Absolute quantitative PCR for detection of molecular biomarkers in melanoma patients: a preliminary report.

BACKGROUND: Malignant melanoma is the most malignant tumours of skin and mucous membranes mainly due to its aggressive biological behaviour and tendency to generate early metastases. Unfortunately, the mechanisms underlying the development, progression and the expression of an aggressive melanoma phenotype still remain largely unknown. OBJECTIVES: The purpose of this study was to determine whether a multi-panel of molecular transcripts can be predictive for risk of recurrent disease in malignant melanoma patients. RESULTS: Peripheral blood was collected from 31 malignant melanoma patients in follow-up for melanoma and from 30 healthy volunteers randomly selected. Each specimen was examined by qRT-PCR analysis for the expression of six markers: PAX3d, TYR, MITFm, MCAM, TGFß2 and ABCB5. Malignant melanoma patients expressed an important number of markers, with a median value of four markers. Only PAX3d displayed a trend in terms of differences when the levels of gene expression were made in function of Breslow index. Furthermore, PAX3d showed the best diagnostic capacity among the remaining residual markers or in combination with TGFß2 and MTIF. CONCLUSIONS: We demonstrated the usefulness of multimarker qRT-PCR to detect circulating melanoma cells in blood and to potentially assessing patient disease status or progression, especially when PAX3d was used in combination with MTIFm and TGFß2.

Differences in clinicopathological features and distribution of risk factors in Italian melanoma patients.

BACKGROUND: No studies are available in the literature on the distribution of different melanoma features and risk factors in the Italian geographical areas. OBJECTIVE: To identify the differences in clinical-pathological features of melanoma, the distribution of risk factors and sun exposure in various Italian macro-areas. METHODS: Multicentric-observational study involving 1,472 melanoma cases (713 north, 345 centre, 414 south) from 26 referral centres belonging to the Italian Multidisciplinary Group for Melanoma. RESULTS: Melanoma patients in northern regions are younger, with thinner melanoma, multiple primaries, lower-intermediate phototype and higher counts of naevi with respect to southern patients; detection of a primary was mostly connected with a physician examination, while relatives were more involved in the south. Northern patients reported a more frequent use of sunbeds and occurrence of sunburns before melanoma despite sunscreen use and a lower sun exposure during the central hours of the day. CONCLUSIONS: The understanding of differences in risk factors distribution could represent the basis for tailored prevention programmes.

Melanoma detection in Italian pigmented lesion clinics.

AIM: Accuracy in melanoma detection is important to recognize early curable melanomas and to minimize the unnecessary excision of benign lesions. The aim of this paper was to evaluate melanoma screening accuracy of Italian pigmented lesion clinics in terms of number needed to excise (NNE), melanoma thickness, and number of melanomas diagnosed during patient follow-up. METHODS: Information on all skin tumors excised in 2011 were extracted from the databases of the participating centers. Information whether the lesion was excised at the baseline examination or during patient follow-up was recorded, as well as the overall number of patients examined in each center in 2011. RESULTS: After e-mail solicitation, 22 of 40 centers agreed to participate. A total of 8229 excised lesions were collected. The overall number of examined patients was 86.564, thus 9.5% of screened patients had a lesion removed. Of the excised lesions, 866 were diagnosed as melanoma (1% of examined patients) and 5311 (88.9%) were melanocytic nevi. Three NNE were calculated giving values of 7.9 excised lesions to find 1 melanoma, 7.1 melanocytic lesions to find 1 melanoma, and 3.7 lesions to find 1 skin malignancy. The median melanoma thickness was 0.6 mm, with only 15.1% of melanomas ≥ 1 mm of thickness. Melanomas detected over time were 96 (11.1%; mean thickness, 0.3 mm), with 15.6% of lesions excised after short-term follow-up and 84.4% after long-term follow-up. CONCLUSION: The NNE values comparable to those achieved in specialized clinical settings and the high number of early melanomas diagnosed at the baseline examination or during patient follow-up indicate a high level of accuracy in melanoma screening achieved by Italian pigmented lesion clinics.

Evaluation of the safety of C-1311 (SYMADEX) administered in a phase 1 dose escalation trial as a weekly infusion for 3 consecutive weeks in patients with advanced solid tumours.

