Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma.

BACKGROUND: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. METHODS: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and -5p transfection in cutaneous melanoma cell lines are investigated. RESULTS: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. CONCLUSIONS: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients.

A phase II study of the L19IL2 immunocytokine in combination with dacarbazine in advanced metastatic melanoma patients.

Engineered cytokine products represent promising agents for the treatment of immunogenic tumors, such as malignant melanoma, in addition to immune checkpoint inhibitors. Here we describe the results of a controlled, randomized phase II clinical trial, aimed at assessing the therapeutic potential of L19IL2, a fully human fusion protein consisting of the L19 antibody specific to the alternatively spliced extra-domain B of fibronectin, fused to human interleukin-2 in advanced metastatic melanoma. In one arm, patients received dacarbazine (DTIC; 1000 mg/m2 of body surface on day 1 of 21-day cycles) as single agent, while in two other arms L19IL2 (22.5 million international units of IL2 equivalents) was added, based on two different schedules of administration. In total, 69 patients with stage IV melanoma were enrolled (24 in the dacarbazine arm, 23 and 22 in the other combination arms, respectively) and 67 received treatment. Analyses of efficacy results show a statistically significant benefit in terms of overall response rate and median progression-free survival for patients receiving L19IL2 in combination with DTIC, compared to DTIC as single agent. In light of these results, further clinical investigations with L19IL2 (alone or in combination with other agents) are warranted.

Anticancer Activity of Euplotin C, Isolated from the Marine Ciliate Euplotes crassus, Against Human Melanoma Cells.

Cutaneous melanoma is the most serious type of skin cancer, so new cytotoxic weapons against novel targets in melanoma are of great interest. Euplotin C (EC), a cytotoxic secondary metabolite of the marine ciliate Euplotes crassus, was evaluated in the present study on human cutaneous melanoma cells to explore its anti-melanoma activity and to gain more insight into its mechanism of action. EC exerted a marked cytotoxic effect against three different human melanoma cell lines (A375, 501Mel and MeWo) with a potency about 30-fold higher than that observed in non-cancer cells (HDFa cells). A pro-apoptotic activity and a decrease in melanoma cell migration by EC were also observed. At the molecular level, the inhibition of the Erk and Akt pathways, which control many aspects of melanoma aggressiveness, was shown. EC cytotoxicity was antagonized by dantrolene, a ryanodine receptor (RyR) antagonist, in a concentration-dependent manner. A role of RyR as a direct target of EC was also suggested by molecular modelling studies. In conclusion, our data provide the first evidence of the anti-melanoma activity of EC, suggesting it may be a promising new scaffold for the development of selective activators of RyR to be used for the treatment of melanoma and other cancer types.

Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma.

Melanoma is a devastating disease with few therapeutic options in the advanced stage and with the urgent need of reliable biomarkers for early detection. In this context, circulating microRNAs are raising great interest as diagnostic biomarkers. We analyzed the expression profiles of 21 selected microRNAs in plasma samples from melanoma patients and healthy donors to identify potential diagnostic biomarkers. Data analysis was performed using global mean normalization and NormFinder algorithm. Linear regression followed by receiver operating characteristic analyses was carried out to evaluate whether selected plasma miRNAs were able to discriminate between cases and controls. We found five microRNAs that were differently expressed among cases and controls after Bonferroni correction for multiple testing. Specifically, miR-15b-5p, miR-149-3p, and miR-150-5p were up-regulated in plasma of melanoma patients compared with healthy controls, while miR-193a-3p and miR-524-5p were down-regulated. Receiver operating characteristic analyses of these selected microRNAs provided area under the receiver operating characteristic curve values ranging from 0.80 to 0.95. Diagnostic value of microRNAs is improved when considering the combination of miR-149-3p, miR-150-5p, and miR-193a-3p. The triple classifier had a high capacity to discriminate between melanoma patients and healthy controls, making it suitable to be used in early melanoma diagnosis.

AM251 induces apoptosis and G2/M cell cycle arrest in A375 human melanoma cells.

