Extraskeletal myxoid chondrosarcoma (EMC) is a rare mesenchymal neoplasm, rarely reported in the genitourinary tract with only 5 cases reported in the vulva. We investigated 2 cases of vulvar sarcomas whose morphologic appearance and immunohistochemical profiles were consistent with EMC using fluorescence in situ hybridization (FISH), reverse-transcription polymerase chain reaction, and a whole genome expression array. FISH and reverse-transcription polymerase chain reaction assays showed no EWSR1 and NR4A3 loci rearrangements. Microarray-based analysis also revealed no changes in NR4A3 and EWSR1 gene transcription levels. Microarray data showed a significant downregulation of the muscle-related genes (eg, myosin heavy chain family, actins, myoglobin, desmin, creatine kinase, troponins) and cytokeratins (KRT6A, 6B, 13, 14, and 78), upregulation of several neuron-specific genes [neural cell adhesion molecule 1 (NCAM-1/CD56), neurofilament (NEFH)], along with some well-characterized tumor biomarkers [carbonic anhydrase IX (CA-9), topoisomerase II&agr; (TOP2A), matrix metalloproteinases (MMP-7, MMP-9), CDKN2 gene (p16-INK4a), checkpoint homolog 2 (CHEK2)]. Notably, both tumors showed upregulation of the pleomorphic adenoma gene 1 (PLAG1), and in 1 case PLAG1 gene rearrangement was detected by break-apart FISH. Some vulvar tumors with morphologic and immunohistochemical characteristics of EMC may represent a molecular genetic entity separate from EMCs arising in other locations. PLAG1 gene activation appears to be involved in the development of these neoplasms.

Cancer cells expressing PD-1 ligands (PD-L1/PD-L2) inhibit immune-modulatory T-cell activation facilitating disease progression. Preliminary clinical trials exploring interruption of PD-1/PD-L1 signaling showed benefit in several cancer types. We analyzed the distribution of PD-1–positive tumor-infiltrating lymphocytes (TIL) and cancer cells' expression of PD-L1 in a molecularly profiled cohort of 437 malignancies (380 carcinomas, 33 sarcomas, and 24 melanomas). We showed that the presence of PD-1+ TILs significantly varied among cancer types (from 0% in extraskeletal myxoid chondrosarcomas to 93% in ovarian cancer), and was generally associated with the increased number of mutations in tumor cells (P = 0.029). Cancer cell expression of PD-L1 varied from absent (in Merkel cell carcinomas) to 100% (in chondro- and liposarcomas), but showed the inverse association with the number of detected mutations (P = 0.004). Both PD-1 and PD-L1 expression were significantly higher in triple-negative breast cancers (TNBC) than in non-TNBC (P < 0.001 and 0.017, respectively). Similarly, MSI-H colon cancers had higher PD-1 and PD-L1 expression than the microsatellite stable tumors (P = 0.002 and 0.02, respectively). TP53-mutated breast cancers had significantly higher PD-1 positivity than those harboring other driver mutations (e.g., PIK3CA; P = 0.002). In non–small cell lung cancer, PD-1/PD-L1 coexpression was identified in 8 cases (19%), which lacked any other targetable alterations (e.g., EGFR, ALK, or ROS1). Our study demonstrated the utility of exploring the expression of two potentially targetable immune checkpoint proteins (PD-1/PD-L1) in a substantial proportion of solid tumors, including some aggressive subtypes that lack other targeted treatment modalities. Cancer Epidemiol Biomarkers Prev; 23(12); 2965–70. ©2014 AACR.

