Hereditary leiomyomatosis and renal cell carcinoma syndrome-associated renal cell carcinomas (RCC) are difficult to diagnose prospectively. We used immunohistochemistry (IHC) to identify fumarate hydratase (FH)-deficient tumors (defined as FH negative, 2-succinocysteine [2SC] positive) in cases diagnosed as "unclassified RCC, high grade or with papillary pattern," or "papillary RCC type 2," from multiple institutions. A total of 124 tumors (from 118 patients) were evaluated by IHC for FH and 2SC. An FH deficiency was found in 24/124 (19%) cases. An indeterminate result (only 1 marker abnormal) was found in 27/124 (22%) cases. In a tissue microarray of 776 RCCs of different types, only 2 (0.5%) tumors, initially considered papillary type 2, were FH deficient. FH mutations were found in 19/21 FH-deficient tumors (with confirmed germline mutations in 9 of 9 tumors in which germline status could be assessed) and in 1/26 FH-indeterminate tumors identified by IHC. No FH mutations were found in 2/21 FH-deficient RCCs, 25/26 FH-indeterminate RCCs, and 10/10 RCCs demonstrating FH expression by IHC. Patients with FH-deficient RCC had a median age of 44 years (range, 21 to 65 y). Average tumor size was 8.2 cm (range, 0.9 to 18 cm). FH-deficient RCCs were characterized by at least focal macronucleoli and demonstrated 2 or more growth patterns in 93% cases. Papillary was the most common (74%) and dominant (59%) pattern, whereas other common patterns included: solid (44%), tubulocystic (41%), cribriform (41%), and cystic (33%). At presentation, 57% were stage ≥pT3, 52% had positive nodes, and 19% had distant metastases. After a mean follow-up of 27 months (range, 1 to 114 mo), 39% of patients were dead of disease, and 26% had disease progression. We conclude that FH and 2SC are useful IHC ancillary tools, which allow recognition of FH-deficient RCC.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome secondary to germline fumarate hydratase (FH) mutation presents with cutaneous and uterine leiomyomas, and a distinctive aggressive renal carcinoma. Identification of HLRCC patients presenting first with uterine leiomyomas may allow early intervention for renal carcinoma. We reviewed the morphology and immunohistochemical (IHC) findings in patients with uterine leiomyomas and confirmed or presumed HLRCC. IHC was also performed on a tissue microarray of unselected uterine leiomyomas and leiomyosarcomas. FH-deficient leiomyomas underwent Sanger and massively parallel sequencing on formalin-fixed paraffin-embedded tissue. All 5 patients with HLRCC had at least 1 FH-deficient leiomyoma: defined as completely negative FH staining with positive internal controls. One percent (12/1152) of unselected uterine leiomyomas but 0 of 88 leiomyosarcomas were FH deficient. FH-deficient leiomyoma patients were younger (42.7 vs. 48.8 y, P=0.024) and commonly demonstrated a distinctive hemangiopericytomatous vasculature. Other features reported to be associated with FH-deficient leiomyomas (hypercellularity, nuclear atypia, inclusion-like nucleoli, stromal edema) were inconstantly present. Somatic FH mutations were identified in 6 of 10 informative unselected FH-deficient leiomyomas. None of these mutations were found in the germline. We conclude that, while the great majority of patients with HLRCC will have FH-deficient leiomyomas, 1% of all uterine leiomyomas are FH deficient usually due to somatic inactivation. Although IHC screening for FH may have a role in confirming patients at high risk for hereditary disease before genetic testing, prospective identification of FH-deficient leiomyomas is of limited clinical benefit in screening unselected patients because of the relatively high incidence of somatic mutations.