Human cutaneous melanoma is an aggressive and chemotherapy-resistant type of cancer. AM251 is a cannabinoid type 1 (CB1) receptor antagonist/inverse agonist with off-target antitumor activity against pancreatic and colon cancer cells. The current study aimed to characterize the in-vitro antimelanoma activity of AM251. The BRAF V600E mutant melanoma cell line, A375, was used as an in-vitro model system. Characterization tools included a cell viability assay, nuclear morphology assessment, gene expression, western blot, flow cytometry with Annexin V-FITC/7-AAD double staining, cell cycle analyses, and measurements of changes in intracellular cAMP and calcium concentrations. AM251 exerted a marked cytotoxic effect against A375 human melanoma cells with potency comparable with that observed for cisplatin without significant changes in the human dermal fibroblasts viability. AM251, at a concentration that approximates the IC50, downregulated genes encoding antiapoptotic proteins (BCL2 and survivin) and increased transcription levels of proapoptotic BAX, induced alteration of Annexin V reactivity, DNA fragmentation, chromatin condensation in the cell nuclei, and G2/M phase arrest.AM251 also induced a 40% increase in the basal cAMP levels, but it did not affect intracellular calcium concentrations. The involvement of GPR55, TRPA1, and COX-2 in the AM251 mechanism of action was excluded. The combination of AM251 with celecoxib produced a synergistic antitumor activity, although the mechanism underlying this effect remains to be elucidated. This study provides the first evidence of a proapoptotic effect and G2/M cell cycle arrest of AM251 on A375 cells. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma.

Diagnostic and therapeutic approaches in Italian hospitals: adjuvant and metastatic therapy in melanoma.

Melanoma incidence and mortality rates are rising in Italy, indicating that more effective treatments are required both in the adjuvant and metastatic settings. We analyzed clinical practices in the adjuvant and metastatic settings by conducting a nationwide survey of clinicians responsible for managing melanoma treatment and follow-up in a representative sample of Italian hospitals. 95% of participating hospitals completed the panel of questions on adjuvant and metastatic treatment, making it likely that these results give a realistic picture of treatment and follow-up of melanoma patients in Italy. In low-volume hospitals (<25 new melanoma diagnoses yearly) adjuvant therapy was significantly more used than in large-volume hospitals for patients in stage III and IV (82 versus 66% and 56 versus 30%, respectively), and only 11% of patients were enrolled in clinical trials. In the metastatic setting dacarbazine was the preferred first-line treatment (32%) followed by polychemotherapy (23%); 12% of patients were enrolled in clinical trials and less than 10% received interleukin-2, usually subcutaneously. The information provided by this study was used by the Italian Melanoma Intergroup to improve the quality of care and to redirect financial resources.

Transcription regulators and ultra-rare and other rare translocation-related sarcomas treated with trabectedin: A proof of principle from a post-hoc analysis.

Background: Among sarcomas, which are rare cancers with an incidence of <6 per 100.000/year cases, ultra-rare sarcomas have an incidence of approximately ≤1/1,000,000/year cases and altogether account for ~20% of all soft tissue sarcomas (STS) and bone sarcomas. The Italian Sarcoma Group has recently performed a non-interventional, retrospective TrObs study with data from 512 anthracycline-pretreated patients with advanced multiple STS histologies and treated with trabectedin (Palmerini, Cancers 2021; ClinicalTrials.gov Identifier: NCT02793050). Methods: A post-hoc analysis of case series to evaluate the efficacy and safety of trabectedin on patients with ultra-rare and other rare translocation-related sarcomas included in TrObs study was performed. Main outcomes comprised investigator-assessed overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and safety. Results: Thirty-six patients (18 women) with ultra-rare and other rare sarcoma and a median age of 53.0 years (range: 22-81) were included. Most patients had solitary fibrous tumor (SFT; n=11) followed by epithelioid sarcoma (n=5), malignant peripheral nerve sheath tumor (MPNST; n=4), extraskeletal myxoid chondrosarcoma (EMC; n=3), desmoplastic small round cell tumor (DSRCT; n=3), and alveolar soft part sarcoma (ASPS), rhabdomyosarcoma and clear cell sarcoma (n=2 each). Thirty-five patients had metastatic disease and 23 patients received trabectedin as a second-line treatment. Among 35 patients evaluable for response, two patients with SFT and ASPS had a partial response and one patient with DSRCT obtained a complete response, reaching an ORR of 8.6% (95% CI: 2.8-23.4%). Among patients with an ORR, 6-months PFS was 100% in patients with ASPS, 45.7% in patients with SFT and 33.3% in those with DSRCT. Two patients with epithelioid sarcoma and myoepithelioma had disease stabilization lasting >24 months. Nine patients had at least one grade 3/4 adverse event, mostly being bone marrow toxicity (n=6). Conclusions: Trabectedin has some anti-tumor activity in some ultra-rare and other rare sarcomas, particularly translocation-related sarcomas, with the well-known manageable safety profile.

