Dear Sir, Desmoplastic melanoma (DM) is a spindle cell melanoma that represents less than 4% of all cutaneous melanomas (Quinn et al., 1998). Because of its spindle cell morphology and its frequent lack of clinical and histologic features associated with conventional melanoma, DM is often not identified (Mccarthy et al., 2004). Diagnosis of DM by immunohistochemistry (IHC) may be difficult because primary and metastatic lesions are often negative for relatively specific melanocyte biomarkers such as HMB-45 (human melanoma black-45/gp100) and MART-1 (melanoma antigen recognized by T-cells). DM usually expresses the S-100p protein, but this protein can also be detected in Langerhans cells, dendritic cells, macrophages, Schwann cells, and a wide range of tumors, including benign and malignant nerve sheath tumors, some carcinomas, and myoepithelial tumors. The lack of a DM-specific IHC stain confounds diagnosis, particularly when DM recurs within scars (where distinction of the spindle-shaped DM cells from reactive fibroblasts can be extremely difficult due to their morphologic similarities and the presence of S-100p in some scars) or in the regional lymph nodes (LN). Other proposed IHC markers of DM, such as microphthalmia transcription factor (Mitf) (Busam et al., 2001; Granter et al., 2001) and procollagen 1, show promise, but their sensitivity has not been verified. A large study from the Melanoma Institute Australia challenged the assumption that DM has a worse prognosis than other melanomas (Quinn et al., 1998). In this study of 280 patients, there was no difference in survival between patients with desmoplastic versus non-desmoplastic melanoma, but DM was associated with a lower rate of regional LN metastasis and a higher rate of local recurrence. High molecular weight–melanoma-associated antigen (HMW–MAA), also known as the melanoma chondroitin sulfate proteoglycan, is expressed in >85% of primary and metastatic melanoma lesions, with limited interlesional and intralesional heterogeneity (Campoli et al., 2004). HMW–MAA is a membrane-bound chondroitin sulfate proteoglycan found in the melanoma cell membrane; by contrast, MART-1 co-localizes and forms a complex with the P100 form of gp100 (antigen of HMB-45) in early subcellular compartments of melanocytic cells. HMW–MAA can be used as a detection biomarker for cutaneous metastatic melanoma (Goto et al., 2008). The present study examines HMW–MAA as a potential biomarker for DM. The expression level of HMW–MAA in primary and metastatic DM lesions was assessed and compared to that of MART-1 and HMB-45 by utilizing IHC and qRT-PCR (See Supporting Information section). Immunohistochemistry with HMW–MAA monoclonal antibodies (mAb) or MART-1 mAbs was optimized for paraffin-embedded archival tissues (PEAT) in our previous study (Goto et al., 2008). IHC with HMB-45 mAb was assessed using PEAT specimens of primary melanoma and metastatic melanoma in lung, LN and skin. Each assay was used to analyze 40 DM primaries, 23 DM metastases, nine tumor-negative LNs, and 13 tumor-negative skin specimens (see Appendix S1). As shown in Figure 1, HMW–MAA -specific mAbs stained the membrane and (to a lesser degree) the cytoplasm of melanoma cells. Table S1A,B summarize the HMW–MAA expression level in primary and metastatic DM tumors, by showing the percentage of stained cells and the staining intensity. IHC staining of primary DM for HMW–MAA, MART-1, and HMB-45 IHC. (A) HMW–MAA (+) (×200). (B) HMW–MAA (+) (×75). The cells immunoreactive for HMW–MAA show purple membranous and (to a lesser degree) cytoplasmic staining. (C) MART-1 (−) (×200). (D) MART-1 (−) (×75). (E) HMB-45 (−) (×200). (F) HMB-45 (−) (×75). IHC staining of LN DM metastases for HMW–MAA, MART-1, and HMB-45. (G) HMW–MAA (+) (×200). (H) HMW–MAA (+) (×75). The cells immunoreactive for HMW–MAA show membranous and (to a lesser degree) cytoplasmic staining. (I) MART-1 (−) (×200). (J) MART-1 (−) (×75). (K) HMB-45 (−) (×200). (L) HMB-45 (−) (×75). In primary DM, staining intensity was stronger for HMW–MAA than for MART-1 or HMB-45 (Table S2A, P < 0.0001 and P < 0.0001, respectively). Of the 40 DM primaries, 38 (95%) stained for HMW–MAA but only one (3%) stained for MART-1 and only one (3%) stained for HMB-45 (Table S2A). In primary DM, the percentage of stained cells was significantly higher for HMW–MAA than for MART-1 or HMB-45 (Table S2B, P < 0.0001 and P < 0.0001, respectively). Of the 40 primary lesions, 35 (87%) showed more than 50% staining for HMW–MAA, whereas none of 40 primaries stained for MART-1 and only one (3%) stained for HMB-45. In metastatic DM, staining intensity was stronger for HMW–MAA than for MART-1 or HMB-45 (Table S2C, P = 0.007 and P < 0.0001, respectively). Of the 23 DM metastases, 20 (87%) stained for HMW–MAA, whereas four (18%) stained for MART-1 and two (9%) stained for HMB-45 (Table S2C). The frequency of staining was significantly higher for HMW–MAA than for MART-1 or HMB-45 (Table S2D, P < 0.0001 and P < 0.0001, respectively). Of the 23 DM metastases, 14 (61%) showed more than 50% staining for HMW–MAA; none of the metastases stained for MART-1 or HMB-45. The HMW–MAA mAb cocktail stained 12 of 14 (86%) nodal metastases (Table S1B) but did not stain any tumor-negative LN. The intensity of nodal staining was significantly stronger for HMW–MAA (12 of 14) than for MART-1 (3 of 14) or HMB-45 (1 of 14) (P = 0.007 and P = 0.0023, respectively). These results indicate that IHC staining of primary and metastatic DM was significantly more sensitive with HMW–MAA-specific mAbs than with MART-1-specific mAb or HMB-45-specific mAb. To investigate the potential of HMW–MAA as a molecular biomarker to detect DM, we assessed HMW–MAA mRNA by qRT-PCR level in both PEAT primary and metastatic DM. MART-1 mRNA and GAPDH mRNA were used as controls. The qRT-PCR assay for each biomarker was optimized in PEAT specimens. The range of relative mRNA copies for DM was 0–37.4 for HMW–MAA and 0–6.39 for MART-1. The relative mRNA copy number for HMW–MAA was higher in 40 DM primaries than in tumor-free skin specimens (median 3.35 versus 1.45), and significantly higher in 23 DM LN metastases than in tumor-free LNs (median 0.47 versus 0.013; P = 0.019, Figure 2A,C). HMW–MAA positivity was 25 (63%) of 40 DM primaries and 16 (70%) of 23 DM metastases expressed HMW–MAA mRNA. Assessment of the same PEAT specimens showed that MART-1 was expressed in 9 (23%) of 40 primaries and 5 (22%) of 23 metastases (P = 0.0029 and P = 0.0023, respectively). Relative MART-1 copy number was not significantly different between 40 DM primaries and tumor-free skin specimens, or between 23 DM metastases and tumor-free LNs (P = 0.15, Figure 2B,D). Moreover, HMW–MAA mRNA was expressed in 8 (57%) of 14 nodal metastases, whereas MART-1 mRNA was expressed in 3 (21%) of 14 nodal metastases. HMB-45 (gp100) was not assessed by qRT-PCR as many types of normal cells express this pigment biomarker (Hoon et al., unpublished). These results indicate that HMW–MAA is more sensitive than MART-1 for qRT-PCR assessment of DM. Higher expression level of HMW–MAA mRNA than of MART-1 mRNA in primary DM lesions. (A) HMW–MAA mRNA