Clear Cell Odontogenic Carcinoma: Occurrence of EWSR1-CREB1 as Alternative Fusion Gene to EWSR1-ATF1.

Clear cell odontogenic carcinoma (CCOC) is a rare, low-grade malignant epithelial neoplasm, occurring in the jawbones, mainly affecting the mandible of elderly patients. In addition to hyalinizing clear cell carcinoma of the salivary gland, it is one of the epithelial neoplasms known to harbor an EWSR1-ATF1 fusion. Therefore, a link between these tumors seems plausible. We describe six cases of CCOC showing EWSR1 rearrangements, with two cases being positive for the ATF1 partner gene using FISH analysis. In one case, an EWSR1-CREB1 fusion was identified using RT-PCR, which we report for the first time in this tumor type. The other three cases investigated by FISH were negative for ATF1, CREB1 and CREB3L2. In conclusion, our data show that EWSR1-CREB1 is an alternative fusion gene to EWSR1-ATF1 in CCOC.

Soft tissue angiofibroma: Clinicopathologic, immunohistochemical and molecular analysis of 14 cases.

Soft tissue angiofibroma is rare and has characteristic histomorphological and genetic features. For diagnostic purposes, there are no specific antibodies available. Fourteen lesions (6 females, 8 males; age range 7-67 years) of the lower extremities (12) and trunk (2) were investigated by immunohistochemistry, including for the first time NCOA2. NCOA2 was also tested in a control group of other spindle cell lesions. The known fusion-genes (AHRR-NCOA2 and GTF2I-NCOA2) were examined using RT-PCR in order to evaluate their diagnostic value. Cases in which no fusion gene was detected were additionally analysed by RNA sequencing. All cases tested showed nuclear expression of NCOA2. However, this was not specific since other spindle cell neoplasms also expressed this marker in a high percentage of cases. Other variably positive markers were EMA, SMA, desmin and CD34. STAT6 was negative in the cases tested. By RT-PCR for the most frequently observed fusions, an AHRR-NCOA2 fusion transcript was found in 9/14 cases. GTF2I-NCOA2 was not detected in the remaining cases (n = 3). RNA sequencing revealed three additional positive cases; two harbored a AHRR-NCOA2 fusion and one case a novel GAB1-ABL1 fusion. Two cases failed molecular analysis due to poor RNA quality. In conclusion, the AHRR-NCOA2 fusion is a frequent finding in soft tissue angiofibroma, while GTF2I-NCOA2 seems to be a rare genetic event. For the first time, we report a GAB1-ABL1 fusion in a soft tissue angiofibroma of a child. Nuclear expression of NCOA2 is not discriminating when compared with other spindle cell neoplasms.

Solitary fibrous tumor - clinicopathologic, immunohistochemical and molecular analysis of 28 cases.

BACKGROUND: Solitary fibrous tumor is a mesenchymal tumor of fibroblastic type, which can affect any region of the body. Recently, a recurrent gene fusion NAB2-STAT6 has been identified as molecular hallmark. The NAB2-STAT6 fusion leads to EGR1 activation and transcriptional deregulation of EGR1-dependent target genes and is a driving event in initiation of SFT. In this study, we report the clinicopathologic and RT-PCR findings and evaluated expression of STAT6 and EGR1 protein in a cohort of 28 SFTs. METHODS: 28 patients with a median age of 54 years were included with SFTs originating at different sites, most occurring in the lung and pleura (9, 32%), 5 in soft tissues of the lower extremities (18%) and 5 in the head and neck (18%). For detection of the NAB2-STAT6 fusion gene, RT-PCR was performed using RNA extracted from formalin-fixed and paraffin-embedded tissues. Immunohistochemistry was performed on all cases with antibodies against STAT6 and EGR1. RESULTS: All patients were treated by surgery, 3 with adjuvant chemo- or radiotherapy. Follow-up data of 18 patients could be obtained of which 2 patients died of metastatic disease 13 months and 52 years after first diagnosis. Sixteen patients have no evidence of disease with a median follow up of 29.5 months (range 7 - 120 months). NAB2-STAT6 fusion transcripts were found in 19/28 cases (68%). The most common fusion was between NAB2 exon 4 and STAT6 exon 3 (11/19, 58%), mainly occurring in pleuropulmonary lesions. All cases showed strong nuclear expression of STAT6 (28/28, 100%) while EGR1 showed low-level variable nuclear expression in all samples, comparable with the EGR1 expression results of the control group. CONCLUSIONS: The identification of the NAB2-STAT6 fusion in SFTs can provide important diagnostic information, especially in cases with aberrant morphology or when biopsy material is limited. STAT6 immunohistochemistry is another useful tool in diagnosing SFT. EGR1 immunohistochemistry indicates low-level protein expression in accordance with EGR1 activation due to distorted NAB2 activity. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_224.

