obtained with reference to different ki67 values. The analysis on patients under 50 years old resulted in a p-value < 0.001, highlighting how the behaviors of patients characterized by a ki67 ranging from 10% to 20% and greater than 20% were statistically significantly similar. Conversely, patients over 50 years old characterized by a ki67 ranging from 10% to 20% showed an IDE-free survival probability significantly greater than patients with a ki67 greater than 20%, with a p-value of 0.01. Our work shows that the adoption of two different ki67 values, namely, 10% and 20%, might be discriminant in designing personalized treatments for patients under 50 years old and over 50 years old, respectively.

Case Report: Autoimmune Pemphigus Vulgaris in a Patient Treated With Cemiplimab for Multiple Locally Advanced Cutaneous Squamous Cell Carcinoma.

BACKGROUND: Pemphigus vulgaris (PV) is a rare and severe autoimmune blistering disorder affecting the skin and mucous membranes, characterized by the production of autoantibodies against two desmosomal adhesion proteins, desmoglein 1 and 3. In patients with advanced squamous cell carcinoma of the skin unfit for surgery and radiotherapy, immune check-point inhibitors, including the anti-Programmed Death-1 (PD-1) agent cemiplimab have been successfully employed proving relevant clinical outcomes. Cemiplimab is a monoclonal antibody capable of inhibiting PD-1 signalling that has recently been approved for the treatment of patients with metastatic or locally advanced cutaneous squamous cell carcinoma. Although the peculiar setting of advanced CSCC involving elderly patients, rare and unusual skin immune-related adverse events such as PV could be observed in cemiplimab treated patients. CASE REPORT: A 95-year-old man without a history of autoimmune disease was treated with cemiplimab for multiple and advanced squamous cell carcinomas of the head obtaining a complete response to therapy. After seven cycles of cemiplimab administered every 21 days, the patient developed a mucocutaneous blistering eruption. Clinical diagnosis of PV was suspected on the basis of the diffuse involvement of trunk and extremities with large blisters and necrotic eschar. It was carried out an ELISA test, that showed high level of circulating antibodies against desmoglein 1, thus confirming the diagnosis of PV. For this reason, cemiplimab infusion was discontinued and complete resolution of skin lesions was obtained using oral prednisone 0,8 mg/kg/daily for four weeks. Once remission was achieved, a maintenance dose of 10 mg/day was administered, observing a good control of bullous disease and low value of desmoglein 1. Response to CSCC persisted also during cemiplimab discontinuation, until obtaining a complete remission still persisting at 9 months after the last cycle of therapy. CONCLUSION: The case we observed is the first description of PV revealed from cemiplimab therapy, thus suggesting that cemiplimab could allow the arise of underlying autoimmune PV, through a mechanism both T and B-cell-mediated.

Electrochemotherapy as a Trigger to Overcome Primary Resistance to Anti-PD-1 Treatment: A Case Report of Melanoma of the Scalp.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors is one of the main therapies for advanced melanoma. Nevertheless, albeit remarkable, immunotherapy results are still unsatisfactory as more than half of patients progress, and resistance to treatment still has a dramatic impact on clinical outcomes. Local treatments such as radiotherapy or electrochemotherapy (ECT), in addition to local control with palliative intent, have been shown to release tumoral neoantigens that can stimulate a robust systemic antitumor immune response. CASE PRESENTATION: We report the case of a patient with multiple nodular melanoma lesions of the scalp initially treated with local ECT. Soon after the procedure, multiple new lesions appeared close to the treated ones, therefore the patient started a systemic treatment with the anti-PD-1 nivolumab. The lesions of the scalp did not respond to immunotherapy, presenting a loco-regional spreading. To control the bleeding and painful lesions, we performed a second ECT, while continuing systemic immunotherapy. The treated lesions responded to the second procedure, while the other lesions continued progressing in number and dimension. Unexpectedly, after 2 months from the second ECT, the patient presented a progressive shrinkage of both treated and untreated lesions until complete remission. Concomitantly, he developed immune-related adverse events including grade 4 thyroid toxicity, grade 2 vitiligo-like depigmentation and grade 2 pemphigoid. At present, after 18 months from the first ECT and 14 months from the starting of anti-PD-1 immunotherapy, the patient is in good clinical condition and complete remission of disease still persists. CONCLUSION: This case highlights the potential role of ECT in increasing tumor immunogenicity and consequently in inducing a powerful immune response overcoming primary resistance to checkpoint inhibitor immunotherapy.

