Nima Mesbah Ardakani

Molecular Profiling Reveals a Clonal Relationship Between Ovarian Mucinous Tumors and Corresponding Mural Carcinomatous Nodules

Benign or malignant mural nodules rarely occur in mucinous tumors (MTs) of the ovary and malignant nodules can show mesenchymal or epithelial differentiation. The histogenesis of mural nodules is unclear and it has been suggested that these may evolve through divergent differentiation of the mucinous neoplasm or alternatively represent a collision phenomenon. To test these possibilities we compared the molecular profile of 7 ovarian MTs with their matched mural carcinomatous nodules (MCNs) by next-generation sequencing. We found identical KRAS mutations in paired MTs and MCNs in 6 cases, one of which also showed identical CDH1 mutations in both components. In 1 tumor a KRAS mutation was detected in the mucinous neoplasm but not in the MCN; however, identical p53 mutations were present in both tumor elements. Unpaired p53 and PTEN mutations were detected only in the MCN in 2 cases, while mutations in p53 and PIK3CA genes were observed only in the MT in 2 cases. The overall comparative genomic profile was consistent with the neoplastic nature of the MCNs and strongly supported their clonal relationship with the more differentiated mucinous neoplasms. MCNs possibly develop through the acquisition of additional genomic alterations, such as p53 and PTEN mutations, resulting in an anaplastic morphologic phenotype. Our findings also suggest that ovarian MTs with MCNs often arise in KRAS mutant neoplasms. However, mutations in other genes such as PIK3CA and CDH1 may play a role in the neoplastic evolution of a subset of these tumors.

Clinical and therapeutic implications of BRAF mutation heterogeneity in metastatic melanoma

Heterogeneity of BRAF mutation in melanoma has been a controversial subject. Quantitative data on BRAF allele frequency (AF) are sparse, and the potential relationship with response to BRAF inhibitors (BRAFi) in patients with metastatic melanoma is unknown. We quantitatively measured BRAF AF in a cohort of treatment naïve metastatic melanoma samples by pyrosequencing and correlated with survival data in patients treated with BRAFi as part of their clinical care. Fifty‐two samples from 50 patients were analysed. BRAF V600E mutations were detected in 71.1% of samples followed by V600K (25%) and V600R (3.9%). There was a wide range of AF from 3.9% to 80.3% (median 41.3%). In 33 patients treated with BRAFi, there was no difference in overall or progression‐free survival when the patients were categorized into high or low AF groups. There was no correlation between AF and degree of response, and no difference in survival based on genotype.

Detection of copy number variations in melanocytic lesions utilising array based comparative genomic hybridisation

Distinction between melanocytic naevi and melanoma occasionally poses a diagnostic challenge in ambiguous cases showing overlapping histological features. Melanomas are characterised by the presence of multiple genomic copy number variants (CNVs), while this is not a feature of naevi. We assessed the feasibility and utility of array-based comparative genomic hybridisation (aCGH) to assess CNVs in melanocytic lesions. DNA was extracted from formalin fixed, paraffin embedded (FFPE) sections of unambiguous naevi (n=19) and melanomas (n=19). The test DNA and gender mismatched human reference DNA were differentially labelled with fluorophores. Equal quantities of the two DNA samples were mixed and co-hybridised to a SurePrint G3 Human CGH 8x60K array, and digitally scanned to capture and quantify the relative fluorescence intensities. The ratio of the fluorescence intensities was analysed by Cytogenomics software (Agilent). Frequent large CNVs were identified in 94.7% of melanoma samples, including losses of 9p (73.6%), 9q (52.6%), 10q (36.8%), 11q (36.8%), 3p (21%), and 10p (21%), and gains of 6p (42.1%), 7p (42.1%), 1q (36.8%), 8q (31.5%) and 20q (21%). Only one naevus showed two large copy number changes. Overall aCGH showed a specificity and sensitivity of 94.7% in separating naevi from melanomas. Based on our results, aCGH can be successfully used to analyse CNVs of melanocytic lesions utilising FFPE derived biopsy samples, providing a potentially useful adjunctive test for the classification of diagnostically challenging melanocytic proliferations.