Impaired function of gamma-delta lymphocytes in melanoma patients.

BACKGROUND: Melanoma is an immunogenic tumour but, despite the wide range of immunotherapies tested, only few promising results have been reported to date. Both in vitro and in xenograft models, γδ lymphocyte-mediated cytotoxicity against melanoma cells has been reported. IL-2/zoledronate treatment can expand γδ cells in vitro and in animal models. This could represent an immunotherapeutic strategy against melanoma. To evaluate the feasibility of this approach, we studied γδ lymphocyte phenotype from patients with melanoma, their ability to be expanded by IL-2/zoledronate and their cytotoxic activity against SK-MEL-30 cell line. MATERIALS AND METHODS: Peripheral blood samples were collected from 30 patients with melanoma and 10 healthy donors. Percentage of γδ lymphocytes and CD45RO+CD27+, CD45RA+CD27-, CD57+, Vγ9Vδ2 subpopulations were evaluated by flow cytometry. IL-2/zoledronate γδ cell expansion rate and their cytotoxicity against SK-MEL-30 cell line were studied. RESULTS: A percentage decrease in circulating Vγ9Vδ2 and an increase in CD45RA+CD27- and CD57+ γδ lymphocytes were observed in melanoma. IL-2/zoledronate expansion rate did not differ between controls and patients with melanoma but cytotoxicity against SK-MEL-30 appeared reduced. CONCLUSIONS: Our results show that γδ cell function is impaired in patients with advanced melanoma and suggest a possible role in tumour progression.

Melanoma Prevention: Comparison of Different Screening Methods for the Selection of a High Risk Population.

BACKGROUND: Guidelines recommend limiting melanoma screening in a population with known risk factors, but none indicates methods for efficient recruitment. The purpose of this study is to compare three different methods of recruiting subjects to be screened for melanoma to detect which, if any, is the most efficient. METHODS: From 2010 to 2019, subjects were recruited as follows: (1) regular skin examinations (RS), mainly conducted through the Associazione Contro il Melanoma network; (2) occasional melanoma screening (OS), during annual public campaigns; (3) and selective screening (SS), where people were invited to undergo a skin check after filling in a risk evaluation questionnaire, in cases where the assigned outcome was intermediate/high risk. Melanoma risk factors were compared across different screening methods. Generalized Linear Mixed Models were used for multivariable analysis. RESULTS: A total of 2238 subjects (62.7% women) were recruited, median age 44 years (2-85), and 1094 (48.9 %) records were collected through RS, 826 (36.9 %) through OS, and 318 (14.2 %) through SS. A total of 131 suspicious non-melanoma skin cancers were clinically diagnosed, 20 pathologically confirmed, and 2 melanomas detected. SS performed significantly better at selecting subjects with a family history of melanoma and I-II phototypes compared to OS. CONCLUSIONS: Prior evaluation of melanoma known risk factors allowed for effective selection of a population to screen at higher risk of developing a melanoma.

Trabectedin for Patients with Advanced Soft Tissue Sarcoma: A Non-Interventional, Retrospective, Multicenter Study of the Italian Sarcoma Group.