Epithelioid Hemangioendothelioma: clinicopathologic, immunhistochemical, and molecular genetic analysis of 39 cases.

BACKGROUND: Epithelioid hemangioendothelioma is a malignant, often indolent vascular tumor which occurs at various anatomic sites. Based on a reciprocal translocation t (1;3)(p36;q25), a consistent WWTR1-CAMTA1 fusion gene has been found. An alternate YAP1-TFE3 fusion has been detected in a small and distinct subset of cases. METHODS: Thirty-nine tumors, from 24 females and 15 males with an age range 9-85 years, were located in soft tissue (head and neck [8], trunk [5], upper extremities [3], lower extremities [2], mediastinal [1], and paratesticular [1]), lymph node (1), breast (1), skin (2), bone (6), lung (7), and liver (2). The cases were investigated using a panel of immunohistochemical markers. The aforementioned fusion-genes were examined using RT-PCR and/or FISH in order to validate their diagnostic value. RESULTS: Follow-up available for 17 patients ranged from 3 months to 7 years (median interval 1.5 years). Eleven patients were alive without disease, 2 patients were alive with disease after 1.5 and 2 years, respectively. Four patients died of disease after 4 months (n = 1), 5 months (n = 2), and 1.5 years (n = 1).The size, known for 30 lesions, was >3 cm in 9 of them. Histologically, all lesions had classical features, at least focally. Four tumors counted >3 mitoses/50 HPF. Immunohistochemically, all cases tested stained positive for ERG (21), FLI1 (5) and CD31 (39). CD34 and D2-40 positivity was seen in 81% and 71% of the examined cases, respectively. 11/35 cases expressed pan-keratin and 6/20 cases CK8.18. TFE3 showed a nuclear reaction in 21/24 cases, irrespective of TFE3 rearrangement.Molecular genetically, 35/35 cases revealed one of the fusion genes by FISH and/or RT-PCR with WWTR1-CAMTA1 in 33 cases and YAP1-TFE3 in 2 cases. CONCLUSIONS: These results demonstrate the high diagnostic value of FISH and RT-PCR in detecting the fusion genes of EHE. The immunohistochemical utility of TFE3 appears questionable in this study. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4010279141259481.

Presence of C11orf95-MKL2 fusion is a consistent finding in chondroid lipomas: a study of eight cases.

AIMS: Chondroid lipomas are benign adipose tissue tumours. Their rarity and peculiar morphology can lead to misinterpretation, especially in small biopsies. Based on a recurrent translocation t(11;16)(q13;p13), the C11orf95-MKL2 fusion gene has been found in a few cases. Therefore, it seemed appropriate to look for this fusion gene in a larger cohort. METHODS AND RESULTS: We describe eight further cases from four females and four males with an age range of 21-81 years (median 49 years). The tumours were situated in the lower arm (three), lower leg (two), thigh (one), back (one) and head (one); seven lesions were deep-seated and one was located subcutaneously. Sizes ranged from 3 to 12 cm (median 6.3 cm). All patients were treated by simple excision, and follow-up, available for six patients (range 2 months-12 years; median 15 months), demonstrated recurrence in one case. Histologically, the circumscribed and lobulated tumours showed a variable composition of adipocytes, lipoblasts, hibernoma-like cells and chondroblast-like cells embedded in a chondroid matrix. Immunohistochemistry, performed in four cases, revealed positivity for S-100 and pancytokeratin in two of three neoplasms stained for each marker. A C11orf95-MKL2 fusion gene was shown by RT-PCR analysis in seven of the eight cases. CONCLUSIONS: Molecular analysis can be used to support the diagnosis of chondroid lipoma, especially in small samples. This may be helpful in planning treatment when the differential diagnosis includes malignant lesions.

NR4A3 rearrangement reliably distinguishes between the clinicopathologically overlapping entities myoepithelial carcinoma of soft tissue and cellular extraskeletal myxoid chondrosarcoma.

