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Distal-less homeobox-1 (DLX1) is a well-established non-invasive biomarker for prostate cancer (PCa) diagnosis, however, its mechanistic underpinnings in disease pathobiology are not known. Here, we reveal the oncogenic role of DLX1 and show that abrogating its function leads to reduced tumorigenesis and metastases. We observed that ~60% of advanced-stage and metastatic patients display higher DLX1 levels. Moreover, ~96% of TMPRSS2-ERG fusion-positive and ~70% of androgen receptor (AR)-positive patients show elevated DLX1, associated with aggressive disease and poor survival. Mechanistically, ERG coordinates with enhancer-bound AR and FOXA1 to drive transcriptional upregulation of DLX1 in ERG-positive background. However, in ERG-negative context, AR/AR-V7 and FOXA1 suffice to upregulate DLX1. Notably, inhibiting ERG/AR-mediated DLX1 transcription using BET inhibitor (BETi) or/and anti-androgen drugs reduce its expression and downstream oncogenic effects. Conclusively, this study establishes DLX1 as a direct-target of ERG/AR with an oncogenic role and demonstrates the clinical significance of BETi and anti-androgens for DLX1-positive patients., Distal-less homeobox 1 (DLX1) is reported as a prostate cancer (PCa) diagnostic biomarker, but the mechanism for its upregulation in PCa is unclear. Here the authors show that ERG, AR and FOXA1 transcriptionally regulates DLX1 expression in PCa, and the inhibition of this ERG/AR transcriptional circuitry with a BET inhibitor reduces the oncogenic effects of DLX1.

Background Ataxia Telangiectasia Mutated (ATM) serine/threonine protein kinase is a known tumor suppressor, involved in DNA damage repair. It has prognostic and predictive therapeutic implications and is associated with aggressive prostate cancer (PCa). Objective To investigate the prognostic value of ATM protein expression in PCa patients and assessed the combined value of ATM, ERG, and PTEN status. Design, setting, and participants This study consisted of 303 patients with incidental, locally advanced, and castrate-resistant PCa by transurethral resection of the prostate (TURP). Outcome measurements and statistical analysis TURP samples from 303 PCa patients were assessed by immunohistochemistry (IHC for ATM, ERG, and PTEN. Individual and combined marker status were correlated with International Society of Urological Pathology Gleason grade group, overall survival (OS), and PCa-specific mortality (PCSM). Results and limitations Decreased ATM expression (negative/weak intensity) occurred in 164/303 (54.1%) patients, and was associated with shorter OS and higher PCSM (p = 0.015 and p = 0.001, respectively). Negative/weak ATM expression was significantly associated with PCSM with a hazard ratio of 2.09 (95% confidence interval 1.34–3.27, p = 0.001). Assessment of Combined ATM/PTEN expression showed improved prognostic power to predict OS and PCSM, independent of Gleason grade groups. Conclusions Decreased ATM protein expression is associated with poor outcomes in advanced PCa patients. Patients with combined low ATM/PTEN negative expression are at the highest risk for reduced OS and PCSM. Assessing the combined status of ATM/PTEN by IHC in PCa patients may aid in risk stratification relative to OS and PCSM. Moreover, since ATM plays an integral role in DNA damage response pathways, future studies will enhance our understanding of how outcomes of patients with altered ATM and PTEN expression can be improved further with poly-ADP ribose polymerase inhibitors (PARPi), combinations of PARPi and androgen receptor–targeted therapies, as well as platinum-based chemotherapies. Patient summary Lower ATM intensity is associated with increased cancer-specific mortality in prostate cancer patients. Patients with lower ATM and PTEN negative expression showed decreased overall survival and increased cancer mortality compared with controls., Take Home Message In prostate cancer, lower ATM intensity is associated with increased cancer-specific mortality. Combining ATM with PTEN, patients with lower ATM and negative PTEN expression showed lower overall survival and prostate-specific mortality independent of Gleason grade group. Lower ATM/PTEN-negative tumors showed 2.5 times higher risk for cancer mortality compared with controls. Lower ATM/PTEN negative expression showed a decrease in overall survival and an increase in cancer mortality of about 3 and 4 yr, respectively.