PURPOSE: C-1311 is a member of the novel imidazoacridinone family of anticancer agents. This phase 1 trial was designed to investigate the safety, tolerability and preliminary anti-tumour activity of C-1311. PATIENTS AND METHODS: This was a phase 1, inter-subject dose escalating and pharmacokinetic study of intravenous (IV) C-1311, administered weekly during 3consecutive weeks followed by 1week rest (constituting 1 cycle) in subjects with advanced solid tumours. RESULTS: Twenty-two (22) patients were treated with C-1311, the highest dose given was 640mg/m(2). All subjects experienced one or more treatment-related adverse events (AEs). The most frequently observed treatment-related AEs were neutropaenia and nausea (50% each), followed by vomiting (27%), anaemia (23%), asthenia (23%) and diarrhoea (18%). Most treatment-related AEs were of Common Terminology Criteria for Adverse Events (CTCAE) grades 1-2, except for the blood and lymphatic system disorders, which were primarily of grades 3-4. The recommended dose (RD) of C-1311 administered as once weekly IV infusions for 3weeks every 4weeks is 480mg/m(2), with the dose limiting toxicity (DLT) being grade 4 neutropaenia lasting more than 7days. Treatment at this dose offers a predictable safety profile and excellent tolerability. CONCLUSION: The safety profile and preliminary anti-tumour efficacy of C-1311, observed in this broad-phase dose-finding study, warrants further evaluation of the compound.

Association between cutaneous melanoma, Breslow thickness and vitamin D receptor BsmI polymorphism.

BACKGROUND: Literature data report an association between some vitamin D receptor (VDR) polymorphisms and different kinds of tumours, including malignant melanoma (MM). Only three VDR polymorphisms (FokI, TaqI and A-1012G) have been investigated in association with the presence of cutaneous MM or the development of metastases. OBJECTIVES: The present paper analyses for the first time the association between BsmI polymorphism and MM prevalence together with Breslow thickness. In addition, the FokI single nucleotide polymorphism was also determined. METHODS: One hundred and one patients with MM and 101 healthy donors matched for age and sex were enrolled. Molecular VDR typing was performed by means of restriction fragment length polymorphism analysis. RESULTS: All cases and controls were in Hardy-Weinberg equilibrium for BsmI, FokI and A-1012G. Significant associations were found between the BsmI bb genotype frequency and MM (P = 0.02) along with Breslow thickness (P = 0.001). This same behaviour was not observed for the FokI or A-1012G polymorphisms. Multivariate logistic regression analysis confirmed these significant results after correction for age, gender, skin type and MM localization. CONCLUSIONS: Although the biological meaning of the effects exerted by BsmI polymorphism is still under debate, the statistical association found in the present study suggests that further work should be done to verify this variant as a possible risk marker for MM and its aggressiveness, also considering that the real association may be due to other unknown genes linked to the BsmI b allele.

Hepatocellular carcinoma after systemic chemotherapy: gadolinium-enhanced mr measurement of necrosis by volume histogram.

As a preliminary study, we measured the necrosis of advanced hepatocellular carcinoma (HCC) by volume histogram after systemic chemotherapy and correlated it with clinical data. Five patients with advanced HCC secondary to chronic hepatitis and cirrhosis underwent pretreatment and posttreatment MR examination on a 1.5 T MR scanner following systemic chemotherapy. MR sequences included dynamic enhanced fast spoiled gradient echo 3D images. Clinical response to chemotherapy, as determined by MR images, was measured as changes of both the total tumor volume and the percent of tumor necrosis by volume histogram algorithm. Four of five patients had clinical improvement. Three of these patients had no or minimal change of tumor volume; however, there was an increase in tumor necrosis in follow-up MR image. One patient of five with no change in tumor necrosis had no response and died at 3 months. Serial MR images showed increased irregular necrosis of advanced HCC after systemic chemotherapy, but stable volume, in patients who responded clinically to systemic chemotherapy.

Huriez syndrome: case report with a detailed analysis of skin dendritic cells.