expression in primary DM and tumor-negative skin specimens. HMW–MAA mRNA expression was measured as relative mRNA copy number (absolute mRNA copy number of HMW–MAA/absolute mRNA copy number of GAPDH). The cut-off for HMW–MAA positivity was 2.37, corresponding to the mean relative HMW–MAA copy number plus 1 SD in tumor-negative skin. (B) MART-1 mRNA expression was measured as relative mRNA copy number (absolute mRNA copy number of MART-1/absolute mRNA copy number of GAPDH). The cut-off for MART-1 positivity was 4.74 × 10−2, corresponding to the mean relative MART-1 copy number plus 1 SD in tumor-negative skin. The red bar indicates mean copy numbers. Higher expression level of HMW–MAA mRNA than of MART-1 mRNA in metastatic DM. (C) HMW–MAA mRNA expression was scored as relative mRNA copies (absolute mRNA copies of HMW–MAA/absolute mRNA copies of GAPDH). The cut-off for HMW–MAA positivity was 2.95, corresponding to the mean relative HMW–MAA copy number plus one SD in tumor-negative skin. (D) MART-1 mRNA expression in primary DM and tumor-negative skin specimens. MART-1 mRNA expression was scored as relative mRNA copies (absolute mRNA copies of MART-1/absolute mRNA copies of GAPDH). MART-1 mRNA expression was negative in all tumor-negative LN. The red bar indicates mean copy numbers. As most DM primaries are slow growing and almost half lack pigmentation, melanoma may not be considered in the clinical differential diagnosis of cutaneous tumors (Mccarthy et al., 2004). Histopathological features are also subtle. DM stains for S-100p, but S-100p also can be detected in non-neoplastic cells and in a wide range of tumors (Longacre et al., 1996). Melanocytic differentiation biomarkers such as MART-1 and HMB-45 are commonly used for detection of both primary and metastatic melanoma, including LN metastasis, but have limited value in DM. More sensitive and specific IHC biomarkers of DM are needed to avoid misdiagnosis of primary DM, reduce the risk of local persistence or recurrent DM, and prevent understaging of metastatic DM. A cocktail of HMW–MAA-specific mAbs was more sensitive than HMB-45 mAb or MART-1 mAb for IHC DM assessment. HMW–MAA was more sensitive than MART-1 for qRT-PCR of DM primary and metastasis. Recently, several groups have reported results of sentinel lymph node (SLN) biopsy in patients with primary DM (Gyorki et al., 2003; Pawlik et al., 2006; Thelmo et al., 2001). These studies found a very low incidence of regional LN metastases (0–12.1% but usually below 4%), as compared with approximately 21% in patients with other forms of melanoma (Morton et al., 2006). The lower rate of LN positivity in DM reflects the biology of the tumor, which metastasizes less often via lymphatic routes than non-DM; however, this does not decrease the importance of detecting metastatic DM in LNs. S-100p antibody stains DM cells but may also stain non-neoplastic cells, such as cells of histiocytic derivation, which may be numerous in LNs. More sensitive and specific IHC biomarkers would be useful in the detection of metastatic DM cells in LNs. In our study, the HMW–MAA-specific mAb cocktail stained DM cells in 86% of nodal metastases; only 21% of nodal metastases were stained by either MART-1 mAb or HMB-45 mAb. We believe that assessment of SLN specimens using HMW–MAA-specific mAbs may improve the identification of metastatic DM in regional LNs. Although reports have suggested that Mitf-specific mAb D5 is highly sensitive and specific for DM (Busam et al., 2001; Koch et al., 2001), not all investigators agree (Granter et al., 2001). We therefore performed IHC analysis with Mitf-specific mAb D5 (Neomarker, Fremont, CA, USA) in seven of the LN metastases from the present study. Only three specimens (43%) stained with Mitf-specific mAb; all three were among the five (71%) that stained with HMW–MAA mAbs. Interestingly, MART-1 and HMB-45 Abs stained only one specimen, which was negative for HMW–MAA and Mitf. Combining these two IHC biomarkers might optimize the sensitivity for detection and identification of DM more accurately. In summary, HMW–MAA is more sensitive than HMB-45 and MART-1 for IHC diagnosis of primary DM. Moreover, the HMW–MAA mAb cocktail is valuable for detecting occult DM metastases, especially in regional tumor-draining LNs. The study indicates that HMW–MAA is more sensitive than MART-1 as an mRNA biomarker for primary DM. Because HMW–MAA mRNA was detected in metastatic DM that did not express MART-1 mRNA, combining these biomarkers for qRT-PCR assay might increase the sensitivity of mRNA assessment for high-risk DM that may metastasize to LNs. Thus HMW–MAA has potential value as a component of a multimarker probe for qRT-PCR assessment of DM metastases. Table S1. (A) HMW–MAA expression level in 40 primary DM lesions. (B) HMW–MAA expression level in 23 metastatic DM lesions from the regional nodes or distant sites. Table S2. (A) IHC staining intensity of HMW–MAA, MART-1 and HMB-45 of primary DM (n = 40). (B) Distribution of staining in 40 primary DM lesions assessed by IHC for HMW–MAA, MART-1 and HMB-45. (C) IHC staining intensity of HMW–MAA, MART-1 and HMB-45 of metastatic DM (n = 23). (D) Distribution of staining of metastatic DM lesions assessed by IHC for HMW–MAA, MART-1 and HMB-45 (n = 23). Appendix S1. Materials and Methods. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The authors reviewed 143 cases (87 benign, 37 borderline, and 19 malignant) of mammary phyllodes tumors (PTs) and used immunohistochemistry to detect p53 protein product semi-quantitatively as negative, weak, moderate and strong (scored 0 to 3). For all PTs, an increasing trend of tumor size and malignancy was detected with increasing age. For p53 staining, 60 cases (42%) were negative, 55 (38%) stained weakly, 28 (13%) stained moderately, and 10 (7%) stained strongly. Of the 87 benign PTs, 41 (47%) were negative, 37 (43%) stained weakly, and 9 (10%) stained moderately. For the 37 borderline PTs, 16 (43%) were negative, 14 (38%) stained weakly, 6 (16%) stained moderately, and 1 (3%) stained strongly. Of the 19 malignant PTs, 3 (16%) were negative, 4 (21%) stained weakly, 3 (16%) stained moderately, and 9 (47%) stained strongly. The mean intensity score for p53 staining increased progressively from benign to borderline to malignant PT, with established statistical significance (P <.0001). This is significantly correlated with mitotic count but not stromal cellularity, pleomorphism, margin, and stromal overgrowth. When considering strong staining alone (score, 3), 47% of malignant, 3% of borderline, and none of the benign PTs were positive. The use of strong positive staining for diagnosing malignant PT gave positive and negative predictive values, specificity, and sensitivity of 90%, 92.5%, 99%, and 47%, respectively. Thus diffuse strong p53 protein staining can be used as a soft sign in assisting the diagnosis of malignant PT. Conversely, negative or weak staining of p53 protein in PT is of little discriminatory value. The role of p53 gene mutation in the malignant transformation of PT is unclear; but this may not be the sole mechanism as many malignant PT were p53 protein negative.

