Epigenetic mechanisms play a crucial role in cellular proliferation, migration and differentiation in both normal and neoplastic development. One of the key signaling pathways whose components are altered through the epigenetic mechanisms is the Wnt signaling pathway. In this review, we briefly discuss the key concepts of epigenetics and focus on the recent advances in the Wnt signaling pathway research and its potential diagnostic and therapeutic implications.

Background Cancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Despite extensive laboratory and imaging efforts, the primary site usually cannot be unequivocally confirmed, and the treatment for the most part remains empirical. Recently, identification of common cancer pathway alterations in diverse cancer lineages has offered an opportunity to provide targeted therapies for patients with CUP, irrespective of the primary site. Patients and Methods 1806 cancers of unknown primary were identified among more than 63,000 cases profiled at Caris Life Sciences. Multiplatform profiling of the tumor samples included immunohistochemistry, gene sequencing and in situ hybridization methods in an effort to identify changes in biomarkers that are predictive of drug responses. Results Biomarkers associated with a potential drug benefit were identified in 96% of cases. Biomarkers identified included those associated with potential benefit in nearly all classes of approved cancer drugs (cytotoxic, hormonal, targeted biological drugs). Additionally, biomarkers associated with a potential lack of benefit were identified in numerous cases, which could further refine the management of patients with CUP. Conclusion Comprehensive biomarker profiling of CUP may provide additional choices in treatment of patients with these difficult to treat malignancies.

Secreted frizzled-related proteins 1 and 3 (SFRP1 and SFRP3) act as Wnt signaling pathway antagonists and play an important role in embryonic development and carcinogenesis. The aim of the present study was to analyze immunohistochemically the distribution of 2 SFRP family proteins, SFRP1 and SFRP3, in an experimental rat model, in normal and intrauterine growth-restricted (IUGR) human placentas, and in a subset of the corresponding human trophoblastic tumors (pure choriocarcinomas and mixed germ cell tumors with choriocarcinoma component). In rats, expression of both SFRP1 and SFRP3 was pronounced in the perimetrium and myometrium, whereas decidual cells showed only occasional positive cytoplasmic staining. The most prominent expression of both proteins was found in blood vessel endothelial cells. Stereological variable of volume density (Vv, mm0) showed statistically higher expression of SFRP1 and SFRP3 in human IUGR placentas than in normal pregnancy placentas (P<0.0001). Compared with adjacent normal/benign tissues, reduced expression of SFRP1 and SFRP3 was observed in human trophoblastic tumors (58.5% and 31.25%, respectively), although none of the examined tumors exhibited complete loss of either protein. Our study indicates that increased expression of both SFRP1 and SFRP3 may contribute to the pathogenesis of IUGR placental dysfunction, whereas the loss of these proteins may be involved in the development of human trophoblastic tumors.

We report two new cases of cystic fibroepithelioma of Pinkus together with immunohistochemical features and analyze the presence of cystic changes in a series of 16 classical fibroepitheliomas of Pinkus. Our findings show that the formation of cystic spaces is most probably caused by ischemic degeneration of stromal fenestrations, rather than by central tumor cell necrosis. This finding 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. Therefore, 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 fibroepitheliomas 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.

ABSTRACT Aim: 5-FU based treatment remains the standard of care for the adjuvant treatment of colorectal carcinoma (CRC), in combination with oxaliplatin and irinotecan. CRC is molecularly heterogenous disease and patients with microsatellite stable (MSS) CRC have been previously shown to benefit from 5-FU based therapies while patients with high microsatellite instability (MSI-H) CRC did not. We investigated the expression thymidylate synthase, the targeted enzyme for 5-FU and topoisomerase I (Topo1), the targeted enzyme for irinotecan, in molecularly defined cohort of CRC. Methods: 106 patients with CRC (86 MSS and 20 MSI-H) were molecularly profiled using multiplatform approach [Caris Life Sciences] including immunohsitochemistry, PCR microsatellite analysis and gene sequencing (NGS) . Results: TS over-expression (over the ≥1+ and ≥10% threshold, and frequently >2+ and >50%) was observed in all 20 MSI-H cases (100%). In contrast, MSS cases overexpressed TS in only 31/86 (36%) of the cases (p Conclusions: Two most common molecular subtypes of colorectal cancer exhibit different expression patterns of TS and Topo1; overexpression of TS may explain the observed lack of benefit of 5-FU based therapy in MSI-H CRC patients. Additional genetic and molecular biomarkers could provide guidance to alternative chemotherapy modalities. Disclosure: Z. Gatalica: Stock options Caris Life Sciences; D. Bryant: Stock options Caris Life Sciences All other authors have declared no conflicts of interest.