Purpose: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine carcinoma that can be divided into two classes: virus-positive (VP) MCC, associated with oncogenic Merkel cell polyomavirus (MCPyV); and virus-negative (VN) MCC, associated with photodamage. Experimental Design: We classified 346 MCC tumors from 300 patients for MCPyV using a combination of IHC, ISH, and qPCR assays. In a subset of tumors, we profiled mutation status and expression of cancer-relevant genes. MCPyV and molecular profiling results were correlated with disease-specific outcomes. Potential prognostic biomarkers were further validated by IHC. Results: A total of 177 tumors were classified as VP-MCC, 151 tumors were VN-MCC, and 17 tumors were indeterminate. MCPyV positivity in primary tumors was associated with longer disease-specific and recurrence-free survival in univariate analysis, and in multivariate analysis incorporating age, sex, immune status, and stage at presentation. Prioritized oncogene or tumor suppressor mutations were frequent in VN-MCC but rare in VP-MCC. TP53 mutation developed with recurrence in one VP-MCC case. Importantly, for the first time we find that VP-MCC and VN-MCC display distinct sets of prognostic molecular biomarkers. For VP-MCC, shorter survival was associated with decreased expression of immune markers including granzyme and IDO1. For VN-MCC, shorter survival correlated with high expression of several genes including UBE2C. Conclusions: MCPyV status is an independent prognostic factor for MCC. Features of the tumor genome, transcriptome, and microenvironment may modify prognosis in a manner specific to viral status. MCPyV status has clinicopathologic significance and allows for identification of additional prognostic subgroups.

Background Serine-arginine protein kinase 1 (SRPK1) has been implicated in prostate cancer (PCa) progression. However, its prognostic value and association with ERG and PTEN expression, two of the most common genetic alterations, have not been explored fully. Objective We assessed the prognostic value of SRPK1 in association with ERG and PTEN in a cohort of patients managed nonsurgically by androgen deprivation therapy (ADT) for advanced disease. Design, setting, and participants The study cohort consisted of men diagnosed with PCa by transurethral resection of the prostate (TURP; n = 480). The patients were divided into three main groups: incidental (patients with Gleason score [GS] ≤7 with no prior ADT), advanced (patients with GS ≥8 with no prior ADT), and castrate-resistant PCa (patients with prior ADT). Outcome measurements and statistical analysis A total of 480 TURP samples were assessed by immunohistochemistry for SRPK1, ERG, and PTEN, and results were correlated with Gleason grade group (GG), overall survival (OS), and PCa-specific mortality (PCSM). Results and limitations High SRPK1 expression was noted in 105/455 (23%) available patient cores. Expression of SRPK1 was associated with Gleason grade grouping (p, Take Home Message The expression of serine-arginine protein kinase 1 (SRPK1) is associated with prostate cancer progression, overall survival, and cause-specific mortality. Assessing combined status of SRPK1 and PTEN is of added prognostic significance compared with either marker alone. Patients with high SRPK expression/PTEN loss show the highest prognostic value among all combinations.

Prostate cancer is frequently multifocal. Although there may be morphological variation, the genetic underpinnings of each tumor are not clearly understood. To assess the inter and intra tumor molecular heterogeneity in prostate biopsy samples, we developed a combined immunohistochemistry and RNA in situ hybridization method for the simultaneous evaluation of ERG, SPINK1, ETV1, and ETV4. Screening of 601 biopsy cores from 120 consecutive patients revealed multiple alterations in a mutually exclusive manner in 37% of patients, suggesting multifocal tumors with considerable genetic differences. Furthermore, the incidence of molecular heterogeneity was higher in African Americans patients compared to Caucasian American patients. About 47% of the biopsy cores with discontinuous tumor foci showed clonal differences with distinct molecular aberrations. ERG positivity occurred predominantly in low Gleason grade cancer, whereas ETV4 expression was observed mostly in high Gleason grade cancer. Further studies revealed correlation between the incidence of molecular markers and clinical and pathologic findings, suggesting potential implications for diagnostic pathology practice, such as defining dominant tumor nodules and discriminating juxtaposed but molecularly different tumors of different grade patterns.