To the Editor: T-cell factor 1 (TCF-1) protein is a member of the LEF1/TCF family of transcription factors, which are constituents of the Wnt signaling pathway, that plays an important role in embryonal development, adult stem cell maintenance and tumor growth. TCF-1 binds to the DNA through a high mobility group and at least eight protein isoforms. The canonical Wnt operates in two modes, on and off. In the off mode, the Wnt ligand is absent and cytoplasmic complex, composed of axin, adenomatous polyposis coli (APC), Casein kinase 1 (Ck1) and Glycogen Synthase Kinase (Gsk3), stimulates the destruction of β-catenin. Consequently, as β-catenin fails to enter the nucleus and activates T-cell transcription factors, the TCFs instead bind to a group of co-repressor proteins and inhibit transcription of β-catenin dependent genes. In the on mode, Wnt ligand binding with membrane receptor inhibits cytoplasmic destruction complex, which allows translocation of β-catenin into nucleus and binding with amino-terminal end of TCF. As a consequence of this interaction, TCFs become transcriptional activators which bind to Wnt response elements of target genes, and increased activation of this signaling pathway may lead to development of cancer. Previous studies have indicated that the Wnt signaling pathway plays an important role in balancing cell proliferation, apoptosis and differentiation during the trophoblast differentiation. The β-catenin-induced TCF4 protein activates the GCM1/syncictin signaling pathway, which leads to fusion of the human choriocarcinoma cells. Expression of Wnt antagonist Wnt5a, found normally in human cytotrophoblast cells, is completely absent from choriocarcinoma cell line JAR, whereas exogenous recombinant Wnt5a applied to choriocarcinoma cells decreases their proliferation and induces apoptosis, suggesting it acts as a tumor suppressor for placental choriocarcinomas. Hypermethylation of the APC suppressor gene has been associated with all human choriocarcinoma cell lines. Dickkopf-related protein 1 (DKK1), another Wnt signaling pathway antagonist, is abundantly present in human cytotrophoblast cells, but it is absent from choriocarcinoma cells lines JAR and JEG3. When given exogenously to cells, it reduces proliferation of both choriocarcinoma cells lines and induces apoptosis of JAR cells. This tumor suppressor effect is believed to be accomplished through c-Jun N-terminal kinase, without directly involving Wnt signaling pathway. TCF-1 stimulates the growth of human malignant hematopoietic cells independently of β-catenin binding, i.e. its activity is accomplished through binding to ATF2 [activating transcription factor 2] family of proteins. It is thought that this alternative mechanism is responsible for development of some hematopoietic tumors. Increased expression of TCF-1 protein has been reported recently in various human malignancies including renal cell carcinoma and hepatocellular tumors (adenoma and carcinoma). The status of TCF-1 protein has not been analyzed in germ cell tumors of gonads including the tumors with trophoblastic differentiation. In the present report using immunohistochemistry (Clone: sc-8589, Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:50) we explored TCF-1 expression in a subset of germ cell tumors with trophoblastic differentiation and compared it with adjacent normal/benign reproductive tissues (negative control) and tumor-infiltrating T-lymphocytes (positive control). TCF-1 protein was scored for the intensity and percentage in the choriocarcinoma cells. Tumor-infiltrating lymphocytes retained a strong, nuclear expression of TCF-1 protein (Fig. 1c), whereas normal and benign tissues (testis, ovary, endometrium and myometrium) were negative (Fig. 1a,c). Pure ovarian choriocarcinomas (n = 3) exhibited predominantly strong TCF-1 expression (score 3+) in the range 20–70% of the tumors cells (Fig. 1b). In mixed germ cell tumors of the testis (n = 5), choriocarcinoma cells also showed intense TCF-1 protein expression (Fig. 1d), whereas other germ cell compartments (seminoma, embryonal carcinoma, yolk sac tumor and teratoma) exhibited only weak (score 1+) and focal TCF-1 positivity (<10% of the cells) (Fig. 1c). Our preliminary data indicate that TCF-1 protein may be actively involved in pathogenesis of a subset of germ cell tumors with trophoblastic differentiation. Further functional and clinical studies should validate the relevance of TCF-1 protein in these tumors.

311 Background: Infiltrating urothelial carcinoma (UC) is the most common variant of urinary bladder cancer. The prognosis for muscle infiltrating or metastatic UC of the bladder is poor with no major advances made in the last 20 years. We investigated a large cohort of such patients for specific genetic/biomarker alterations and compared them to other, less common urothelial malignancies. Methods: We reviewed 602 cases; 518 cases (86%) were locally advanced or metastatic UCs of the bladder and the remaining 84 cases (14%) were non-bladder UCs. Multiple methodologies for optimal assessment of biomarker expression (Caris Molecular Intelligence, Caris Life Sciences, Phoenix, AZ) were employed: Mutation analysis (Next-generation sequencing, Sanger, pyrosequencing, qPCR, RFLP), in-situ hybridization (fluorescent and chromogenic), immunohistochemistry, and RNA fragment analysis. Results: Bladder UC showed slightly higher rates of HER2/neu gene amplification (12% in bladder vs. 6% non-bladder, p=0.32) and EGFR ...