Abstract Although many studies in recent years have uncovered an important role of receptor-interacting protein kinase 1 (RIP1) in mediating cell survival and death signaling, its potential part in the pathogenesis of cancer remains less understood. Here we report that RIP1 functions as an oncogenic regulator in human melanoma. While the expression of RIP1 was commonly upregulated in melanoma, knockdown of RIP1 inhibited melanoma cell proliferation in vitro, and retarded melanoma growth in a xenograft model. Conversely, despite induction of apoptosis in a small proportion of melanoma cells, overexpression of RIP1 enhanced proliferation in the remaining cells. The promoting effect of RIP1 on melanoma cell proliferation was mediated by activation of NF-κB, as blockade of NF-κB activation eliminated RIP1 overexpression-triggered increase in cell proliferation, whereas hyperactivation of NF-κB abolished inhibition of cell proliferation caused by RIP1 knockdown. In support, RIP1 knockdown led to reduction, whereas its overexpression caused an increase, in NF-κB activation. Strikingly, ectopic expression of RIP1 enhanced melanocyte proliferation and triggered anchorage-independent growth of the cells similarly in a NF-κB-dependent manner. While upregulation of RIP1 was associated with DNA copy number gain in a subset of melanomas, constitutive ubiquitination and subsequent stabilization of the RIP1 protein driven by TNFα autocrine appeared to be another mechanism commonly responsible for upregulation of RIP1 in melanoma cells. Collectively, these results identify RIP1 as an oncogenic regulator in melanoma, and points to the possibility of targeting the NF-κB activating mechanism of RIP1 as a novel approach in the treatment of the disease. Citation Format: Lei Jin, Xiao Ying Liu, Fritz Lai, Xu Guang Yan, Chen Chen Jiang, Su Tang Guo, Chun Yan Wang, Amanda Croft, Hsin-Yi Tseng, James S. Wilmott, Richard A. Scolyer, Xu Dong Zhang. Receptor-Interacting protein kinase 1 functions as an oncogenic regulator in human melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 56. doi:10.1158/1538-7445.AM2015-56