Microcystic adnexal carcinoma is a locally aggressive sweat gland carcinoma characterized by its infiltrative growth and histopathologic overlap with benign adnexal tumors, often posing challenges to both diagnosis and management. Understanding the molecular underpinnings of microcystic adnexal carcinoma may allow for more accurate diagnosis and identify potential targetable oncogenic drivers. We characterized 18 microcystic adnexal carcinomas by targeted, multiplexed PCR-based DNA next-generation sequencing of the coding sequence of over 400 cancer-relevant genes. The majority of cases had relatively few (

To investigate the incidence and prognostication of ERG, PTEN and SPINK1 protein expressions in prostate cancer cohort of Middle Eastern descent in comparison to published data from Western population. Immunohistochemistry for ERG, PTEN and SPINK1 was performed in a cohort of localized PCA (n = 340). The data were correlated to pathological and clinical outcomes and compared to Western populations. ERG expression and PTEN loss were noted in 123/288 (42.7%) and 91/297 (30.6%) of patients, respectively. SPINK1 expression was assessed in a subset of cases, noted in 6/150 (4%) of patients. Only ERG expression was associated with grade groups, being more common in the lower grade groups (1–3 vs 4–5; p = 0.04). In contrast to the Western population, PTEN loss foci were more likely to be ERG negative, observed in 81% of tumor foci and patients with PTEN neg/ERG pos were more likely to exhibit biochemical recurrence (OR 2.831; 95% CI 1.10–726, p = 0.03). This association remained significant in multivariate analysis (OR 2.68; 95% CI 0.98–7.33, p = 0.05), after adjusting for GG, path stage and surgical margin. This study documents significant differences in key molecular events in PCA in Middle Eastern population compared to Western populations that could explain differences in PCA incidence, progression and prognostication. ERG, PTEN and SPINK1 genomic alteration occur less frequently and the enrichment of ERG for PTEN loss is not observed. Additionally, patients with combined PTEN loss/ERG positive are at highest risk for BCR vs North American Caucasian population where PTEN loss alone seems to be associated with the worst clinical outcome. The data presented here further support differences in clonal evolution between Middle Eastern and Western population in relation to PCA and add further insight to understanding PCA molecular pathways.

Cranial fasciitis is an uncommon benign fibroblastic tumor, generally histologically identical to nodular fasciitis. It develops almost exclusively in children. Cranial fasciitis manifests clinically as a painless rapidly growing solitary nodule in the head and neck area, frequently eroding the underlying bone. Thus, this entity is often confused with aggressive lesions such as sarcomas, both clinically and radiologically. Histopathologic examination is essential to differentiate between cranial fasciitis and fibrohistiocytic or even sarcomatous lesions observed in children. In this article, we present a case of cranial fasciitis with intracranial extension in a 2-year-old boy. Although USP6 rearrangement has recently been recognized as a recurring alteration in nodular fasciitis, we present a novel COL1A1-CAMTA1 fusion in this lesion.