Malignant Melanocytic Matricoma: A Report of 2 Cases and Review of the Literature

Background:To describe 2 cases of melanocytic matricoma with malignant histological features and systematically review previously reported cases of malignant melanocytic matricoma. Methods:Two cases of malignant melanocytic matricoma were identified from the practice of the authors. Additional 3 cases were identified in the literature. The clinical and pathological features of these 5 cases are described. Results:Malignant melanocytic matricoma occurs predominantly in sun-damaged skin of elderly individuals. The tumor is composed of atypical epithelial cells with brisk mitotic activity showing evidence of matrical keratinization and widespread positivity with beta-catenin. There is an admixed cytologically bland dendritic melanocytic component. To date, local recurrence has occurred in one case, but no cases of disseminated disease or death have been reported. Conclusions:Recognition of this rare tumor and separation from a range of diagnostic mimics is important to ensure adequate treatment by local excision and to allow further cases to be identified to better define the biological potential of this lesion.

Molecular profiling study of a recurrent ovarian mucinous tumour with a mural nodule of anaplastic carcinoma, providing supportive evidence of dedifferentiation

S S143 directional sequencing (SBS) has been used as the gold standard for EGFR mutational analysis, however there are emerging new assays utilizing targeted real time PCR technology. In this study we compared the performance of ‘Cobas 4800’ (COBAS) against Sanger sequencing. Methods: 480 consecutive formalin fixed paraffin embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations by SBS and COBAS. Mutational results were catogorised as positive, negative and invalid. Unweighted Kappa test was utilsed to compare the concordance between two assays. Results: After exclusion of invalid results (n=16), 477 samples from 458 patients (47.2% male, 52.8% female) underwent statistical analysis. There was an excellent observed percentage agreement of 98.3% (kappa value 0.944, SE 0.0194) between the two methods. The combined mutation detection rate (19%) was superior to either SBS (18.4%) or COBAS (18%). EGFR mutation frequency was significantly higher in women (23%) compare to men (12%). Discussion: COBAS assay is a diagnostically robust platform comparable with Sanger with very high analytical sensitivity and short turns around time. COBAS failure is due to lower sensitivity to samples with low DNA quality and its limited primer detection range, while Sanger is mostly affected by its lower analytic sensitivity related to low volume of diagnostic material. Consequently, the higher combined mutation detection rate necessitates a dual testing strategy to guarantee detection of novel mutations and mutations outside the COBAS detection range, and avoiding false negative results due to lower analytical sensitivity of SBS. 78. MOLECULAR PROFILING STUDY OF A RECURRENT OVARIAN MUCINOUS TUMOUR WITH A MURAL NODULE OF ANAPLASTIC CARCINOMA, PROVIDING SUPPORTIVE EVIDENCE OF DEDIFFERENTIATION Nima Mesbah Ardakani, Anup Naran, Fabienne Grieu-Iacopetta, Colin Stewart 1Anatomical Pathology Department, PathWest, QEII, and 2Anatomical Pathology Department, PathWest, KEMH, Perth,

Pilomatrical carcinosarcoma: report of a case with comparative genomic hybridisation analysis

Sir, Cutaneous carcinosarcomas are a heterogenous group of unusual tumours. While the majority include an epithelial component of ‘epidermal-type’ carcinoma, carcinosarcoma with an adnexal epithelial component is well recognised. A limited number of recent studies have shown that, despite the striking morphological differences in the appearance of the components, cutaneous carcinosarcomas frequently show evidence of a clonal origin for both components. We report a case of pilomatrical carcinosarcoma, including array based comparative genomic hybridisation (aCGH) comparing the pilomatrical and sarcomatous components. An 87-year-old female presented with a 16 mm nodule on her left hand. Clinically the nodule was dermal and subcutaneous with no surface changes. The patient reported that the lesion had been slowly growing for 4 years. She had previously had several foci of Bowen’s disease from the left cheek, right upper arm and left forearm removed, with no other relevant history. Histopathology of the lesion showed severely sun damaged skin with a dermally based tumour showing biphasic morphology (Fig. 1A). The first area was a circumscribed solid and cystic nodule, composed of a peripheral rim of matrical cells surrounding a central zone of matrical keratinisation with numerous ghost cells (Fig. 1B). The matrical cells showed mitoses up to 7/mm, with a proliferation index of 20% on MIB1 immunohistochemistry. A foreign body giant cell reaction and focal calcification was also noted. A small amount of cellular spindled stroma surrounded this nodule (Fig. 1C). The nodule merged with a