Examining the Relationship between Circulating CD4- CD8- Double-Negative T Cells and Outcomes of Immuno-Checkpoint Inhibitor Therapy-Looking for Biomarkers and Therapeutic Targets in Metastatic Melanoma.

BACKGROUND: The role of circulating CD4-/CD8- double-negative T cells (DNTs) in the immune response to melanoma is poorly understood, as are the effects of checkpoint inhibitors on T cell subpopulations. METHODS: We performed a basal and longitudinal assessment of circulating immune cells, including DNTs, in metastatic melanoma patients treated with checkpoint blockade in a single-center cohort, and examined the correlations levels of immune cells with clinical features and therapy outcomes. RESULTS: Sixty-eight patients (48 ipilimumab, 20 PD1 inhibitors) were enrolled in the study. Our analysis indicated that better outcomes were associated with normal LDH, fewer than three metastatic sites, an ECOG performance status of 0, M1a stage, lower WBC and a higher lymphocyte count. The increase in lymphocyte count and decrease of DNTs were significantly associated with the achievement of an overall response. The median value of DNT decreased while the CD4+ and NK cells increased in patients that responded to treatment compare to those who did not respond to treatment. CONCLUSIONS: DNT cells change during treatment with checkpoint inhibitors and may be adept at sensing the immune response to melanoma. The complementary variation of DNT cells with respect to CD4+ and other immune actors may improve the reliability of lymphocyte assessment. Further investigation of DNT as a potential target in checkpoint inhibitor resistant melanoma is warranted.

No Impact of NRAS Mutation on Features of Primary and Metastatic Melanoma or on Outcomes of Checkpoint Inhibitor Immunotherapy: An Italian Melanoma Intergroup (IMI) Study.

AIMS: It is debated whether the NRAS-mutant melanoma is more aggressive than NRAS wildtype. It is equally controversial whether NRAS-mutant metastatic melanoma (MM) is more responsive to checkpoint inhibitor immunotherapy (CII). 331 patients treated with CII as first-line were retrospectively recruited: 162 NRAS-mutant/BRAF wild-type (mut/wt) and 169 wt/wt. We compared the two cohorts regarding the characteristics of primary and metastatic disease, disease-free interval (DFI) and outcome to CII. No substantial differences were observed between the two groups at melanoma onset, except for a more frequent ulceration in the wt/wt group (p = 0.03). Also, the DFI was very similar in the two cohorts. In advanced disease, we only found lung and brain progression more frequent in the wt/wt group. Regarding the outcomes to CII, no significant differences were reported in overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) or overall survival (OS) (42% versus 37%, 60% versus 59%, 12 (95% CI, 7-18) versus 9 months (95% CI, 6-16) and 32 (95% CI, 23-49) versus 27 months (95% CI, 16-35), respectively). Irrespectively of mutational status, a longer OS was significantly associated with normal LDH, <3 metastatic sites, lower white blood cell and platelet count, lower neutrophil-to-lymphocyte (N/L) ratio. Our data do not show increased aggressiveness and higher responsiveness to CII in NRAS-mutant MM.

Cemiplimab in an Elderly Frail Population of Patients With Locally Advanced or Metastatic Cutaneous Squamous Cell Carcinoma: A Single-Center Real-Life Experience From Italy.