We report two new cases of cystic fi broepithelioma of Pinkus together with immunohistochemical features and analyze the presence of cystic changes in a series of 16 classical fi broepitheliomas of Pinkus. Our fi ndings show that the formation of cystic spaces is most probably caused by ischemic degeneration of stromal fenestrations, rather than by central tumor cell necrosis. Th is fi nding is supported by lack of CD34 positive blood vessels in edematous and hyalinized stromal fenestrations undergoing transformation into cystic spaces, as opposed to the uninvolved stromal fenestrations. Th erefore, it is probably more accurate to refer to this process as pseudocystic stromal degeneration rather than true cyst formation. Also, two out of 16 classical Pinkus fi broepitheliomas exhibited focal pseudocystic changes in 50 and 10 of the tumor, respectively, demonstrating that this degenerative process can be found, rarely and focally, in classical cases as well.

Metastases to gastrointestinal tract are uncommon. In particular, metastases to the ampulla of Vater are very rare and may represent a significant diagnostic challenge. Metastases from the uterine cervix to the ampulla of Vater are exceedingly rare and only one case has been described in the available literature. We describe here a second case of metastatic squamous cell carcinoma of the cervix to the ampulla of Vater in a 45-year-old woman. Poorly differentiated squamous cell carcinoma presented as an isolated metastasis to the ampulla of Vater, two years after the initial diagnosis. While the squamous cell carcinoma could occur as primary ampullary carcinoma, albeit very rare, it is necessary to exclude the possibility of metastatic cancer.

Apocrine carcinoma of the breast is a rare, special type of breast carcinoma showing distinct morphologic, immunohistochemical and molecular genetic features. Apocrine epithelium has a characteristic steroid receptor profile that is estrogen receptor and progesterone receptor negative and androgen receptor positive. This combination of morphologic and immunohistochemical characteristics is essential for the proper recognition of the apocrine carcinomas. Strictly defined, apocrine carcinomas express either Her-2/neu or EGFR, which along with androgen receptor positivity make patients with the apocrine carcinoma eligible for targeted therapies.

Apocrine differentiation occurs in a variety of breast lesions, both benign and malignant. The molecular classification of breast cancer defined an apocrine molecular subtype of breast cancer. In general, apocrine carcinomas do not present clinical behavior distinct from the observed in ductal carcinomas of no special type. However, these carcinomas are frequently triple negative (ER-/PgR-/HER2-) with no targeted therapy and poor prognosis. Still they are unique in the aspect of the expression of a different nuclear receptor - androgen receptor (AR). In this study we thought to perform a comprehensive genetic/molecular characterization of this type of lesions. For that, a series of 44 apocrine lesions were studied. These comprised 9 apocrine metaplasia, 8 apocrine adenosis, 13 apocrine in situ carcinoma and 18 apocrine invasive carcinoma. Importantly four samples contained different lesions in different stages in close proximity. Immunohistochemistry was performed to evaluate the ER, PgR, HER2 and AR status of these lesions. Genomic DNA was microdissected from FFPE slides and used both to identify chromosomal copy number aberrations using aCGH and to monitor 739 mutations from 46 oncogenes and tumor suppressor genes using the Ion PGM system. The aCGH study allowed the identification of recurrent chromosomal aberrations. These were rare in benign apocrine lesions, mostly comprised of small gains and deletions, and dramatically increased in malignant lesions up to complete loss and gain of full chromosome arms. Also the analysis of the different lesions within the same sample indicates a putative existence of an apocrine tumor progression. Moreover the profiling of mutational status of the different tumor suppressor/oncogene allowed the identification of mutations that may provide new avenues for the development of new targetable therapeutically options and also corroborated the putative apocrine tumor progression. The study of apocrine lesions in different differentiation stages allowed a deeper insight on the molecular complexity of these lesions. Although further studies are required to independently validate this work, this study identifies recurrent genomic alteration that may open targeted treatment opportunities that may be used independently or in combination with anti-androgen drugs. Citation Format: Jose L. Costa, Ana Justino, Madalena Gomes, Cesar Augusto Alvarenga, Rene Gerhard, Semir Vranic, Zoran Gatalica, Jose Carlos Machado, Fernando Schmitt. Comprehensive genetic characterization of apocrine lesions of the breast. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2013. doi:10.1158/1538-7445.AM2013-2013

p53 is one of the most frequently mutated genes in human tumors including head and neck tumors like oral squamous cell carcinoma. It might be responsible for more than 50% of all relapses in patients with surgically treated oral carcinoma and clean margins. The aim of the present study was to explore p53 protein expression in peritumoral tissue and correlate it with relapse of the disease. The study included 25 patients (17 males and 8 females) with oral squamous cell carcinoma in the period August 2006 till August 2008. For immunohistochemical assay, a monoclonal antibody against p53 protein was applied (clone DO-7, DAKO Glostrup, Denmark). Peritumoral expression of p53 was as follows: 10 out of 25 cases (40%) were negative, 2 cases (8%) showed weak, 5 cases (20%) moderate and 8 cases (32%) strong p53 positivity. No significant correlation between peritumoral expression of p53 protein and patient's relapse was found. In contrast, we found a trend toward association between intratumoral p53 expression and patient's relapse (p = 0.07). There was also trend toward higher peritumoral p53 expression in females comparing with p53 expression in males (52.9% of males did not have p53 expression while 87.5% females had mild, moderate or high p53 expression, p = 0.088). Peritumoral expression of p53 protein is frequently seen in oral squamous cell carcinoma and merits further research.