deficiency may co-exist ina small percentage of nevus cells toenhance melanocyte proliferation anddrive neoplastic transformation. Theabsence of a mutant-specific B-RAFantibody has so far precluded theexecution of such an analysis. It is wellestablished that activated B-RAF canpromote nevus formation in murine andfish melanoma models (Patton et al.,2005; Goel et al., 2009), and thearrested state of nevi does not appearto require p16

9008 Background: Acquired resistance (AR) to BRAF inhibitors (BRAFi) in melanoma is a near-universal phenomenon driven by numerous genetic and non-genetic alterations. Clinical implications of these AR mechanisms have not been described in a large cohort. We assessed the spectrum of BRAFi AR mechanisms and their associated timing of onset, pattern of disease progression (DP), and clinical outcomes. Methods: We compiled clinical and genetic data from 100 patients (pts) with 132 melanoma samples obtained at BRAFi DP from three previously published studies of BRAFi resistance. Whole exome sequencing and/or PCR-based genetic testing were performed on all samples. Associations between AR mechanisms and clinical features/outcomes were assessed with multivariate logistic regression models. Results: In 132 DP samples, putative AR mechanisms were identified in 58%, including NRAS or KRAS mutations (20%), BRAF splice variants (16%), BRAFV600E/K amplifications (13%), MEK1/2 mutations (7%), and non-MAPK pathway alterations (11%). Marked heterogeneity was observed within tumors and patients. BRAFV600E/K amplifications and non-MAPK alterations often co-occurred with other genetic changes, whereas NRAS mutations, MEK1/2 mutations, and BRAF splice variants largely arose in isolation (p = 0.02). Of 19 pts with ≥ 2 DP biopsies, identified AR mechanisms were concordant in only 1 pt (5%). NRAS mutations were associated with vemurafenib use (p = 0.045) and baseline intracranial metastases (p = 0.036). Progression-free survival and patterns of DP were similar across AR mechanisms. The median survival after DP was 6.9 months, and subsequent responses to combined BRAF/MEK inhibition were uncommon (2/15; 13%); no patients responded to ipilimumab (0/24). Post-progression outcomes did not correlate with specific BRAFi AR mechanisms. Conclusions: This is the largest study of acquired BRAFi resistance in pts with BRAF mutant melanoma. Despite marked heterogeneity of AR mechanisms within pts and tumors, NRAS mutations were associated with vemurafenib use and intracranial disease. Further investigation into non-genetic AR mechanisms and immune features of BRAFi progression is warranted.