BACKGROUND: Cutaneous squamous cell carcinoma (CSCC) is the second most common skin cancer whose incidence is growing parallel to the lengthening of the average lifespan. Cemiplimab, an antiPD-1 monoclonal antibody, is the first approved immunotherapy for patients with locally advanced CSCC (laCSCC) or metastatic CSCC (mCSCC) thanks to phase I and II studies showing high antitumor activity and good tolerability. Nevertheless, at present, very few data are available regarding cemiplimab in real-life experience and in frail, elderly, and immunosuppressed patients as well as regarding biomarkers able to predict response so as to guide therapeutic choices. PATIENTS AND METHODS: We built a retroprospective cohort study including 30 non-selected patients with laCSCC (25) and mCSCC (five) treated with cemiplimab from August 2019 to November 2020. Clinical outcomes, toxicity profile, and correlations with disease, patients, and peripheral blood parameters are explored. RESULTS: The median age was 81 years (range, 36-95), with 24 males and five patients having an immunosuppressive condition, while the frailty prevalence was 83% based on index derived from age, Eastern Cooperative Oncology Group performance status, and Charlson Comorbidity Index. We reported 23 responses (76.7%) with nine complete responses (30%). A statistically significant higher response rate was observed in head and neck primary tumors and in patients with hemoglobin level >12 g/dl. No difference was observed with respect to frailty, median age, sex, and body mass index. The baseline low neuthophil/lymphocyte ratio and low platelet/lymphocyte ratio resulted to be also correlated with a better response. Moreover, lymphocyte, neutrophil, and monocyte behaviors had an opposite trend in responders and non-responders. An overall response was reported in four of five immunosuppressed patients. Seventeen patients (57.6%) have an ongoing response and are still alive. Six responders had interrupted treatment (two for toxicity and four for personal choice) but maintained their response. The treatment was well tolerated by the majority of patients. The most common adverse events were fatigue in seven patients (23.3%) and skin toxicity in 10 patients (33.3%), including pruritus in six patients, rash in three patients, and bullous erythema in one patient. CONCLUSIONS: In our real-life experience, cemiplimab showed a high antitumor activity with acceptable safety profile similar to those in trials with selected patients. Moreover, its antitumor activity resulted to be not impaired in very elderly patients and in those with immunocompromised status.

Neoangiogenesis in Melanoma: An Issue in Biology and Systemic Treatment.

Neoangiogenesis is a recognized hallmark of cancer, granting tumor cells to dispose of metabolic substrates through a newly created vascular supply. Neoangiogenesis was also confirmed in melanoma, where vascular proliferation is associated with increased aggressiveness and poorer prognosis. Furthermore, melanoma cells show the so-called vascular mimicry, consisting in the assumption of endothelial-like features inducing the expression of pro-angiogenic receptors and ligands, which take part in the interplay with extracellular matrix (ECM) components and are potentiated by the ECM remodeling and the barrier molecule junction alterations that characterize the metastatic phase. Although neoangiogenesis was biologically proven and clinically associated with worse outcomes in melanoma patients, in the past anti-angiogenic therapies were employed with poor improvement of the already unsatisfactory results associated with chemotherapic agents. Among the novel therapies of melanoma, immunotherapy has led to previously unexpected outcomes of treatment, yet there is a still strong need for potentiating the results, possibly by new regimens of combination therapies. Molecular models in many cancer types showed mutual influences between immune responses and vascular normalization. Recently, clinical trials are investigating the efficacy of the association between anti-angiogenetic agents and immune-checkpoint inhibitors to treat advanced stage melanoma. This paper reviews the biological bases of angiogenesis in melanoma and summarizes the currently available clinical data on the use of anti-angiogenetic compounds in melanoma.

DAXX mutations as potential genomic markers of malignant evolution in small nonfunctioning pancreatic neuroendocrine tumors.

Management of localized well-differentiated pancreatic neuroendocrine tumors (panNETs) is controversial and primarily dependent on tumor size. Upfront surgery is usually recommended for tumors larger than 2 cm in diameter since they frequently show metastatic potential, whereas smaller panNETs are generally characterized by an indolent clinical course, with a rate of relapse or metastasis below 15%. To explore whether increased tumor size is paralleled by genomic variations, we compared the rate and the mutational patterns of putative driver genes that are recurrently altered in these tumors by investigating differential cohorts of panNET surgical specimens smaller (n = 27) or larger than 2 cm (n = 29). We found that the cumulative number of mutations detected in panNETs >2 cm was significantly higher (p = 0.03) relative to smaller tumors, while mutations of DAXX were significantly more frequent in the cohort of larger tumors (p = 0.05). Moreover, mutations of DAXX were associated with features of malignancy including increased grade, nodal involvement and lymphovascular invasion, and independently predicted both relapse after surgery (p = 0.05) and reduced DFS in multivariable analysis (p = 0.02). Our data suggest that alterations of the DAXX/ATRX molecular machinery increase the malignant potential of panNETs, and that identification of mutations of DAXX/ATRX in small, nonfunctioning tumors can predict the malignant progression observed in a minority of them.