We report a 60-year-old man with familial scleroatrophic syndrome of Huriez who developed squamous cell carcinomas on the affected skin of the right palm. Immunohistochemical analysis showed a marked reduction in the number of CD1a+, Lag+ and S100+ epidermal Langerhans cells, but not of CD1b+ and factor XIIIa+ dermal dendritic cells, limited to palmoplantar skin. The Langerhans cell depletion was not associated with an abnormal skin content of mRNA for factors involved in Langerhans cell development or recruitment in the epidermis, including granulocyte/macrophage colony-stimulating factor, transforming growth factor-beta1 and macrophage inflammatory protein-3alpha. The results indicate that other as yet unknown mechanisms may account for the reduced number of Langerhans cells in the affected skin of such patients.

Chemoprotective and radioprotective effects of amifostine: an update of clinical trials.

Amifostine (Ethyol), the first broad-spectrum cytoprotectant approved in many countries for clinical use, is an analog of cysteamine and was originally developed by the U.S. Walter Reed Army Institute of Research in the 1950s as a radioprotective agent. Studies have shown that amifostine selectively protects normal tissues of various organs from the effects of radiation and multiple cytotoxic chemotherapeutic drugs. Amifostine has demonstrated broad-spectrum cytoprotection against myelotoxicity, nephrotoxicity, xerostomia, and mucositis associated with various chemotherapy and radiation modalities. Amifostine has been evaluated in large comparative clinical trials in patients with advanced ovarian cancer, rectal cancer, and head and neck cancer, and in many phase 2 trials in patients with various neoplastic diseases. These trials have shown that amifostine delivers protection from the cytotoxic effects of cisplatin, cyclophosphamide, and radiation on various organs. Pretreatment with amifostine has also improved salivary gland tolerance of high-dose radioiodine treatment. Recent unique observations include improvement in cytopenia in patients with myelodysplastic syndrome. This review summarizes preclinical and clinical data on amifostine and includes trials that evaluated the drug's chemoprotective and radioprotective effects and other potential uses in clinical oncology.

The preclinical basis for broad-spectrum selective cytoprotection of normal tissues from cytotoxic therapies by amifostine.

Administered before cytotoxic chemotherapy or radiation, the aminothiol, amifostine (Ethyol; Alza Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA), provides broad-spectrum cytoprotection of various normal tissues without attenuating antitumor response. The basis for the selectivity of action resides in the anabolism of amifostine at the normal tissue site by membrane-bound alkaline phosphatase. Dephosphorylation to the free thiol, WR-1065, is followed by rapid uptake into normal tissues. In contrast, uptake into tumor tissue is slow to negligible. Pretreatment with amifostine provides protection of normal tissues from the cytotoxic effects of alkylating agents, organoplatinums, anthracyclines, taxanes, and radiation. Additionally, the mutagenic and carcinogenic effects of these modalities are also attenuated. Preclinical studies show significant protection of marrow progenitor cells. Synergistic effects in marrow recovery are noted with the sequential use of amifostine and granulocyte colony-stimulating factor. Protection of kidneys and neural tissues from cisplatin toxicity has been shown, as well as protection of the heart, intestinal crypt cells, and pulmonary tissues from chemotherapy and radiation, and vasculoconnective and musculoconnective tissue in an irradiated field. Comparative in vitro and in vivo studies using murine and human tumor xenografts show no protection of antitumor effects of these same therapies despite the protection of normal organs. The unique preclinical profile of amifostine serves as the basis for the clinical development program for this new broad-spectrum cytoprotective agent.

Amifostine reduces the incidence of cumulative nephrotoxicity from cisplatin: laboratory and clinical aspects.

Cisplatin, a heavy metal complex, is one of the most active drugs used in the treatment of a variety of cancers. One of the major limitations to the maximization of its therapeutic potential is nephrotoxicity. Several preclinical studies have shown that pretreatment of mice or rats with amifostine (WR-2721, Ethyol; Alza Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA) protected against nephrotoxicity induced by both single and repeated doses of cisplatin without affecting the antitumor effects of cisplatin. The preclinical evidence of amifostine's protective effects led to phase I-III clinical studies. A phase III trial was conducted in 242 women with stage III/IV ovarian cancer receiving six cycles of cyclophosphamide/cisplatin (CP) +/- amifostine. Consistent with the cumulative nature of cisplatin-induced nephrotoxicity, by cycles 5 and 6, a significantly greater proportion of patients in the control arm compared with patients in the amifostine-treated arm were not eligible to receive cisplatin as scheduled because their serum creatinine levels had failed to return to < or = 1.5 mg/dL. Amifostine pretreatment did not affect the antitumor effects of CP as assessed by response determined at second-look surgery or overall survival. Phase II trials support these findings. To date, amifostine is the only available therapy that can ameliorate the cumulative nephrotoxic effects of cisplatin without reducing antitumor efficacy.

Clinical status and optimal use of amifostine.

Amifostine (Ethyol) is an analog of cysteamine that selectively protects normal tissues in multiple organ systems against the toxic effects of radiation and various cytotoxic drugs while preserving the antitumor effects of these therapies. Amifostine was evaluated in a multicenter, multinational phase III clinical trial that enrolled women with stage III/IV ovarian cancer. Its effects have also been studied using normal human bone marrow and human breast cancer cells, as well as leukemia cells. Additional clinical trials have shown that amifostine can protect normal tissues from the toxic effects of alkylating agents, organoplatinums, anthracyclines, taxanes, and radiation. Other laboratory and clinical investigations indicate a potential role for this cytoprotective agent in the treatment of the ineffective hematopoiesis characteristic of the myelodysplastic syndromes.

Amifostine stimulates formation of multipotent and erythroid bone marrow progenitors.

Amifostine (WR-2721, Ethyol) is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastics. To further characterize its hematopoietic activities, we investigated the effects of amifostine and its dephosphorylated metabolite, WR1065, on the in vitro growth of human bone marrow progenitors. Preincubation exposure to amifostine or WR1065 stimulated the growth of colony-forming units granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) and erythroid bursts (BFU-E) from bone marrow mononuclear cells in a dose-dependent fashion. Over the concentration range tested (0.1-1000 microM), pretreatment with the aminothiols enhanced formation of CFU-GEMM up to five-fold and BFU-E nine-fold, compared to a three-fold increase in myeloid colony recovery. In CD34+ selected cells, preincubation with amifostine increased formation of CFU-GEMM up to 38-fold and produced macroscopic colonies, exceeding colony number in cultures initiated with optimal concentrations of interleukin-1 (IL-1), IL-3, or kit ligand (KL). When compared with recombinant human cytokines, amifostine enhanced IL-1 and IL-3 induced colony formation, although its stimulatory effect was less than additive. In contrast, pretreatment with amifostine antagonized the stimulatory effects of KL, whereas synergy was observed with concurrent exposure. Ex vivo expansion studies showed that amifostine alone supported and augmented the production of myeloid progenitors in secondary cultures. Similarly, under cytokine-deficient conditions, amifostine promoted cell survival and delayed apoptosis as measured by nucleosome generation. These data indicate that amifostine is a novel multipotent hematopoietic stimulant that augments the formation and survival of bone marrow progenitors.

Amifostine protects normal tissues from paclitaxel toxicity while cytotoxicity against tumour cells is maintained.

The objectives of this study were to evaluate the protective effects of amifostine against paclitaxel-induced toxicity to normal and malignant human tissues. Haematopoietic progenitor colony assays were used to establish the number of CFU-GEMM and BFU-E colonies after incubation with WR-1065 alone, Amifostine alone, paclitaxel (2.5 or 5 microM) +/- WR-1065 or amifostine. MTT and alkaline elution assays evaluated the in vitro growth inhibitory and DNA damaging effects, respectively, of paclitaxel with or without amifostine against normal human fibroblasts and human non-small cell lung cancer (NSCLC) cells. This combination was also evaluated in vivo using severe combined immune deficient (scid) mouse models of early (non-palpable tumours) and advanced (palpable tumours) human ovarian cancer. Human 2780 ovarian cancer cells were inoculated subcutaneously while paclitaxel and amifostine were administered intraperitoneally. A brief exposure (15 min) to amifostine not only protected human haematopoietic progenitor colonies from paclitaxel toxicity, but stimulated the growth of CFU-GEMM and BFU-E beyond control values. Amifostine protected normal human lung fibroblasts from paclitaxel-induced cytotoxicity and DNA single-strand breaks. However, paclitaxel cytotoxicity and DNA single-strand breaks were actually enhanced by pretreatment with amifostine in the NSCLC model. Importantly, amifostine did not interfere with paclitaxel antitumour activity even with prolonged exposure (24.5 h) of the lung cancer cells to high concentrations (1.2 mM) in vitro or following five repetitive high doses (200 mg/kg) given to scid mice with human ovarian cancer xenografts. Indeed, under certain circumstances, amifostine resulted in sensitisation of tumour cells to paclitaxel. Our results confirm previous reports of the ability of amifostine to protect normal tissues from the toxic effects of chemotherapy drugs and now extend these observations to paclitaxel.

Stimulation of hematopoiesis by amifostine in patients with myelodysplastic syndrome.

The aminothiol, amifostine (Ethyol; U.S. Bioscience, West Conshohocken, PA), is a cytoprotective agent that ameliorates the toxicities of anticancer therapy. In vitro, amifostine promotes the formation and survival of primitive hematopoietic progenitors derived from myelodysplastic bone marrow (BM) specimens. To evaluate the hematological effects of amifostine, 18 patients with myelodysplastic syndrome (MDS) and one or more refractory cytopenias received treatment with amifostine in a Phase I/II study. Four cohorts received intravenous treatment with 100, 200, or 400 mg/m2 amifostine three times a week, or 740 mg/m2 weekly for three consecutive weeks followed by 2 weeks observation. Nonresponding patients received a second course of therapy at the next higher dose level depending upon drug tolerance. Bone marrow (BM) progenitor growth was assessed before treatment and after day 21. Diagnoses included refractory anemia (7), refractory anemia with ringed sideroblasts (5), refractory anemia with excess blasts (RAEB) (4), and RAEB-in transformation (RAEB-t) (2). Single- or multi-lineage hematologic responses occurred in 15 patients (83%) treated with the three-times-a-week dose schedule. Fourteen patients had a 50% or greater increase in absolute neutrophil count with amifostine treatment (range, 426 to 11,348/microL). Platelet count increased in 6 (43%) of 14 patients with thrombocytopenia (absolute increase, 16, 000 to 110,000/microL), and 5 of 15 red blood cell transfusion-dependent patients had a 50% of greater reduction in transfusion needs. Assayable hematopoietic progenitors increased in 13 of 15 evaluable patients; including CFU-GEMM (12), BFU-E (8), and CFU-GM (6). Amifostine doses less than or equal to 200 mg/m2 were well tolerated, whereas grade II nausea, vomiting, and fatigue was limiting at higher doses. Three patients with excess blasts before enrollment experienced an increase in BM blast percentage and two patients had evolution to acute leukemia that persisted after treatment withdrawal. We conclude that amifostine administered at doses

Randomized trial of carboplatin plus amifostine versus carboplatin alone in patients with advanced solid tumors.

BACKGROUND: To test the hypothesis that the cytoprotectant amifostine attenuates the thrombocytopenia produced by carboplatin, the authors performed a randomized trial comparing treatment with carboplatin alone versus the combination of amifostine and carboplatin. METHODS: Patients with refractory or carboplatin-sensitive malignancies were randomized to receive either carboplatin, 500 mg/m2 alone or carboplatin, 500 mg/m2 in conjunction with 2 doses of amifostine of 910 mg/m2 each. RESULTS: Fifty-five patients with a variety of malignancies were entered on this study. One patient withdrew from each arm prior to the administration of any therapy, leaving 30 evaluable patients treated with carboplatin alone and 23 treated with the combination of amifostine and carboplatin. For 82 cycles of therapy with amifostine plus carboplatin, the median platelet nadir was 127 x 10(9)/L while the median platelet nadir was 88 x 10(9)/L over the 80 courses of therapy with carboplatin alone (P = 0.023). The median platelet nadir after the first cycle of therapy was 144 x 10(9)/L for patients treated with amifostine plus carboplatin and 85 x 10(9)/L for patients treated with carboplatin alone (P = 0.24). The median survival for 9 patients with advanced nonsmall cell lung carcinoma treated with carboplatin alone was 39 weeks whereas the median survival for 12 such patients treated with amifostine plus carboplatin was 52 weeks (P = 0.116). CONCLUSIONS: These data support the hypothesis that amifostine attenuates the myelosuppression of carboplatin. Additional studies of amifostine in combination with carboplatin-containing chemotherapy regimens are warranted.