Our knowledge of prostate cancer (PCa) genomics mainly reflects European (EUR) and Asian (ASN) populations. Our understanding of the influence of Middle Eastern (ME) and African (AFR) ancestry on the mutational profiles of prostate cancer is limited. To characterize genomic differences between ME, EUR, ASN, and AFR ancestry, fluorescent in situ hybridization (FISH) studies for NKX3-1 deletion and MYC amplification were carried out on 42 tumors arising in individuals of ME ancestry. These were supplemented by analysis of genome-wide copy number profiles of 401 tumors of all ancestries. FISH results of NKX3-1 and MYC were assessed in the ME cohort and compared to other ancestries. Gene level copy number aberrations (CNAs) for each sample were statistically compared between ancestry groups. NKX3-1 deletions by FISH were observed in 17/42 (17.5%) prostate tumors arising in men of ME ancestry, while MYC amplifications were only observed in 1/42 (2.3%). Using CNAs called from arrays, the incidence of NKX3-1 deletions was significantly lower in ME vs. other ancestries (20% vs. 52%, p = 2.3 × 10−3). Across the genome, tumors arising in men of ME ancestry had fewer CNAs than those in men of other ancestries (p = 0.014). Additionally, the somatic amplification of 21 specific genes was more frequent in tumors arising in men of ME vs. EUR ancestry (two-sided proportion test, Q &lt, 0.05). Those included amplifications in the glutathione S-transferase family on chromosome 1 (GSTM1, GSTM2, GSTM5) and the IQ motif-containing family on chromosome 3 (IQCF1, IQCF2, IQCF13, IQCF4, IQCF5, IQCF6). Larger studies investigating ME populations are warranted to confirm these observations.

Pigmented epithelioid melanocytoma (PEM) represents a group of rare, heavily pigmented melanocytic tumors encompassing lesions previously designated as "animal-type melanomas" and "epithelioid blue nevi." Despite the association of multiple such tumors in the setting of Carney complex, most cases of PEM occur spontaneously as solitary neoplasms in otherwise healthy patients. PEM may arise in both children and adults, and has a known propensity to spread to the regional lymph nodes. Despite this latter finding, recurrence at the biopsy site or spread beyond the lymph node basin is exceptionally uncommon. Although the molecular basis for PEM continues to be characterized, findings to date suggest that this category of melanocytic neoplasia has genetic alterations distinct from those seen in common nevi, dysplastic nevi, Spitz nevi, and melanoma. Herein, we present an in-depth clinical, histopathologic, and molecular analysis of a case of PEM occurring on the scalp of a young African American girl found to have a novel NTRK3-SCAPER gene fusion.

Trefoil Factor 3 (TFF3) has been implicated in Prostate Cancer (PCa) progression. However, its prognostic value and association with other biomarkers have not been fully explored. We assessed the combined value of TFF3 and PTEN in two cohorts: one is managed surgically for localized PCa and the second is managed non-surgically by androgen deprivation therapy for advanced disease. 228 radical prostatectomies (RP) and 318 transurethral resection of prostate (TURP) samples were assessed by immunohistochemistry (IHC) for TFF3 and by IHC and fluorescent in situ hybridization (FISH) for PTEN. Results of biomarkers expression were correlated with various pathological and clinical outcome parameters including biochemical recurrence (BCR) in the RP cohort and cancer-specific mortality (PCSM) and overall survival (OS) in the TURP cohort. TFF3 expression was detected in 131/226 (57.9%) RP samples and 148/318 (46.5%) of TURP cases. In general, TFF3 positivity was less frequently observed with advanced Gleason Groups. TFF3 expression was also assessed in relation to PTEN expression. Only 15–16% of TFF3-expressed cases were present in association with complete loss of PTEN expression in the TURP and localized cohorts, respectively. Loss of TFF3 expression was not related to BCR after RP, but was prognostic in the non-surgical cohort and associated with decrease OS and PCSM (HR 2.31, CI: 1.67–3.18, p

Aim Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distinguished morphologically from small-cell neuroendocrine carcinomas (SmCC) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node (SLN) metastases of MCC. Cytokeratin 20 (CK20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK20 in MCC compared with SmCC from other sites. Methods and results We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK20-negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK20-negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus-positive tumours and five of 10 (50%) virus-negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK20 expression was also assessed in 61 extracutaneous SmCC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK20 for MCC versus non-cutaneous SmCC was 96.7% and 59.0%, respectively. Conclusions Neurofilament has superior specificity to CK20 in distinguishing MCC from non-cutaneous SmCC. Neurofilament is frequently expressed in CK20- and virus-negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non-cutaneous SmCC.