Liquid biopsy of cancer: a multimodal diagnostic tool in clinical oncology.

Over the last decades, the concept of precision medicine has dramatically renewed the field of medical oncology; the introduction of patient-tailored therapies has significantly improved all measurable outcomes. Liquid biopsy is a revolutionary technique that is opening previously unexpected perspectives. It consists of the detection and isolation of circulating tumor cells, circulating tumor DNA and exosomes, as a source of genomic and proteomic information in patients with cancer. Many technical hurdles have been resolved thanks to newly developed techniques and next-generation sequencing analyses, allowing a broad application of liquid biopsy in a wide range of settings. Initially correlated to prognosis, liquid biopsy data are now being studied for cancer diagnosis, hopefully including screenings, and most importantly for the prediction of response or resistance to given treatments. In particular, the identification of specific mutations in target genes can aid in therapeutic decisions, both in the appropriateness of treatment and in the advanced identification of secondary resistance, aiming to early diagnose disease progression. Still application is far from reality but ongoing research is leading the way to a new era in oncology. This review summarizes the main techniques and applications of liquid biopsy in cancer.

ALK gene alterations in cancer: biological aspects and therapeutic implications.

ALK was first reported in 1994 as a translocation in anaplastic large cell lymphoma and then described with different abnormalities in a number of tumors. Recently, a shortly accumulated biomedical research clarified the numerous biological processes underlying its ability to support cancer development, growth and progression. Advent of precision medicine has finally provided unexpected advances, leading to the development of ALK-targeting inhibitors with superior efficacy as compared with standard chemotherapy regimens, as well as the identification of resistance mechanisms and the creation of 'next-generation' treatments. This review summarizes the current understanding of ALK-driven cancers from the oncogenesis and mutation frequency by The Cancer Genome Atlas database through the diagnostic approach, to an updated portrait of available tyrosine kinase inhibitors, considering their effectiveness in cancer treatment, the molecular reasons of therapeutic failure, and the actual and future ways to overcome resistances.

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA).

BACKGROUND: Isolation and genotyping of circulating tumor cells (CTCs) is gaining an increasing interest by clinical researchers in oncology not only for investigative purposes, but also for concrete application in clinical practice in terms of diagnosis, prognosis and decision treatment with targeted therapies. For the mutational analysis of single CTCs, the most advanced biotechnology methodology currently available includes the combination of whole genome amplification (WGA) followed by next-generation sequencing (NGS). However, the sequence of these molecular techniques is time-consuming and may also favor operator-dependent errors, related to the procedures themselves that, as in the case of the WGA technique, might affect downstream molecular analyses. MATERIALS AND METHODS: A preliminary approach of molecular analysis by NGS on a model of CTCs without previous WGA procedural step was performed. We set-up an artificial sample obtained by spiking the SK-MEL-28 melanoma cell line in normal donor peripheral whole blood. Melanoma cells were first enriched using an AutoMACS® (Miltenyi) cell separator and then isolated as single and pooled CTCs by DEPArray™ System (Silicon Biosystems). NGS analysis, using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies) with the Ion Torrent PGM™ system (Life Technologies), was performed on the SK-MEL-28 cell pellet, a single CTC previously processed with WGA and on 1, 2, 4 and 8 recovered CTCs without WGA pre-amplification. RESULTS: NGS directly carried out on CTCs without WGA showed the same mutations identified in SK-MEL-28 cell line pellet, with a considerable efficiency and avoiding the errors induced by the WGA procedure. CONCLUSION: We identified a cost-effective, time-saving and reliable methodological approach that could improve the analytical accuracy of the liquid biopsy and appears promising in studying CTCs from cancer patients for both research and clinical purposes.