The spectrum of the renal oncocytic tumors has been expanded in recent years to include several novel and emerging entities. We describe a cohort of novel, hitherto unrecognized and morphologically distinct high-grade oncocytic tumors (HOT), currently diagnosed as "unclassified" in the WHO classification. We identified 14 HOT by searching multiple institutional archives. Morphologic, immunohistochemical (IHC), molecular genetic, and molecular karyotyping studies were performed to investigate these tumors. The patients included 3 men and 11 women, with age range from 25 to 73 years (median 50, mean 49 years). Tumor size ranged from 1.5 to 7.0 cm in the greatest dimension (median 3, mean 3.4 cm). The tumors were all pT1 stage. Microscopically, they showed nested to solid growth, and focal tubulocystic architecture. The neoplastic cells were uniform with voluminous oncocytic cytoplasm. Prominent intracytoplasmic vacuoles were frequently seen, but no irregular (raisinoid) nuclei or perinuclear halos were present. All tumors demonstrated prominent nucleoli (WHO/ISUP grade 3 equivalent). Nine of 14 cases were positive for CD117 and cytokeratin (CK) 7 was either negative or only focally positive in of 6/14 cases. All tumors were positive for AE1-AE3, CK18, PAX 8, antimitochondrial antigen, and SDHB. Cathepsin K was positive in 13/14 cases and CD10 was positive in 12/13 cases. All cases were negative for TFE3, HMB45, Melan-A. No TFEB and TFE3 genes rearrangement was found in analyzable cases. By array CGH, complete chromosomal losses or gains were not found in any of the cases, and 3/9 cases showed absence of any abnormalities. Chromosomal losses were detected on chromosome 19 (4/9), 3 with losses of the short arm (p) and 1 with losses of both arms (p and q). Loss of chromosome 1 was found in 3/9 cases; gain of 5q was found in 1/9 cases. On molecular karyotyping, 3/3 evaluated cases showed loss of heterozygosity (LOH) on 16p11.2-11.1 and 2/3 cases showed LOH at 7q31.31. Copy number (CN) losses were found at 7q11.21 (3/3), Xp11.21 (3/3), Xp11.22-11.21 (3/3), and Xq24-25 (2/3). CN gains were found at 13q34 (2/3). Ten patients with available follow up information were alive and without disease progression, after a mean follow-up of 28 months (1 to 112 months). HOT is a tumor with unique morphology and its IHC profile appears mostly consistent. HOT should be considered as an emerging renal entity because it does not meet the diagnostic criteria for other recognized eosinophilic renal tumors, such as oncocytoma, chromophobe renal cell carcinoma (RCC), TFE3 and TFEB RCC, SDH-deficient RCC, and eosinophilic solid and cystic RCC.

Aim To describe a group of distinct low‐grade oncocytic renal tumours that demonstrate CD117 negative/cytokeratin (CK) 7‐positive immunoprofile. Methods and results We identified 28 such tumours from four large renal tumour archives. We performed immunohistochemistry for: CK7, CD117, PAX8, CD10, AMACR, e‐cadherin, CK20, CA9, AE1/AE3, vimentin, BerEP4, MOC31, CK5/6, p63, HMB45, melan A, CD15 and FH. In 14 cases we performed array CGH, with a successful result in nine cases. Median patient age was 66 years (range 49–78 years) with a male‐to‐female ratio of 1:1.8. Median tumour size was 3 cm (range 1.1–13.5 cm). All were single tumours, solid and tan‐brown, without a syndromic association. On microscopy, all cases showed solid and compact nested growth. There were frequent areas of oedematous stroma with loosely arranged cells. The tumour cells had oncocytic cytoplasm with uniformly round to oval nuclei, but without significant irregularities, and showed only focal perinuclear halos. Negative CD117 and positive CK7 reactivity were present in all cases (in two cases there was focal and very weak CD117 reactivity). Uniform reactivity was found for PAX8, AE1/AE3, e‐cadherin, BerEP4 and MOC31. Negative stains included CA9, CK20, vimentin, CK5/6, p63, HMB45, Melan A and CD15. CD10 and AMACR were either negative or focally positive; FH was retained. On array CGH, there were frequent deletions at 19p13.3 (seven of nine), 1p36.33 (five of nine) and 19q13.11 (four of nine); disomic status was found in two of nine cases. On follow‐up (mean 31.8 months, range 1–118), all patients were alive with no disease progression. Conclusion Low‐grade oncocytic tumours that are CD117‐negative/CK7‐positive demonstrate consistent and readily recognisable morphology, immunoprofile and indolent behaviour.

A unique renal neoplasm characterized by eosinophilic cytoplasm and solid and cystic growth was recently reported in patients with tuberous sclerosis complex (TSC). We searched multiple institutional archives and consult files in an attempt to identify a sporadic counterpart. We identified 16 morphologically identical cases, all in women, without clinical features of TSC. The median age was 57 years (range, 31 to 75 y). Macroscopically, tumors were tan and had a solid and macrocystic (12) or only solid appearance (4). Average tumor size was 50 mm (median, 38.5 mm; range, 15 to 135 mm). Microscopically, the tumors showed solid areas admixed with variably sized macrocysts and microcysts that were lined by cells with a pronounced hobnail arrangement. The cells had voluminous eosinophilic cytoplasm with prominent granular cytoplasmic stippling and round to oval nuclei with prominent nucleoli. Scattered histiocytes and lymphocytes were invariably present. Thirteen of 16 patients were stage pT1; 2 were pT2, and 1 was pT3a. The cells demonstrated a distinct immunoprofile: nuclear PAX8 expression, predominant CK20-positive/CK7-negative phenotype, patchy AMACR staining, but no CD117 reactivity. Thirteen of 14 patients with follow-up were alive and without disease progression after 2 to 138 months (mean: 53 mo; median: 37.5 mo); 1 patient died of other causes. Although similar to a subset of renal cell carcinomas (RCCs) seen in TSC, we propose that sporadic "eosinophilic, solid, and cystic RCC," which occurs predominantly in female individuals and is characterized by distinct morphologic features, predominant CK20-positive/CK7-negative immunophenotype, and indolent behavior, represents a novel subtype of RCC.