The Italian Sarcoma Group performed this retrospective analysis of patients with advanced soft tissue sarcoma, pretreated with ≥1 anthracycline-based treatment, and treated with trabectedin every three weeks. Primary endpoint was to describe real-life use of trabectedin across Italy. Secondary endpoints included objective response rate (ORR) and safety. Overall, 512 patients from 20 Italian centers were evaluated. Leiomyosarcoma (37.7%)/liposarcoma (30.3%) were the most prevalent histological types (abbreviated as L-sarcoma). Patients received a median of four trabectedin cycles (range: 1-40), mostly as a second-line treatment (~60% of patients). The ORR was 13.7% superior (p < 0.0001) in patients with L-sarcoma compared with patients with non-L-sarcoma (16.6% vs. 9.0%). Median progression-free survival (PFS) was 5.1 months, whereas median overall survival (OS) was 21.6 months. Significantly better PFS and OS were observed in patients with L-sarcoma, those with objective responses and/or disease stabilization, treated in an early line and treated with reduced dose. Bone marrow toxicity (61.4%) and transaminase increases (21.9%) were the most common grade 3/4 adverse events. The results of this real-life study suggest that trabectedin is an active treatment, which is mostly given as a second-line treatment to patients with a good performance status and high-grade, metastatic L-sarcoma (clinical trial information: NCT02793050).

Circulating microRNAs as biomarkers for early diagnosis of cutaneous melanoma.

Introduction: Cutaneous melanoma is the deadliest form of skin cancer, with a dramatic increase in the incidence rate worldwide over the past decade. Early detection has been shown to improve the outcome of melanoma patients. The identification of noninvasive biomarkers able to identify melanoma at an early stage remains an unmet clinical need. Circulating miRNAs (c-miRNAs), small non-coding RNAs, appear as potential ideal candidate biomarkers due to their stability in biological fluids and easy detectability. Moreover, c-miRNAs are reported to be heavily deregulated in cancer patients.Areas covered: This review examines evidence of the specific c-miRNAs or panels of c-miRNAs reported to be useful in discriminating melanoma from benign cutaneous lesions.Expert opinion: Although the interesting reported by published studies, the non-homogeneity of detection and normalization methods prevents the individuation of single c-miRNA or panel of c-miRNAs that are specific for early detection of cutaneous melanoma. In the future, prospective wide and well-designed clinical trials will be needed to validate the diagnostic potential of some of the c-miRNA candidates in clinical practice.

Clinical genetic testing for familial melanoma in Italy: a cooperative study.

BACKGROUND: The Italian Society of Human Genetics' (SIGU) recommendations on genetic counseling and testing for hereditary melanoma state that clinical genetic testing can be offered to Italian melanoma families with at least two affected members. OBJECTIVE: In the framework of a cooperative study, we sought to establish the frequency of cyclin-dependent kinase inhibitor 2A mutations in melanoma families that underwent clinical genetic counseling and testing in accordance with the SIGU recommendations at 9 centers in different Italian regions. METHODS: Cyclin-dependent kinase inhibitor 2A testing was conducted by direct sequencing and multiplex ligation-dependent probe amplification analysis in melanoma families with at least two affected members. RESULTS: A total of 33% (68/204) of the families harbored cyclin-dependent kinase inhibitor 2A mutations. In the 145 families with two affected members the mutation frequency was 25%. Three novel mutations, L94P, A86T, and c.407dupG, were identified among the cases and not in 200 controls. LIMITATIONS: We were unable to perform separate analyses for individual centers, as in some cases the number of families was too small. CONCLUSIONS: The availability of clinical genetic testing for melanoma to families with just two affected members in the same branch is justified in Italy in terms of the likelihood of identifying a mutation.

The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches.

