Angana Mitra

Prognosis in Patients With Sentinel Node–Positive Melanoma Is Accurately Defined by the Combined Rotterdam Tumor Load and Dewar Topography Criteria

PURPOSE Prognosis in patients with sentinel node (SN)-positive melanoma correlates with several characteristics of the metastases in the SN such as size and site. These factors reflect biologic behavior and may separate out patients who may or may not need additional locoregional and/or systemic therapy. PATIENTS AND METHODS Between 1993 and 2008, 1,080 patients (509 women and 571 men) were diagnosed with tumor burden in the SN in nine European Organisation for Research and Treatment of Cancer (EORTC) melanoma group centers. In total, 1,009 patients (93%) underwent completion lymph node dissection (CLND). Median Breslow thickness was 3.00 mm. The median follow-up time was 37 months. Tumor load and tumor site were reclassified in all nodes by the Rotterdam criteria for size and in 88% by the Dewar criteria for topography. RESULTS Patients with submicrometastases (< 0.1 mm in diameter) were shown to have an estimated 5-year overall survival rate of 91% and a low nonsentinel node (NSN) positivity rate of 9%. This is comparable to the rate in SN-negative patients. The strongest predictive parameter for NSN positivity and prognostic parameter for survival was the Rotterdam-Dewar Combined (RDC) criteria. Patients with submicrometastases that were present in the subcapsular area only, had an NSN positivity rate of 2% and an estimated 5- and 10-year melanoma-specific survival (MSS) of 95%. CONCLUSION Patients with metastases < 0.1 mm, especially when present in the subcapsular area only, may be overtreated by a routine CLND and have an MSS that is indistinguishable from that of SN-negative patients. Thus the RDC criteria provide a rational basis for decision making in the absence of conclusions provided by randomized controlled trials.

Gene Expression Profiling of Paraffin-Embedded Primary Melanoma Using the DASL Assay Identifies Increased Osteopontin Expression as Predictive of Reduced Relapse-Free Survival

Purpose: Gene expression studies in melanoma have been few because tumors are small and cryopreservation is rarely possible. The purpose of this study was to evaluate the Illumina DASL Array Human Cancer Panel for gene expression studies in formalin-fixed melanoma primary tumors and to identify prognostic biomarkers. Experimental Design: Primary tumors from two studies were sampled using a tissue microarray needle. Study 1: 254 tumors from a melanoma cohort recruited from 2000 to 2006. Study 2: 218 tumors from a case-control study of patients undergoing sentinel node biopsy. Results: RNA was obtained from 76 of blocks; 1.4 of samples failed analysis (transcripts from <250 of the 502 genes on the DASL chip detected). Increasing age of the block and increased melanin in the tumor were associated with reduced number of genes detected. The gene whose expression was most differentially expressed in association with relapse-free survival in study 1 was osteopontin (SPP1; P = 2.11 106) and supportive evidence for this was obtained in study 2 used as a validation set (P = 0.006; unadjusted data). Osteopontin level in study 1 remained a significant predictor of relapse-free survival when data were adjusted for age, sex, tumor site, and histologic predictors of relapse. Genes whose expression correlated most strongly with osteopontin were PBX1, BIRC5 (survivin), and HLF. Conclusion: Expression data were obtained from 74 of primary melanomas and provided confirmatory evidence that osteopontin expression is a prognostic biomarker. These results suggest that predictive biomarker studies may be possible using stored blocks from mature clinical trials. (Clin Cancer Res 2009;15(22):693946)

Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma

The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8–30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels.