Giant proliferating pilomatricoma; report of a rare entity

Proliferating pilomatricoma is a benign tumour and a rare variant of pilomatricoma that has the potential for local recurrence if incompletely excised. We report a case of giant proliferating pilomatricoma on the forearm of a 66‐year‐old woman. This tumour was unusually large and the presence of ulceration and rapid growth raised clinical suspicion of malignancy. The identification of shadow or ghost cells is a good clue to matrical differentiation, which can be confirmed by β‐catenin immunostaining.

Cutaneous Atypical Neurofibroma: A Case Report and Review of Literature

To the Editor: Neurofibroma (NF) is a benign peripheral neural neoplasm, which occurs in the skin and subcutis as well as in deeper soft tissue. These tumors are often sporadic; however, a small proportion is associated with type I neurofibromatosis (NF-1). There are several histological variants including solitary, plexiform, diffuse, cellular, and atypical NF, all of which show indolent clinical behavior. Atypical NF is an uncommon subtype, often located in the deep soft tissue, and is characterized by worrisome histological features such as nuclear atypia and low level of mitotic activity, falling short of a diagnosis of malignant peripheral nerve sheath tumor (MPNST).1,2 In cutaneous examples, separation of atypical NF from desmoplastic melanoma and MPNST may present a diagnostic challenge. In this report, we present an elderly woman with a cutaneous atypical NF showing prominent cytological atypia but an absence of chromosomal aberrations on array comparative genomic hybridization (aCGH). A 16 · 14-mm polypoid nodule was excised from the left side of the neck of a 71-year-old woman. The patient did not have any significant history; in particular, there was no history or clinical stigmata of NF-1 such as café-au-lait spots, axillary freckling, multiple cutaneous NFs, or iris hamartomas (Lisch nodules). On histological examination, there was a wellcircumscribed polypoid mass, predominantly located in the dermis, with extension to the subcutaneous tissue (Fig. 1A). The background skin showed mild solar elastosis. The lesion appeared nonencapsulated, with an overlying grenz zone separating the tumor from the epidermis. There were occasional entrapped adnexal structures, including hair follicles and eccrine ducts (Fig. 1B). The constituent lesional cells showed spindled morphology and were randomly distributed in a stroma containing wiry collagen fibers. The tumor cells showed random but prominent cytologic atypia and nuclear pleomorphism with hyperchromatic lobated and giant nuclei and occasional eosinophilic intranuclear pseudoinclusions (Fig. 1C). The nuclear chromatin had a smudgy and finely granular/fragmented appearance, mimicking mitosis, in a subset of cells; however, no definite mitotic activity was observed (Fig. 1D). There were scattered intralesional mast cells. There was no significant peritumoral or intratumoral lymphocytic infiltrate. There were no hypercellular areas and no necrosis. There was no overlying intraepidermal melanocytic proliferation. The lesion extended to the edges of the biopsy. By immunohistochemistry, a large proportion of both atypical and nonatypical lesional cells demonstrated diffuse positive nuclear staining with S100 (Fig. 2A) and SOX10. P16 expression was retained in most tumor nuclei (Fig. 2B). There was patchy focal staining with EMA and CD34, the latter focally displaying a “fingerprint” pattern (Fig. 2C). The tumor cells were negative for MelanA, HMB45, MiTF, SMA, desmin, CD68, and multiple cytokeratins including HMWCK, AE1/3, and MNF116. The proliferation index (estimated by Ki67) was less than 5% (Fig. 2D), and P53 was positive in less than 10% of tumor cells. No mitotic figures were identified by phosphohistone H3 staining. Immunostaining with H3K27me3 antibody showed a normal pattern with retained nuclear staining in the lesional cells (Fig. 3). Molecular testing using aCGH was performed on DNA extracted from the formalin-fixed, paraffin-embedded tissue sections. aCGH showed a stable genome, with no significant gains or losses of genomic material. Based on the overall clinical, histological, immunohistochemical, and molecular findings, a diagnosis of “atypical neurofibroma” was rendered. On clinical follow-up, there was no recurrence or metastasis after a period of 18 months. Atypical NF is a rare variant of NF, histologically defined by proliferation of spindle Schwann cells with random cytological atypia and occasional mitoses, and accompanying perineurial cells and fibroblasts in a collagenous to myxoid stroma.1,2 However, it lacks other morphological and clinical characteristic features of MPNST such as rapid and infiltrative growth, large size (often more than 5 cm), high cellularity, diffuse marked cytological atypia, high mitotic activity and atypical mitoses, and tumor necrosis.3 Lin et al studied 14 cases of soft tissue atypical NF from 6 patients, 3 of whom had NF-1.1 They observed mildto-severe degree of cytological atypia but absent or low mitotic activity (#1 mitosis per 10 high-power field), no necrosis, and very low proliferation index as judged by immunohistochemistry for Ki67 proliferation marker (average 2.5% in 13 tumors and 10% in 1 tumor). They described areas of hypercellularity in 35.7% of cases. In addition, most cases in their cohort exhibited absence or low level of P53 expression by immunohistochemistry (mostly absent or less than 5%) and a diploid cytogenetic profile. They compared these lesions with 3 cases of MPNST, which showed high expression of P53 (range 10%–16%, mean 12%), high proliferation index (32%–42%, mean 38%), and other attributes of malignancy including necrosis, high cellularity, and high mitotic rate. On follow-up ranging from 8 months to 6 years, the cases of atypical NF showed no recurrence or metastasis. They postulated that small NFs (less than 5 cm) with cytological atypia, even if showing low mitotic activity or focal hypercellularity, most likely pursue an indolent clinical course and can be treated conservatively. Most other authors have supported this view.2 The largest series of cutaneous atypical NF, reported by Jokinen et al, comprises 11 cases including 9 women and 2 men with an average age of 38, 1 of whom had NF-1.3 The tumors were located in the dermis (n = 3), subcutis The authors declare no conflicts of interest.