Myoepithelial carcinoma of soft tissue (MEC) and cellular extraskeletal myxoid chondrosarcoma (cEMC) share striking similarities. In this paper, we compare ten MECs with five cEMCs. MEC patients had an equal gender distribution. The age range was 15-76 years (mean, 42 years). Tumours were located on extremities, pelvic girdle, vulva and neck. Follow-up, available for nine patients, ranged from 4 to 85 months (mean, 35 months). Five patients were alive without evidence of disease, two were alive with disease and two died 8 months after the initial diagnosis. cEMCs were from three males and two females with an age range of 37-82 years (mean, 57 years); they presented in extremities, shoulder and paravertebral/cervical. Follow-up, available for four patients, ranged from 6 to 220 months (mean, 61 months). All patients were alive, two with recurrences and/or metastases and two without evidence of disease. Morphologically, the distinction between these two entities was difficult since all cases exhibited features typically seen in myoepithelial tumours. Immunohistochemically, MECs expressed pan-keratin (80 %), epithelial membrane antigen (EMA; 57 %), S100 (50 %), alpha-smooth muscle actin (ASMA; 75 %), calponin (67 %) and p63 (25 %). S100 and EMA were expressed in 40 % of cEMC cases respectively with additional immunoreactivity for p63, ASMA and glial fibrillary acidic protein in one case. Pan-keratin was negative in all neoplasms. NR4A3 rearrangement was present in four of four cEMCs and in none of the MECs. In contrast, three of nine (33 %) MECs and four of five (80 %) cEMCs showed an EWSR1 rearrangement. In summary, MECs and cEMCs share clinical, morphological, immunohistochemical and genetic characteristics. The pathognomic rearrangement of NR4A3 is a useful diagnostic feature in identifying cEMCs.

EWSR1-ATF1 chimeric transcript in a myoepithelial tumor of soft tissue: a case report.

Soft tissue myoepithelial tumors, a recently defined entity, include benign and malignant lesions showing a considerable morphological and immunohistochemical heterogeneity. EWSR1 rearrangements are well recognized in this tumor type, and some of the partner genes have been identified. Herein we describe a soft tissue myoepithelioma arising in the pelvis with an EWSR1-ATF1 fusion, therefore extending the spectrum of partner genes of EWSR1. In addition, this case indicates that there are overlapping genetic features of myoepithelial tumors, clear cell sarcoma, angiomatoid fibrous histiocytoma, and hyalinizing clear-cell carcinoma of the salivary gland.

CDKN2A but not TP53 mutations nor HPV presence predict poor outcome in metastatic squamous cell carcinoma of the skin.

Genetic alterations in metastatic cutaneous squamous cell carcinoma (CSCC) which might serve as prognostic biomarkers are not well investigated. We investigated the mutation status and protein expression of the CDKN2A (INK4a-ARF) and TP53 genes in metastatic CSCCs and correlated this with clinicopathological variables, HPV presence, and survival data. Sequence analysis was performed on formalin-fixed and paraffin-embedded tissue of 35 metastases and their primary tumors, and was correlated with immunohistochemical stainings for p53, p16 and p14. Beta-PV and alpha-PV DNA was detected using PCR-based assays. Kaplan-Meier and Cox regression methods were used for survival assessment. CDKN2A was mutated in 31% of the metastases and their primary tumors, while the TP53 gene was mutated in 51% of the metastases. P53 protein expression was significantly associated with missense type of mutations (p = 0.002). No persistent HPV types were detected. CDKN2A mutations were significantly associated with disease-specific death (p = 0.001). A significant difference was observed in disease-specific survival between patients with or without a CDKN2A mutation (p = 0.010), while this was not the case for TP53. At univariate Cox's regression analysis tumor size (p = 0.010), invasion depth (p = 0.030) and CDKN2A mutations (p = 0.040) were significantly related to shorter disease-specific survival. At multivariate Cox's regression only tumor size had an adverse effect on survival (p = 0.002). In conclusion, our study indicates that the CDKN2A mutation status might be of prognostic value in metastatic CSCCs. In most cases, CDKN2A and TP53 mutations are early genetic events in CSCC tumorigenesis. The possible role of HPV in metastatic CSCC needs further exploration.

Occurrence of ocular melanoma thirteen years after skin melanoma: two separate primaries or metastatic disease? A case solved with NRAS and CDKN2A (INK4A-ARF) mutational analysis.

The differential diagnosis between primary uveal melanoma and cutaneous melanoma metastasis in the eye may be difficult, both clinically and histologically. We report successful application of combined mutational analysis of the NRAS and the CDKN2A gene to discriminate between these two entities. The patient had a history of a superficial spreading cutaneous melanoma of the left shoulder. Nine years later, she developed a lymph node metastasis in the left axilla, and 13 years later she presented with an atypical, pigmented tumor in the uvea. Histologically, the origin of the uveal melanoma could not be determined with certainty. We performed molecular analysis on the skin melanoma, the lymph node metastasis and the uveal melanoma. We detected an NRAS codon 61 mutation (c.182A>G, p.Gln61Arg) in all three tumor specimens. This mutation was absent in the normal control tissue of the patient, thereby excluding a germline mutation. To confirm a clonal relationship between the tumors, we also performed CDKN2A mutational analysis. We detected a CDKN2A mutation ((p16) c.238C>T, p.Arg80X, (p14) c.404C>T, p.Pro135Leu)) in the tumor samples, but not in the normal control tissue of the patient. We concluded that the uveal melanoma is a metastasis from the cutaneous melanoma removed 13 years before.