Nearly half of the prostate cancer (PCa) cases show elevated levels of ERG oncoprotein due toTMPRSS2-ERGgene fusion. Here, we demonstrate ERG mediated upregulation of Distal-less homeobox-1 (DLX1), an established PCa biomarker. Using series of functional assays, we show DLX1 elicits oncogenic properties in prostate epithelial cells, and abrogating its function leads to reduced tumor burden in mouse xenografts. Clinically, ∼60% of the PCa patients exhibit high DLX1 levels, while ∼50% of these cases also harbor elevated ERG associated with aggressive disease and poor survival probability. Mechanistically, we show that ERG gets recruited ontoDLX1promoter and interacts with its enhancer-bound androgen receptor (AR) and FOXA1 to regulateDLX1expression inTMPRSS2-ERGpositive cases. Alternatively, in ERG-negative cases, DLX1 is regulated by AR/AR-V7 and FOXA1. Importantly, BET bromodomain inhibitors disrupt the transcriptional regulators ofDLX1and its associated oncogenic properties, signifying their efficacy in treatment of DLX1-positive PCa patients.

Purpose: Advances in prostate cancer lag behind other tumor types partly due to the paucity of models reflecting key milestones in prostate cancer progression. Therefore, we develop clinically relevant prostate cancer models. Experimental Design: Since 1996, we have generated clinically annotated patient-derived xenografts (PDXs; the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical prostate cancer. Results: We studied two cell line–derived xenografts and the first 80 PDXs derived from 47 human prostate cancer donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (androgen receptor, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the speckle-type POZ protein-like (SPOPL) gene in PDXs derived from seven human donors of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of prostate cancer. SPOPLdeletions are found in 7% of The Cancer Genome Atlas prostate cancers, which suggests that our cohort is a reliable platform for targeted drug development. Conclusions: The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of prostate cancers progressing under novel treatments and enables optimization of prostate cancer–specific, marker-driven therapy.

Aims To evaluate the molecular underpinnings of the rare aggressive prostate cancer variants adenosquamous carcinoma, pleomorphic giant-cell carcinoma, and sarcomatoid carcinoma. Methods and results We retrieved 19 tumours with one or more variant(s), and performed ERG immunohistochemistry, a next-generation sequencing assay targeting recurrent gene fusions, and fluorescence in-situ hybridisation (FISH) for ERG and BRAF. Divergent differentiation included: sarcomatoid carcinoma (n = 10), adenosquamous carcinoma (n = 7), and pleomorphic giant-cell carcinoma (n = 7). Five patients had more than one variant. Four had variants only in metastases. ERG rearrangement was detected in nine (47%, seven via sequencing, showing TMPRSS2-ERG fusions and one GRHL2-ERG fusion, and two via FISH, showing rearrangement via deletion). ERG was immunohistochemically positive in the adenocarcinoma in eight of nine (89%) patients, but was immunohistochemically positive in the variant in only five of nine patients (56%, typically decreased). One patient had a false-positive ERG immunohistochemical result in the sarcomatoid component despite a negative FISH result. Two (11%) harboured BRAF fusions (FAM131A-BRAF and SND1-BRAF). Conclusions ERG fusions are present in these rare prostate cancer variants with a frequency close to that in conventional prostate cancer (9/19, 47%). ERG immunohistochemistry usually detects rearrangement in the adenocarcinoma, but is less sensitive for the variant histology, with weak to negative staining. Adenosquamous and sarcomatoid variants can, particularly, occur together. Molecular assessment may be an additional tool in selected cases to confirm the prostatic origin of unusual tumours. The presence of two BRAF rearrangements suggests that this gene fusion may be enriched in this setting, as RAF kinase fusions have been previously reported in 1-2% of prostate cancers.