Metastatic Cellular Blue Nevus: A Rare Case With Metastasis Beyond Regional Nodes

Abstract: In this study, we present a rare case of a 35-year-old man with a long-standing blue-black lesion on his left hand with subsequent infraclavicular and axillary lymph node tumor deposits. The hand lesion and lymph nodes were excised revealing histological, immunohistochemical, and molecular findings consistent with cellular blue nevus. Despite nonregional lymph node involvement, there has been no progression at 12-months follow-up. This is an index case of a cellular blue nevus with metastasis to both regional and nonregional lymph nodes. The lack of atypical/malignant features in this lesion makes the metastatic behavior extraordinary, and hence the prognosis of lesions of this type is indeterminate.

Pseudomelanomas Following Stevens-Johnson Syndrome

Abstract: In this report, we present a novel case of pseudomelanoma, similar to that seen in a recurrent/traumatized nevus, in pre-existing nevi in a 36-year-old man a few months after recovering from an episode of severe Stevens–Johnson syndrome. The mechanism responsible for the atypical transformation of these nevi is likely the release of cytokines and growth factors in the microenvironment during the repair/regeneration process. It is important to be aware of this phenomenon, and specific inquiry about potential recent blistering skin disorder in addition to the other causes of trauma should be made when dealing with cases of pseudomelanoma to avoid misdiagnosis.