CDKN2A (INK4A-ARF) mutation analysis to distinguish cutaneous melanoma metastasis from a second primary melanoma.

The histologic differential diagnosis between a second primary cutaneous melanoma and cutaneous melanoma metastasis in a patient with a previous history of melanoma can be very difficult. This case report describes the first application of CDKN2A mutation analysis for discriminating a cutaneous melanoma metastasis from a new primary melanoma. In 2005, we received a skin excision of the right arm of a 38-year-old female patient for second opinion. Histologically, we considered the lesion to be a melanoma. The patient had a history of superficial spreading melanoma in the right subclavicular region, with a Breslow thickness of 1.1 mm, in 1998. The morphology showed resemblance to the present melanoma on the right arm, but the differential diagnosis between metastasis or second primary melanoma could not be made with certainty based on histology alone. We decided to perform TP53 and CDKN2A mutation analysis on both tumors. Molecular analysis revealed that both the melanoma of 1998 and of 2005 contained an identical CDKN2A mutation (a deletion in exon 1alpha, c.95_112del (p.Leu32_Leu37del)), which was absent in normal control tissue of the patient, thereby excluding a germline mutation. TP53 mutations were absent in both tumors and in normal skin. Based on these molecular findings the present melanoma on the right arm was diagnosed as a metastasis. Seven months later the patient died of widespread metastatic disease confirming the metastatic nature of the lesion. This case illustrates that molecular analysis can contribute to the sometimes-difficult differentiation between a second primary melanoma and a melanoma metastasis.

INK4-ARF and p53 mutations in metastatic cutaneous squamous cell carcinoma: case report and archival study on the use of Ink4a-ARF and p53 mutation analysis in identification of the corresponding primary tumor.

So far, histopathologic, immunohistochemical and molecular properties of metastatic cutaneous squamous cell carcinomas (CSCCs) are relatively unexplored. In patients with multiple CSCCs, as for instance renal transplant recipients (RTRs), it might prove difficult to identify the primary tumor responsible for metastasis. We report a case of an RTR with multiple CSCCs, one of which metastasized. By using p53 and INK4a-ARF mutation analysis, we identified the responsible primary tumor due to an identical mutation in exon 2 of the INK4a-ARF locus. Archival study yielded 14 cases of metastatic CSCC (present case included). In only 8 of 14 metastases, DNA quality was sufficient to perform PCR reactions. In 7 of 8 metastases, either an INK4a-ARF (6 of 8 cases) and/or p53 (3 of 8 cases) mutation was present. In 6 of 7 cases, the corresponding primary could be identified by an identical mutation in p53 and/or INK4a-ARF. In conclusion, molecular analysis using a combination of p53 and INK4a-ARF mutation analysis can identify the corresponding primary skin tumor in case of CSCC metastases in the majority of cases. This is facilitated by the high frequency of these mutations in metastatic CSCC when compared with frequency spectra reported in the literature in primary CSCCs. The major limitation was formed by insufficient DNA quality in archival tissue.

Mutual exclusion of t(11;18)(q21;q21) and numerical chromosomal aberrations in the development of different types of primary gastric lymphomas.

Gastric non-Hodgkin's lymphomas can be divided histologically into mucosa-associated lymphoid tissue (MALT) lymphoma (ML) and diffuse large cell lymphoma (DLCL) with or without evidence of preceding/accompanying ML (DLCL + ML). We studied the incidence of the most frequent structural chromosomal aberration in ML, t(11;18)(q21;q21), and numerical aberrations of seven chromosomes in 36 ML, 39 DLCL + ML and ten gastric DLCL cases, by dual-colour interphase fluorescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). t(11;18)(q21;q21) was exclusively detected in ML (FISH 22%; RT-PCR 24%), being completely absent in DLCL + ML and DLCL. No other translocations involving 11q21 or 18q21 and other partner chromosomes were detected by FISH. In lymphomas harbouring t(11;18)(q21;q21), this translocation was the sole genetic abnormality. In contrast, 45% of the t(11;18)(q21;q21)-negative ML showed trisomies, especially of chromosome 3 and 18. In DLCL + ML with separate small and large cell components, trisomies were either detected in both components or occurred exclusively in large tumour cells. Our results suggest that ML can be divided in lymphomas characterized by the t(11;18)(q21;q21), which are unlikely to transform into high-grade tumours, and t(11;18)(q21;q21)-negative ML that may develop into DLCL + ML after the acquisition of additional genetic aberrations.