Characterization of a rare nonpathogenic sequence variant (c.1905C>T) of the dihydropyrimidine dehydrogenase gene (DPYD).

BACKGROUND: In the era of precision medicine, the suitability of fluoropyrimidine therapies in clinical oncology can be checked by pharmacogenetic investigations of single patients, thus optimizing resources and indicating the appropriate drugs to personalize their chemotherapy. For example, the presence of dihydropyrimidine dehydrogenase gene (DPYD) polymorphisms in cancer patients may lead to adverse effects when adopting fluoropyrimidine-based therapies. METHODS: We detected in a cancer patient a rare germline synonymous heterozygous variant of DPYD (c.1905C>T) in proximity to the exon 14 splice donor site. Because in silico analyses hypothesized potential deleterious effects of the splice site, we performed both quantitative and qualitative mRNA analyses to investigate the possible pathogenic nature of the variant. RESULTS: We did not detect any alterations in mRNA expression or in the cDNA sequence of DPYD gene transcript. CONCLUSIONS: Our observations suggest that the c.1905C>T variant of DPYD does not have a pathogenic effect. Therefore, assessment of the clinical significance of rare sequence variants could emphasize the predictive value of DPYD gene alterations in identifying patients at potential risk for fluoropyrimidine-related toxicity.

Osteotropism of neuroendocrine tumors: role of the CXCL12/ CXCR4 pathway in promoting EMT in vitro.

Neuroendocrine tumors (NETs) metastasize to the skeleton in approximately 20% of patients. We have previously shown that the epithelial-mesenchymal transition (EMT) regulates the NET osteotropism and that CXCR4 overexpression predicts bone spreading. Here, we unravel the molecular mechanisms linking the activation of the CXCL12/CXCR4 axis to the bone colonization of NETs using cell lines representative of pancreatic (BON1, CM, QGP1), intestinal (CNDT 2.5), and bronchial origin (H727). By combining flow cytometry and ELISA, BON1, CM and QGP1 cells were defined as CXCR4high/CXCL12low, while H727 and CNDT 2.5 were CXCR4low/CXCL12high. CXCL12 was inert on cell proliferation, but significantly increased the in vitro osteotropism of CXCR4high/CXCL12low cells, as assessed by transwell assays with or without Matrigel membranes. In these cells, CXCL12 induced in vitro a marked EMT-like transcriptional shift with acquirement of a mesenchymal shape. The nuclei of CXCR4high/CXCL12low NET cells were typically enriched in non-phosphorylated CXCR4, particularly upon agonist stimulation. Silencing of CXCR4 via siRNA prevented the CXCL12-induced EMT in CXCR4high/CXCL12low NET cell lines resulting in the abrogation of both migration and transcriptional mesenchymal patterns. Our data suggest that CXCL12 conveys EMT-promoting signals in NET cells through CXCR4, which in turn regulates transcriptional, morphologic and functional modifications resulting in enhanced in vitro osteotropism of NET cells. Unique functions of CXCR4 may be segregated in relation to its subcellular localization and may acquire potential relevance in future in vivo studies.

Desmoid Tumors in Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is a cancer syndrome caused by a germline mutation in the adenomatous polyposis coli (APC) gene. It is characterized by the presence of hundreds of colonic polyps, which have a high tendency to undergo malignant transformation. Among associated lesions in FAP, desmoid tumors represent a common possible life-threatening condition that requires special attention. They are rare tumors occurring with a particularly high incidence in FAP, especially after surgery. In agreement with Knudson's 'two-hit' theory, the inactivation of the residual APC gene in FAP is a critical step in the development of both colorectal cancer and desmoids. Several lines of evidence show that germline mutations affect the functional domains of the APC gene that are responsible for interactions of the transcript with ß-catenin, whereas somatic second mutations involve the downstream region of the gene. Hence, an understanding of the molecular pathways underlying desmoid progression in FAP could be important for research and a valid resource for the early prevention and tailored treatment of this disease. In this review, we provide an updated insight into desmoids in FAP syndrome, from molecular pathogenesis to the main issues in management, with special attention given to genetic and molecular features of these tumors.