BACKGROUND: Because colorectal cancer is a significant cause of morbidity and mortality in the Western population, knowledge of the molecular and biological alterations associated with its development is important. Since primary human colon cancer cultures from fresh tumor tissue are technically difficult to obtain, experiments in most laboratories are performed on colon epithelial cell lines, but these represent just one stage of tumor progression. Only primary cultures of neoplastic colonocytes may reflect the actual responsiveness of tumors at certain developmental stages to antitumor agents. METHODS: This paper analyzes several critical points concerning primary cultures, ranging from cell isolation to culture conditions, and compares different methodological approaches to isolate and cultivate a pure fraction of viable tumor cells. Samples of resected colorectal cancers were collected from 20 patients (stage T3 or T4). We compared in vitro several approaches of tissue disaggregation including mechanical disaggregation and enzymatic dissociation with trypsin or collagenase. Isolated cells were maintained in a short-term serum-free culture system. Evaluation of the purity and tumoral nature of isolated cells was performed by immunochemistry. RESULTS: We established the antibiotic concentration necessary during transport and washing of the specimens to prevent microbial overgrowth. We demonstrated that the number of viable cells was dependent on the dissociation method used. Mechanical disaggregation is not a valid dissociation method because of the high mortality of cells and might be used only in samples for molecular analysis. Comparison of the enzymatic digestion procedures showed that digestion with trypsin allowed the highest recovery of viable cells. CONCLUSION: In this paper we analyzed several critical aspects of cell culture procedures and designed a methodological approach suitable for functional studies of colorectal cancer.

Circulating cell-free microRNAs in cutaneous melanoma staging and recurrence or survival prognosis.

Cutaneous melanoma is a skin cancer with increasing incidence. Identification of novel clinical biomarkers able to detect the stage of disease and suggest prognosis could improve treatment and outcome for melanoma patients. Cell-free microRNAs (cf-miRNAs) are the circulating copies of short non-coding RNAs involved in gene expression regulation. They are released into the interstitial fluid, are detectable in blood and other body fluids and have interesting features of ideal biomarker candidates. They are stable outside the cell, tissue specific, vary along with cancer development and are sensitive to change in the disease course such as progression or therapeutic response. Moreover, they are accessible by non-invasive methods or venipuncture. Some articles have reported different cf-miRNAs with the potential of diagnostic tools for melanoma staging, recurrence and survival prediction. Although some concordance of results is already emerging, differences in analytical methods, normalization strategies and tumour staging still will require further research and standardization prior to clinical usage of cf-miRNA analysis. This article reviews this literature with the aim of contributing to a shared focusing on these new promising tools for melanoma treatment and care.

Detection of Uterine Leiomyosarcoma Peritoneal Lesions by SPECT/CT and ROLL Technique.

We reported the preoperative radio-guided localization of 4 peritoneal metastatic nodules in the case of a 45-year-old woman with uterine leiomyosarcoma. Three lines of chemotherapy were tried, but cardiotoxicity occurred. Within the context of so-called GOSTT (guided intraoperative scintigraphic tumor targeting), preoperative radio-guided localization of peritoneal metastases enabled their subsequent radio-guided excisional biopsy. SPECT/CT allowed for anatomical localization of the hot lesions and generated a 3-dimensional volume-rendering roadmap, facilitating a surgical approach.

MC1R variants in childhood and adolescent melanoma: a retrospective pooled analysis of a multicentre cohort.

BACKGROUND: Germline variants in the melanocortin 1 receptor gene (MC1R) might increase the risk of childhood and adolescent melanoma, but a clear conclusion is challenging because of the low number of studies and cases. We assessed the association of MC1R variants with childhood and adolescent melanoma in a large study comparing the prevalence of MC1R variants in child or adolescent patients with melanoma to that in adult patients with melanoma and in healthy adult controls. METHODS: In this retrospective pooled analysis, we used the M-SKIP Project, the Italian Melanoma Intergroup, and other European groups (with participants from Australia, Canada, France, Greece, Italy, the Netherlands, Serbia, Spain, Sweden, Turkey, and the USA) to assemble an international multicentre cohort. We gathered phenotypic and genetic data from children or adolescents diagnosed with sporadic single-primary cutaneous melanoma at age 20 years or younger, adult patients with sporadic single-primary cutaneous melanoma diagnosed at age 35 years or older, and healthy adult individuals as controls. We calculated odds ratios (ORs) for childhood and adolescent melanoma associated with MC1R variants by multivariable logistic regression. Subgroup analysis was done for children aged 18 or younger and 14 years or younger. FINDINGS: We analysed data from 233 young patients, 932 adult patients, and 932 healthy adult controls. Children and adolescents had higher odds of carrying MC1R r variants than did adult patients (OR 1·54, 95% CI 1·02-2·33), including when analysis was restricted to patients aged 18 years or younger (1·80, 1·06-3·07). All investigated variants, except Arg160Trp, tended, to varying degrees, to have higher frequencies in young patients than in adult patients, with significantly higher frequencies found for Val60Leu (OR 1·60, 95% CI 1·05-2·44; p=0·04) and Asp294His (2·15, 1·05-4·40; p=0·04). Compared with those of healthy controls, young patients with melanoma had significantly higher frequencies of any MC1R variants. INTERPRETATION: Our pooled analysis of MC1R genetic data of young patients with melanoma showed that MC1R r variants were more prevalent in childhood and adolescent melanoma than in adult melanoma, especially in patients aged 18 years or younger. Our findings support the role of MC1R in childhood and adolescent melanoma susceptibility, with a potential clinical relevance for developing early melanoma detection and preventive strategies. FUNDING: SPD-Pilot/Project-Award-2015; AIRC-MFAG-11831.