ETV6 gene abnormalities are well described in tumor pathology. Many fusion partners of ETV6 have been reported in a variety of epithelial, mesenchymal, and hematological malignancies. In salivary gland tumor pathology, however, the ETV6-NTRK3 translocation is specific for (mammary analog) secretory carcinoma, and has not been documented in any other salivary tumor type. The present study comprised a clinical, histologic, and molecular analysis of 10 cases of secretory carcinoma, with typical morphology and immunoprofile harboring a novel ETV6-RET translocation.

We describe a novel gene fusion, EWSR1-CREM, identified in 3 cases of clear cell carcinoma (CCC) using anchored multiplex polymerase chain reaction, a next-generation sequencing-based technique. CCC is a low-grade salivary tumor recently characterized to have EWSR1-ATF1 fusions in the majority of cases. Three cases of malignant tumor presenting in the base of tongue, lung, and nasopharynx were studied. All cases shared a clear cell morphology with hyalinized stroma, presence of mucin and p63 positivity and were initially diagnosed as mucoepidermoid carcinoma but were negative for evidence of any of the expected gene fusions. Anchored multiplex polymerase chain reaction demonstrated a EWSR1-CREM fusion in all 3 cases to confirm a diagnosis of CCC. This finding is biologically justified as CREM and ATF1 both belong to the CREB family of transcription factors. EWSR1-CREM fusions have not been previously reported in CCC and have only rarely been reported in other tumors. We show that the ability to discover novel gene variants with next-generation sequencing-based assays has clinical utility in the pathologic classification of fusion gene-associated tumors.

Brooke-Spiegler syndrome (BSS) is a rare, inherited, autosomal dominant disorder characterized by development of multiple adnexal cutaneous neoplasms including spiradenoma, cylindroma, spiradenocylindroma, and trichoepithelioma. The syndrome of multiple familial trichoepitheliomas (MFT) is considered a phenotypic variant of BSS in which patients present with trichoepitheliomas only. We studied germline and somatic mutations of the CYLD gene by direct sequencing in patients with BSS (n = 49) and MFT (n = 18) using peripheral blood and 90 samples of frozen or formalin-fixed paraffin-embedded tumor tissue selected from 379 available histology specimens. Germline CYLD mutations were found in 51 patients (76%) from 36 families (75%). Germline CYLD mutations were found in 43 of the 49 patients with BSS (88%) but in only 8 of 18 MFT cohort (44%). Twenty-one frameshift, 15 nonsense, 3 missense, and 4 splice site mutations were found in patients with BSS, whereas 1 frameshift, 5 nonsense, and 2 splice site mutations were identified in the MFT cohort. Five novel mutations were identified including 4 frameshift mutations (c.1027dupA/p.T343NfsX7, c.2155dupA/p.M719NfsX5, c.2288_2289delTT/p.F763X, and c.2641delG/p.D881TfsX32) and 1 nonsense mutation (c.2713C>T/p. Q905X). Of the 76 tumors from 32 patients with a germline CYLD mutation, 12 were spiradenomas, 15 spiradenocylindromas, 26 cylindromas, 15 trichoepitheliomas, and 7 were other tumor types. Somatic mutations were detected in 67 specimens of these 76 tumors (88%). Of the 67 somatic mutations, 21 (31%) represented a sequence alteration and 46 (69%) showed loss of heterozygosity. In the remaining 9 cases (12%), the somatic changes remained unknown. A germline CYLD mutation was not detected in 14 tumor samples from 8 patients. In these 14 tumors, somatic mutations were identified in 6 samples (43%), all consisting of sequence alterations (1 sample showed 2 different sequence alterations). In the remaining 8 samples (53%), neither germline nor somatic mutations were found in the lesional tissue. Our study increases the catalog of known CYLD mutations in patients with BSS/MFT to 86 and documents the variability of somatic mutations that may occur in them. We confirm the absence of firm genotype-phenotype correlations and the existence of a subset of patients with BSS/MFT who lack a demonstrable germline CYLD mutation. Further studies are needed to explain the reasons for this phenomenon.