ABSTRACT Aim: Malignant phyllodes tumors constitute 0.1% of all breast tumors, which lack effective treatment options. Comprehensive molecular profiling of rare cancers holds the promise of identifying underlying pathogenetic mechanisms with potentially druggable targets. Methods: Seventeen malignant phyllodes tumors (9 primary, 3 recurrent and 5 metastatic samples) were analyzed (Caris Life Sciences, Phoenix, AZ) using gene sequencing (NGS and Sanger), gene copy number analysis (FISH and CISH), RNA expression (RT-qPCR and whole genome microarrays) and protein expression (immunohistochemistry). Results: Malignant phyllodes tumors showed co-expression of genes involved in angiogenesis (vascular endothelial growth factor A and its receptor KDR) in 6 of 9 cases, and additional 3 cases showed overexpression of VEGFA alone. Amplification of EGFR gene was observed in 4 of 9 tested cases, while EGFR protein overexpression (H-score ≥ 20) was observed in 10 of 11 tested cases. TP53 mutations were identified in the majority of cases tested (8/11). One metastatic tumor harbored increased cMET gene copy number. No other oncogene alterations were identified (e.g. HER2, BRAF, KRAS, PIK3CA, cKIT). DNA repair pathways were altered in 9 out of 17 cases (7/17 low ERCC1; 2/17 low BRCA1) indicating potential response to platinum agents. Low expression of TS and RRM1 (9/17 cases) indicates potential response to fluoropyrimidines and gemcitabine. Steroid receptors were uniformly negative in all cases. Conclusions: Comprehensive tumor profiling identified diverse molecular alterations providing insight into potentially beneficial therapies in all malignant phyllodes tumors. These include novel therapies targeting angiogenesis and the EGFR pathway, as well as biomarkers of the conventional chemotherapy in this rare cancer type. Disclosure: Z. Gatalica: Stock options Caris Life Sciences; R. Bender: Stock options Caris Life Sciences. All other authors have declared no conflicts of interest.

ABSTRACT Aim: Angiosarcoma of the breast is a rare, aggressive soft tissue neoplasm occurring as either a primary or secondary malignancy due to previous radiotherapy for breast carcinoma. There is an unmet medical need for targeted therapies for angiosarcoma. Methods: Seventeen patients with angiosarcoma of the breast were identified (Caris Life Sciences) and profiled for biomarkers of drug response using multiplatform methodologies (Tumor DNA sequencing, gene copy number alterations, RNA/microarray and protein/IHC expression). Results: Eight primary, 5 post-radiation and 4 mammary angiosarcomas of unknown etiology were identified in women (age 31-85 years), all exhibiting multiple biomarker alterations. Eight out of 17 cases (47%) harbored overexpression (mRNA and/or protein) of the genes [VEGFR2 (5/17), PDGFRA/PDGFRB (3/17), c-KIT (2/17)] that can be targeted by multikinase inhibitor Sunitinib. Based on the VEGFR2 overexpression, one patient with recurrent, refractory angiosarcoma was treated with Sunitinib resulting in a complete remission and is disease free at 14 months. Also, multiplatform molecular profiling revealed useful predictors of various other treatment modalities including anthracyclines (overexpression of topoisomerase 2a in 69%), topo 1 inhibitors (topoisomerase 1 overexpression in 44%), and fluoropyrimidines (low levels of thymidylate synthase in 29%). One case devoid of the Sunitinib-associated genetic alterations harbored a KRAS mutation indicating a potential benefit of MEK inhibitors. Conclusions: A comprehensive molecular profiling of breast angiosarcomas enables identification of various molecular alterations that can be treated by both targeted and conventional treatment modalities. Disclosure: All authors have declared no conflicts of interest.

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 ...