The study was undertaken to investigate EGFR and HER-2/neu expression in a cohort of apocrine carcinomas of the breast with emphasis on the classification of the breast carcinomas with apocrine morphology. In total, 55 breast carcinomas morphologically diagnosed as apocrine were evaluated for steroid receptor expression profile characteristic of normal apocrine epithelium (ER-/PR-/AR+), and for the expression of EGFR and Her-2/neu proteins, and the copy number ratios of the genes EGFR/CEP7 and HER-2/CEP17. Another cohort composed of 72 invasive ductal carcinomas of no-special-type was used to further determine the impact of CEP17 polysomy on the interpretation of HER-2/neu testing. Our study confirms that apocrine carcinomas of the breast are molecularly diverse group of carcinomas. Strictly defined, pure apocrine carcinomas (ER-, PR-, AR+) (38 cases, 69%) are either HER-2 overexpressing breast carcinomas (52%) or triple-negative breast carcinomas (48%). Apocrine-like carcinomas (ER+/-, PR+/-, AR+/-) (17 cases, 31%) belong predominantly to the luminal phenotype (76%). Pure apocrine carcinomas show consistent over-expression of either EGFR or Her-2/neu. EGFR gene amplification was observed in two pure apocrine carcinomas and one apocrine-like carcinoma. CEP7 polysomy (defined as three or more CEP7 signals) was seen in 61% pure apocrine carcinomas and 27% of apocrine-like carcinomas and showed a weak positive correlation with EGFR protein expression. HER-2/neu gene amplification is the primary mechanism of Her-2/neu activation and is found in 52% of all apocrine carcinomas. CEP17 polysomy (defined as three or more CEP17 signals) was observed in 10 pure apocrine carcinomas (32%) and 8 apocrine-like carcinomas (50%). CEP17 polysomy may be seen without HER-2/neu gene amplification. Further exploration on a cohort of invasive ductal carcinomas of no-special-type confirmed that increased CEP17 signals may lead to discordant interpretation of HER-2/neu gene amplification in a significant proportion of the cases, depending on which criterion (ratio versus absolute number) is used for interpretation. However, increased gene dosage (>6 HER-2/neu genes or HER-2/CEP17 ratio>2.2), regardless of the evaluation method, is positively correlated with Her-2/neu protein expression.

Cellular hypoxia is a hallmark of cancer. Hypoxia-inducible factor-1&agr; (HIF-1&agr;) and von Hippel-Lindau protein (pVHL) are the key mediators of cellular response to hypoxia. Little is known about their role in germ cell tumors of the testis. We therefore examined their status in a cohort of germ cell tumors of the testis. Thirty-six primary germ cell tumors of the testis (11 seminomas, 24 mixed germ cell tumors, and 1 case of pure intratubular germ cell neoplasia) were included in the study. HIF-1&agr; and pVHL expression were studied using immunohistochemical (IHC) methods in the tumor and adjacent benign tissue. Selected cases with a low pVHL expression were further tested for genetic alterations using polymerase chain reaction. HIF-1&agr; protein expression was not detectable in adjacent atrophic seminiferous tubules. In contrast, HIF-1&agr; was expressed in one third of the malignancies, but in a low percentage of cells (mean, 3%; range, 0% to 20%). No difference in HIF-1&agr; expression was observed between seminomas and nonseminomas (P=0.71). pVHL was expressed in atrophic tubular epithelium and in the Leydig cells, whereas a substantial loss of pVHL expression was observed in germ cell tumors regardless of the histologic type (mean, 45.6%; range, 0% to 100%). No genetic alterations of the VHL gene were observed in the cases with low pVHL expression. No significant correlation between HIF-1&agr; and pVHL expression was observed (P=0.16). Germ cell tumors of the testis, regardless of the histologic type, are characterized by consistently low HIF-1&agr; protein overexpression and a partial loss of pVHL without underlying VHL gene alterations. Further studies are necessary to clarify the functional importance of such alterations in testicular germ cell tumors.