The development of cutaneous melanoma is attributed mainly to UV-induced DNA damage producing genetic mutations that make melanocytes capable of invasion and metastasis. Early diagnosis and surgical excision are the key to successful treatment. Breslow thickness, ulceration and mitotic rate are the most important prognostic factors. Histological examination of the ‘sentinel’ lymph node indicates prognosis with even greater accuracy, and is now routinely recommended for patients with melanomas ≥1.0 mm in Breslow thickness. Early complete regional node clearance in sentinel node-positive patients may improve melanoma-specific survival. Clinically involved nodes are best treated by surgery, as are isolated systemic metastases. However, new systemic therapies are achieving good short- to-medium term systemic disease control; these include BRAF and MEK inhibitors and immune check-point regulators (e.g. CTLA-4 inhibitors and agents targeting PD-1 and PDL-1). Radiotherapy, regional chemotherapy, topical therapies and intralesional therapies may also be effective in patients with metastatic melanoma.

The 2010 7th International Melanoma Congress sponsored by the Society for Melanoma Research and held in Sydney, Australia, was held together with the International Melanoma and Skin Cancer Centers group and the International Melanoma Pathology Study Group. As a consequence, there were over 900 registrants that included a wide range of clinicians (surgeons, medical oncologists, dermatologists) specialising in the management of melanoma as well as scientists and students carrying out laboratory-based research in melanoma. There was a general consensus that this grouping of clinicians, pathologists and scientists was mutually advantageous and plans are afoot to continue this grouping in future meetings. The meeting was dominated by the advances being made in treatment of melanoma with selective BRAF inhibitors but interest in epithelial mesenchymal transition and phenotypic changes in melanoma was apparent in many of the talks. The authors have attempted to capture many of the new developments in melanoma research but apologize to those speakers and poster presenters who had equally important findings not captured in these summaries.

This data adds to the increasing evidence implicating the SWI/SNF chromatin remodelling complex in tumour development and the association of p16INK4a with chromatin remodelling highlights potentially new functions that may be important in melanoma predisposition and chemoresistance.

The aim of this study was to describe how metastatic melanoma obstructing lymphatic flow to sentinel nodes can result in a false-negative sentinel node biopsy and to show that the use of ultrasound in conjunction with preoperative lymphoscintigraphy can avoid this potential diagnostic pitfall. A series of three patients in whom metastatic melanoma obstructed lymphatic flow to sentinel nodes is reported. In these patients, lymphoscintigraphy failed to identify nodes containing metastatic disease. This resulted in a false-negative sentinel node biopsy result in two patients. A sentinel node biopsy was not carried out in the third patient, but the same dilemma was encountered. These cases provide further insights into the dynamics of lymphatic flow and suggest possible reasons for occasional inaccuracy of sentinel node biopsy. They also highlight the advantages of using ultrasound to assess lymph nodes in any node fields to which lymphatic drainage occurs from a primary tumour site.

OCT has good diagnostic accuracy for diagnosing sBCC and measuring depth in tumours ≤ 0·4 mm. Potentially OCT can reduce the need for biopsy in clinically suspected sBCCs. However, careful follow-up is required in such cases as there is a small risk (5%) of misdiagnosis.

A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALK, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors.