Marguerite Stas

Phase 1/2 study of subcutaneous and intradermal immunization with a recombinant MAGE-3 protein in patients with detectable metastatic melanoma

The purpose of this phase 1/2 study was to evaluate toxicity, tumor evolution and immunologic response following administration of a fixed dose of a recombinant MAGE‐3 protein by subcutaneous and intradermal routes in the absence of immunologic adjuvant. Thirty‐two patients with detectable metastatic melanoma expressing gene MAGE‐3 were included and 30 received at least one injection with a fixed dose of a ProtD‐MAGE‐3 fusion protein. The immunization schedule included 6 intradermal and subcutaneous injections at 3‐week intervals. Afterward, patients without major tumor progression who required other treatments received additional vaccinations at increasing time intervals. The vaccine was generally well tolerated. Among the 26 patients who received at least 4 vaccinations, we observed 1 partial response and 4 mixed responses. For these 5 responding patients, time to progression varied from 3.5 to 51+ months. An anti‐MAGE‐3 CD4 T‐lymphocyte response was detected in 1 out of the 5 responding patients. The majority of patients had no anti‐MAGE‐3 antibody response. The clinical and immunologic responses generated by the vaccine are rather limited. Nevertheless, given the potential antitumor efficacy and the very mild toxicity of vaccinations, further studies combining MAGE proteins and/or peptides with potent immunologic adjuvants are warranted, not only in metastatic melanoma, but also in the adjuvant setting.

Neogenesis of Lymphoid Structures and Antibody Responses Occur in Human Melanoma Metastases

Lymphoid neogenesis, or the development of lymphoid structures in nonlymphoid organs, is frequently observed in chronically inflamed tissues, during the course of autoimmune, infectious, and chronic graft rejection diseases, in which a sustained lymphocyte activation occurs in the presence of persistent antigenic stimuli. The presence of such ectopic lymphoid structures has also been reported in primary lung, breast, and germline cancers, but not yet in melanoma. In this study, we observed ectopic lymphoid structures, defined as lymphoid follicles comprising clusters of B lymphocytes and follicular dendritic cells (DC), associated with high endothelial venules (HEV) and clusters of T cells and mature DCs, in 7 of 29 cutaneous metastases from melanoma patients. Some follicles contained germinal centers. In contrast to metastatic lesions, primary melanomas did not host follicles, but many contained HEVs, suggesting an incomplete lymphoid neogenesis. Analysis of the repertoire of rearranged immunoglobulin genes in the B cells of microdissected follicles revealed clonal amplification, somatic mutation and isotype switching, indicating a local antigen-driven B-cell response. Surprisingly, IgA responses were observed despite the nonmucosal location of the follicles. Taken together, our findings show the existence of lymphoid neogenesis in melanoma and suggest that the presence of functional ectopic lymphoid structures in direct contact with the tumor makes the local development of antimelanoma B- and T-cell responses possible.

Expression of the endothelin-B receptor in pigment cell lesions of the skin

Abstract. Endothelins (ETs) exert several functions in human melanocytes, including proliferation, dendrite formation, and melanin synthesis. Among the ET receptors, the non-selective endothelin-B (ETB) receptor is the major receptor in melanocytes and malignant melanoma (MM) cells. In spite of the important role of ETs and their receptors in the growth and differentiation of melanocytes, the distribution and expression levels of ETB receptors in tissue sections of benign and malignant pigment cell lesions is still unknown. We combined immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) to study ETB receptor expression in benign and malignant pigment cell lesions and in normal skin. Immunohistochemistry on paraffin-embedded tissue sections of 159 cases revealed a significant increase in intensity of ETB receptor expression from common nevi over dysplastic nevi and primary MM to metastatic MM. Quantitative PCR using real-time detection on 75 samples confirmed the immunohistochemical results. These data add the ETB receptor to the growing list of tumor progression markers in MM and suggest that ETs play a role in the progression of MM in the skin.

Lesions of 13q may occur independently of deletion of 16q in spindle cell/pleomorphic lipomas.

Very recent multidisciplinary investigations have allowed for the definition among lipomas of a clinical and histological subtype called spindle cell and/or pleomorphic lipoma, possibly associated with partial monosomy 16 and anomalies of chromosome 13. In order to get nearer to the underlying critical molecular changes further multidisciplinary pathological and genetic research is indicated, to identify which chromosome(s) anomalies are crucial in the development of these tumours.

18-FDG PET scan in the staging of recurrent melanoma: additional value and therapeutic impact

Staging of melanoma patients by means of whole body functional imaging in a single evaluation session using positron emission tomography (PET) with fluorine-18- labelled deoxy-d-glucose (FDG) as a metabolic tracer has created much interest over the last decade. After enthusiastic pilot studies, more attention has been paid to the false-negative and false-positive results of this technique than to its true therapeutic impact. This study aimed to evaluate (1) the sensitivity and specificity of this technique at a single lesion level compared with conventional screening procedures (CSP) – both of these accompanied by careful clinical examination; and (2) the additional value of the PET scan at the level of the individual patient and its therapeutic impact for different types of melanoma recurrence. A consecutive series of 100 PET scans performed on 84 melanoma patients with regional or distant recurrence according to CSP (89 PET scans) or suspicion of recurrence, i.e. inconclusive CSP (11 PET scans), were retrospectively analysed and compared with the CSP results. At the single lesion level, PET scan and CSP showed a sensitivity of 85 and 81%, a specificity of 90 and 87% and an accuracy of 88 and 84%, respectively. PET provided false-negative results for small skin metastases and brain involvement; false-positive results were associated with unrelated benign or malignant tumours and peripheral soft tissue and bone uptake. PET scan showed an additional value over and above CSP at the individual patients level by true upstaging in 10 cases, true downstaging in 24 cases and depiction of more lesions within the same stage of disease in 15 cases. The overall therapeutic impact reached 26%: 17 out of 71 (24%) cases with regional recurrence, one out of 18 cases (5.5%) with distant metastasis and eight out of 11 cases (73%) with suspicion of recurrence where CSP remained doubtful. However, in 19 cases comparison between CSP and PET resulted in discordant findings, suggesting upstaging in one area and downstaging at another site within the same patient. In conclusion, PET scan has an additional value in the staging of recurrent melanoma, providing it is accompanied by careful clinical examination and specific brain imaging. However, in the absence of evidence of metastasis or unrelated conditions at the same site on CSP, PET spots may represent false-positive images, which would falsely upgrade a patient to an incurable state, or they may be early true-positive findings, which will become evident during close follow-up.

Systematic review: Malfunction of totally implantable venous access devices in cancer patients

PurposeMalfunction of totally implantable venous access devices is a common complication. The purpose was to identify definitions used to describe malfunction and to investigate the incidence of malfunction in different types of port and catheter designs.MethodsRelevant studies were identified in PubMed that were published between January 1993 and February 2011. Empirical studies reporting functional outcomes in adults and where, at least 95% of the studied population consisted of onco-hematology patients with a newly inserted chest or arm port, were selected. The following data were extracted: patient and totally implantable venous access devices (TIVAD) characteristics, study design, definitions of malfunction, and functional outcomes. Two independent reviewers assessed the methodological quality of the series.ResultsOf the 4,886 potentially relevant articles, 57 were selected, involving 14,311 TIVADs. Twenty-nine percent of the studies explicitly defined malfunction. Malfunction incidence rates were expressed in six different ways, including the proportion of affected devices per inserted devices (incidence 0–47%); the number of affected devices per 1,000 catheter days (incidence 0–2.24 per 1,000 catheter days); and the number of malfunctions over the total number of accessing attempts (incidence 0–26%).ConclusionsHeterogeneity in the definitions used to describe device malfunction was evident. A broad range in the reported incidence of malfunction and in the kind of calculation and reporting methods was also found. Methodological quality of the studies was often poor. Standardization of definitions and accurate outcome measurement is needed. Calculation and report of malfunction incidence should be based on prospective data collected at the moment of an accession attempt.

Proliferative myositis in a child

Abstract A case of proliferative myositis in the lumbar paraspinal muscles in a 14-year-old boy is presented. Imaging investigations including plain radiograph, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), bone scan and positron emission tomography (PET) were suggestive of an inflammatory process such as myositis ossificans. The diagnosis was made by incisional biopsy. More pronounced edema, more muscle fiber necrosis and a higher cellularity were found compared to adult cases. The karyotype of the lesion was normal. Clinically, the mass disappeared spontaneously. After 24 months, asymptomatic bridging ossification between the third and fourth lumbar vertebrae was noted.

Loss of microRNA-200a and c, and microRNA-203 expression at the invasive front of primary cutaneous melanoma is associated with increased thickness and disease progression

Loss of E-cadherin expression in melanoma correlates with increased tumor thickness and reduced disease-free survival. The molecular mechanisms underpinning its differential expression in melanoma tissue remain elusive. MicroRNAs (miRNAs) have been implicated in tumor progression and regulation of E-cadherin expression. Here, we demonstrate a significant correlation between tumor thickness and loss of expression of miR-200a, miR-200c, and miR-203 in a series of 23 frozen primary melanomas, where it was confirmed in two subsequent validation series (series 1: six nevi, 15 primary melanomas, and 16 metastases; series 2: 11 matched pairs of primary melanomas and metastases). Decreased levels of miR-200a, miR-200c, and miR-203 correlated with increasing thickness in the combined validation series (P = 0.024, 0.033, and 0.031, respectively). In addition, progressive loss of miR-200a expression with disease progression was observed in series 1 (P < 0.001) and in series 2 (P = 0.029). MiR-200 in situ hybridization and E-cadherin immunohistochemistry demonstrated reduced expression of both at the deep invasive margin of the tumor. Furthermore, a functional validation study using an anti-miR200 strategy demonstrated that loss of miR-200 expression in melanoma cell lines reduced E-cadherin expression. Collectively, our data point towards an important role for miR-200 and miR203 expression in regulating E-cadherin during melanoma progression.

The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target.

Genome-wide promoter methylation analysis identifies epigenetic silencing of MAPK13 in primary cutaneous melanoma.

The involvement of epigenetic alterations in the pathogenesis of melanoma is increasingly recognized. Here, we performed genome‐wide DNA methylation analysis of primary cutaneous melanoma and benign melanocytic nevus interrogating 14 495 genes using BeadChip technology. This genome‐wide view of promoter methylation in primary cutaneous melanoma revealed an array of recurrent DNA methylation alterations with potential diagnostic applications. Among 106 frequently hypermethylated genes, there were many novel methylation targets and tumor suppressor genes. Highly recurrent methylation of the HOXA9, MAPK13, CDH11, PLEKHG6, PPP1R3C, and CLDN11 genes was established. Promoter methylation of MAPK13, encoding p38δ, was present in 67% of primary and 85% of metastatic melanomas. Restoration of MAPK13 expression in melanoma cells exhibiting epigenetic silencing of this gene reduced proliferation, indicative of tumor suppressive functions. This study demonstrates that DNA methylation alterations are widespread in melanoma and suggests that epigenetic silencing of MAPK13 contributes to melanoma progression.