The management of patients with uterine sarcoma: a debated clinical challenge.

Uterine sarcomas include a heterogeneous group of rare tumours that usually have an aggressive clinical behaviour and a poor prognosis. Total abdominal hysterectomy and bilateral salpingo-oophorectomy represents the standard surgical treatment. Pelvic and/or para-aortic lymphadenectomy is indicated for carcinosarcoma, but not for leiomyosarcoma and undifferentiated endometrial sarcoma. Some recent data on low numbers of patients with low-grade endometrial stromal sarcoma appear to show an incidence of nodal involvement higher than previously expected, thus suggesting a role for lymphadenectomy in this malignancy. Carcinosarcoma also requires a comprehensive surgical peritoneal staging. Postoperative treatment of uterine sarcomas has been long debated. Adjuvant pelvic radiotherapy appears to improve local control without any significant impact on overall survival. There is little evidence in the literature supporting the use of adjuvant chemotherapy in any gynaecological sarcomas except for carcinosarcomas. However, uterine sarcomas have a high tendency to develop distant recurrences, and recent data on adjuvant chemotherapy in soft tissue sarcomas are promising. As for the drugs to be used, it is worth noting that in a Swiss study, the combination of ifosfamide (IFO) and doxorubicin (DOX) obtained similar response rates in advanced gynaecological sarcomas and in advanced soft tissue sarcomas of other sites. In our decision-making scheme for early-stage disease, patients with leiomyosarcoma or undifferentiated endometrial sarcoma should receive adjuvant doxorubicin/epidoxorubicin (EPIDX)+ifosfamide, and those with carcinosarcoma should be treated with adjuvant cisplatin (CDDP)-based chemotherapy. The same drug regimens are used for the treatment of advanced disease. Sequential pelvic radiotherapy following chemotherapy could be delivered to selected cases. Recurrent disease often requires the integration of different therapeutic modalities, but no curative option is currently available with the possible exception of surgery for lung metastases and hormone therapy with or without debulking surgery for recurrent low-grade endometrial stromal sarcoma. Patients should be encouraged to enter clinical trials designed to identify new active drugs for these malignancies.

Optimal detection of sentinel lymph node metastases by intraoperative radioactive threshold and molecular analysis in patients with melanoma.