Patterns of Expression of DNA Repair Genes and Relapse From Melanoma

Purpose: To use gene expression profiling of formalin-fixed primary melanoma samples to detect expression patterns that are predictive of relapse and response to chemotherapy. Experimental Design: Gene expression profiles were identified in samples from two studies (472 tumors). Gene expression data for 502 cancer-related genes from these studies were combined for analysis. Results: Increased expression of DNA repair genes most strongly predicted relapse and was associated with thicker tumors. Increased expression of RAD51 was the most predictive of relapse-free survival in unadjusted analysis (hazard ratio, 2.98; P = 8.80 × 10−6). RAD52 (hazard ratio, 4.73; P = 0.0004) and TOP2A (hazard ratio, 3.06; P = 0.009) were independent predictors of relapse-free survival in multivariable analysis. These associations persisted when the analysis was further adjusted for demographic and histologic features of prognostic importance (RAD52 P = 0.01; TOP2A P = 0.02). Using principal component analysis, expression of DNA repair genes was summarized into one variable. Genes whose expression correlated with this variable were predominantly associated with the cell cycle and DNA repair. In 42 patients treated with chemotherapy, DNA repair gene expression was greater in tumors from patients who progressed on treatment. Further data supportive of a role for increased expression of DNA repair genes as predictive biomarkers are reported, which were generated using multiplex PCR. Conclusions: Overexpression of DNA repair genes (predominantly those involved in double-strand break repair) was associated with relapse. These data support the hypothesis that melanoma progression requires maintenance of genetic stability and give insight into mechanisms of melanoma drug resistance and potential therapies. Clin Cancer Res; 16(21); 5211–21. ©2010 AACR.

Cutaneous granulomas associated with primary immunodeficiency disorders

Cutaneous granulomas are uncommon in primary immunodeficiency disorders. We report cutaneous granulomas in a child with ataxia telangiectasia (AT) and compare the clinical course with similar lesions in an adult with common variable immunodeficiency (CVI). A 4‐year‐old female with AT developed cutaneous granulomas as erythematous plaques. The largest lesion appeared on her left cheek and continued to progress despite treatment with topical and intralesional steroids. Disease control was obtained initially with oral antibiotics and low‐dose oral steroids. On cessation of oral steroids, significant relapse of the facial granuloma occurred. Pulsed and then oral steroids were required to stop the disease process leaving significant scarring. The second case is of cutaneous granulomas in a 66‐year‐old man, with CVI, who presented with an erythematous reticulate rash on the legs. We consider it useful to report this patient here as disease control was obtained in a similar way with systemic immunosuppression. In this patient a combination of oral steroids and azathioprine was used. These cutaneous granulomas are thought to be a manifestation of immune dysregulation. No infectious cause has been found so far. We recommend the use of broad‐spectrum antibiotics in conjunction with systemic steroids for progressive granulomas, as these patients are immunosuppressed and infection with an unidentified organism cannot be excluded.

Melanoma sentinel node biopsy and prediction models for relapse and overall survival

Background:To optimise predictive models for sentinal node biopsy (SNB) positivity, relapse and survival, using clinico-pathological characteristics and osteopontin gene expression in primary melanomas.Methods:A comparison of the clinico-pathological characteristics of SNB positive and negative cases was carried out in 561 melanoma patients. In 199 patients, gene expression in formalin-fixed primary tumours was studied using Illuminas DASL assay. A cross validation approach was used to test prognostic predictive models and receiver operating characteristic curves were produced.Results:Independent predictors of SNB positivity were Breslow thickness, mitotic count and tumour site. Osteopontin expression best predicted SNB positivity (P=2.4 × 10−7), remaining significant in multivariable analysis. Osteopontin expression, combined with thickness, mitotic count and site, gave the best area under the curve (AUC) to predict SNB positivity (72.6%). Independent predictors of relapse-free survival were SNB status, thickness, site, ulceration and vessel invasion, whereas only SNB status and thickness predicted overall survival. Using clinico-pathological features (thickness, mitotic count, ulceration, vessel invasion, site, age and sex) gave a better AUC to predict relapse (71.0%) and survival (70.0%) than SNB status alone (57.0, 55.0%). In patients with gene expression data, the SNB status combined with the clinico-pathological features produced the best prediction of relapse (72.7%) and survival (69.0%), which was not increased further with osteopontin expression (72.7, 68.0%).Conclusion:Use of these models should be tested in other data sets in order to improve predictive and prognostic data for patients.