UNLABELLED: The aim of this study was to optimize a protocol for radioguided biopsy of the sentinel lymph node (SLN) in patients with melanoma. The protocol was based on a combination of ex vivo counting of the nodes detected intraoperatively and analysis of the harvested nodes by hematoxylin and eosin staining plus immunohistochemistry (conventional histopathology [PATH]) and by molecular biology (reverse-transcriptase polymerase chain reaction [RT-PCR]). METHODS: A total of 124 patients with primary clinical stage I-II (according to the American Joint Committee on Cancer) cutaneous melanoma underwent successful radioguided SLN biopsy. SLNs harvested for analysis included any additional nodes whose ex vivo counting rate exceeded 20% of the hottest node. All removed SLNs were examined by conventional PATH and with RT-PCR analysis for the expression of messenger RNA for tyrosinase and the melanoma antigens recognized by T cells. Complete lymph node dissection (CLND) was performed only in the case of SLN metastasis detected by PATH. Different combinations of the intraoperative parameters (only the hottest node and all nodes harvested) and of analysis (PATH and RT-PCR) were tested as predictors of clinical outcome on the basis of long-term follow-up (12-81 mo; median, 55 mo). RESULTS: A total of 197 SLNs were harvested, 41 of which harbored metastasis as detected by RT-PCR analysis; PATH detected metastasis in only 24 of 41 metastatic SLNs. In 5 of 41 instances, metastasis was not in the hottest SLN. The main factor determining correct classification of the SLN status was RT-PCR, which significantly improved detection of metastasis, even if applied only to the hottest node (P < 0.0001 vs. PATH analysis of either the hottest SLN or all nodes above the 20% threshold). Metastatic disease recurred locally in 5 patients who had not undergone CLND; RT-PCR analysis showed metastasis in 4 of these patients. The false-negative rate of SLN biopsy progressively decreased when applying PATH only to the hottest node (32.1%), additional RT-PCR to the hottest node (21.4%), PATH to all nodes (17.9%), and RT-PCR to all nodes (3.6%, P = 0.015 vs. PATH analysis of only the hottest SLN). CONCLUSION: On the basis of long-term follow-up (the gold standard for final clinical outcome of SLN biopsy), both 20% threshold and RT-PCR analysis should be applied for optimal detection of nodal metastases in patients with melanoma.

Human brain tumors: multidrug-resistance P-glycoprotein expression in tumor cells and intratumoral capillary endothelial cells.

Malignant brain tumor is a lethal disease with currently available treatment options having a limited impact on outcome. Nevertheless, novel therapeutic approaches combined with genetic prediction of chemosensitivity have, in the last decade, significantly improved clinical benefit for the treated patients. The fine characterization of the MDR1 gene encoding for P-glycoprotein (MDR1-Pgp) in brain tumors may be a crucial determinant for evaluating the long-term efficiency of specific anti-cancer compounds. By using a very high specific monoclonal antibody, the MDR1-Pgp was immunodetected in 34 out of 43 grade IV, 6 out of 10 grade III, 4 out of 7 grade II, and 1 out 3 grade I brain tumors. MDR1-Pgp resulted hyper-expressed, both in vessels and in neoplastic cells from the majority of tumors examined, compared to normal parenchyma. This study demonstrates that the MDR1 gene can be detected in all grade tumor brain malignancies and in endothelial cells of newly formed capillaries, thus, impairing drug access at the tumor cell level. Although the role of MDR1-Pgp in tumor blood vessels needs to be further examined and more clearly defined, drug resistance in malignant brain tumors may result from characteristics not only of tumor vasculature but also of neoplastic cells.

Tumor-promoting effects of cannabinoid receptor type 1 in human melanoma cells.

The role of endocannabinoid system in melanoma development and progression is actually not fully understood. This study was aimed at clarifying whether cannabinoid-type 1 (CB1) receptor may function as tumor-promoting or -suppressing signal in human cutaneous melanoma. CB1 receptor expression was measured in human melanoma cell lines by real-time PCR. A genetic deletion of CB1 receptors in selected melanoma cells was carried out by using three different short hairpin RNAs (shRNAs). Performance of target gene silencing was verified by real-time PCR and Western blot. The effects of CB1 receptor silencing on cell growth, clonogenicity, migration capability, cell cycle progression, and activation of mitogenic signals was tested. Lentiviral shRNAs vectors targeting different regions of the human CB1 gene led to a significant reduction in CB1 receptor mRNA and a near complete loss of CB1 receptor protein, compared to control vector (LV-c). The number of viable cells, the colony-forming ability and cell migration were significantly reduced in cells transduced with CB1 lentiviral shRNAs compared to LV-c. Cell cycle analyses showed arrest at G1/S phase. p-Akt and p-ERK expression were decreased in transduced versus control cells. Findings of this study suggest that CB1 receptor might function as tumor-promoting signal in human cutaneous melanoma.