The clinicopathological and gene expression patterns associated with ulceration of primary melanoma

Ulceration of primary melanomas is associated with poor prognosis yet is reported to predict benefit from adjuvant interferon. To better understand the biological processes involved, clinicopathological factors associated with ulceration were determined in 1804 patients. From this cohort, 348 primary tumor blocks were sampled to generate gene expression data using a 502‐gene cancer panel and 195 blocks were used for immunohistochemistry to detect macrophage infiltration and vessel density. Gene expression results were validated using a whole genome array in two independent sample sets. Ulceration of primary melanomas was associated with more proliferative tumors, tumor vessel invasion, and increased microvessel density. Infiltration of tumors with greater number of macrophages and gene expression pathways associated with wound healing and up‐regulation of pro‐inflammatory cytokines suggests that ulceration is associated with tumor‐related inflammation. The relative benefit from interferon reported in patients with ulcerated tumors may reflect modification of signaling pathways involved in inflammation.

Clinicopathologic features of V600E and V600K melanoma--letter.

The presence of a BRAF mutation in a melanoma tumor predicts response to BRAF inhibitors; however, the biological characteristics of tumors with different BRAF mutations have not been investigated until recently. Menzies and colleagues reported that the rarer V600K BRAF mutations were found more

Identification of differentially expressed genes in matched formalin‐fixed paraffin‐embedded primary and metastatic melanoma tumor pairs

To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Identification of differentially expressed genes in matched formalin-fixed paraffin-embedded primary and metastatic melanoma tumor pairs Rosalyn Jewell, Angana Mitra, Caroline Conway, James Iremonger, Christy Walker, Floor de Kort, Martin Cook, Andy Boon, Valerie Speirs and Julia Newton-Bishop

Histopathology of melanocytic lesions in a family with an inherited BAP1 mutation

To the Editor, We read with interest the report by Marusic et al.1 who expanded on the histopathological features of melanocytic neoplasms in a family with an inherited BAP1 mutation. Marusic et al.1 described the presence of nuclear pseudoinclusions and multinucleated melanocytes within five of six nevi from this family. The lesions were mainly intradermal, composed of large epithelioid melanocytes, and showed loss of BAP1 expression by immunohistochemistry. We report similar histopathological findings in melanocytic lesions excised from members of an English family in which a germline pathogenic BAP1 mutation has been identified. The proband was diagnosed with melanoma at 26 years of age that was clinically bland and pink in appearance. This was initially considered to be a melanocytic lesion of uncertain malignant potential (MELTUMP) but on further assessment, showed significant cytological atypia and lacked maturation and was therefore treated as a melanoma. The Breslow thickness was 2.4 mm and there was no evidence of ulceration. This patient later developed a melanoma in situ. He had multiple pink, bland nevi one of which subsequently changed and this was excised in view of his significant history. Histopathological examination showed an intradermal melanocytic neoplasm composed of epithelioid melanocytes with abundant cytoplasm, some of which contained multinucleated cells (Fig. 1A) and nuclear pseudoinclusions (Fig. 1B). The proband’s maternal grandfather had occupational exposure to asbestos and had died of mesothelioma. His maternal uncle had a history of stage IIA melanoma, which later metastasized to a regional lymph node. Striking pleomorphism, multinucleated melanocytes and nuclear pseudoinclusions were readily identified in this nodal metastasis. The proband’s maternal aunt, presented at 44 years of age with a history of a recalcitrant scalp lesion, which had been curetted twice elsewhere in the belief that this was a cyst. The curettings had not been sent for histopathological assessment. Following a further recurrence, approximately 18 months after onset, the lesion was partially excised by general surgeons. Histopathology revealed a malignant blue nevus-like melanoma showing a highly cellular melanocytic tumor, at least 11 mm in thickness; composed of spindle cells and some dendritic-like melanocytes. Nuclear pseudoinclusions were noted, although distributed more sparsely within this lesion. To our knowledge, there is only one other reported case of a 64-year-old female with a BAP1 germline mutation who had a malignant blue nevus-like melanoma of the scalp.2 Our patient was also found to have a meningioma on computed tomography (CT) scanning. Following an invitation for screening, the proband’s maternal cousin had a pink lesion excised, due to a history of change. This was diagnosed as a spitzoid tumor of uncertain malignant potential (STUMP). The tumor consisted of a lobulated, intradermal nodule composed of pleomorphic epithelioid melanocytes, flanked by a more diffuse melanocytic lesion with hyperchromatic nuclei (Fig. 2A). Nuclear