Emergence of an aggressive androgen receptor (AR)-independent neuroendocrine prostate cancer (NEPC) after androgen-deprivation therapy (ADT) is well-known. Nevertheless, the majority of advanced-stage prostate cancer patients, including those with SPINK1-positive subtype, are treated with AR-antagonists. Here, we show AR and its corepressor, REST, function as transcriptional-repressors of SPINK1, and AR-antagonists alleviate this repression leading to SPINK1 upregulation. Increased SOX2 expression during NE-transdifferentiation transactivates SPINK1, a critical-player for maintenance of NE-phenotype. SPINK1 elicits epithelial-mesenchymal-transition, stemness and cellular-plasticity. Conversely, pharmacological Casein Kinase-1 inhibition stabilizes REST, which in cooperation with AR causes SPINK1 transcriptional-repression and impedes SPINK1-mediated oncogenesis. Elevated levels of SPINK1 and NEPC markers are observed in the tumors of AR-antagonists treated mice, and in a subset of NEPC patients, implicating a plausible role of SPINK1 in treatment-related NEPC. Collectively, our findings provide an explanation for the paradoxical clinical-outcomes after ADT, possibly due to SPINK1 upregulation, and offers a strategy for adjuvant therapies., SPINK1 upregulation is found in ~10–25% of prostate cancers. Here, they show that whilst SPINK1 is transcriptionally repressed by androgen receptor and its co-repressor REST, it is upregulated after androgen-deprivation therapy, which leads to neuroendocrine differentiation of prostate cancer cells.

(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45&minus, ) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 &plusmn, 1023 CTCs/mL). EpCAM&minus, CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of &ge, 2 CTCs were observed in 96% of patients&mdash, of which, 75% were EpCAM&minus, CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1 and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM&minus, suggesting that these would have been missed using traditional antibody-based capture methods.

1) Background: One of the most common cancer that affects men worldwide and North American men is prostate cancer. Gleason score is a pathological grading system to examine the potential aggressiveness of the disease in the prostate tissue. The advancement in computing and next-generation sequencing technology now allow us to study the genomic profiles of patients in association with their different Gleason score more accurately and effectively. 2) Methods: In this study, we used a novel machine learning method to analyze gene expression of prostate tumors with different Gleason scores, and identify potential genetic biomarkers for each Gleason group. We obtained a publicly-available RNA-Seq dataset of a cohort of 104 prostate cancer patients from the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) repository, and categorized patients based on their Gleason scores to create a hierarchy of disease progression. A hierarchical model with standard classifiers in different Gleason groups, also known as nodes, was developed to identify and predict nodes based on their mRNA or gene expression. In each node, patient samples were analyzed via class imbalance and hybrid feature selection techniques to build the prediction model. The outcome from analysis of each node is a set of genes that can differentiate each Gleason group from the remaining groups. To validate the proposed method, the set of identified genes are used to classify a second dataset of 499 prostate cancer patients collected from cBioportal [1]. 3) Results: The overall accuracy of applying this novel method to the first dataset was 93.3%, and further validated to 87% accuracy using the second dataset. This method also identified genes that were not previously reported as potential biomarkers for specific Gleason groups. In particular, PIAS3 was identified as a potential biomarker for Gleason score 4+3=7, and UBE2V2 for Gleason score 6. 4) Insight: Previous reports show that the genes predicted by this newly proposed method strongly correlate with prostate cancer development and progression. Furthermore, pathway analysis shows that both PIAS3 and UBE2V2 share similar protein interaction pathways, the JAK/STAT signaling process.

Context Glycogen storage diseases are rare. Increased glycogen in the liver results in increased attenuation. Objective Investigate the association and function of a noncoding region associated with liver attenuation but not histologic nonalcoholic fatty liver disease. Design Genetics of Obesity-associated Liver Disease Consortium. Setting Population-based. Main Outcome Computed tomography measured liver attenuation. Results Carriers of rs4841132-A (frequency 2%-19%) do not show increased hepatic steatosis; they have increased liver attenuation indicative of increased glycogen deposition. rs4841132 falls in a noncoding RNA LOC157273 ~190 kb upstream of PPP1R3B. We demonstrate that rs4841132-A increases PPP1R3B through a cis genetic effect. Using CRISPR/Cas9 we engineered a 105-bp deletion including rs4841132-A in human hepatocarcinoma cells that increases PPP1R3B, decreases LOC157273, and increases glycogen perfectly mirroring the human disease. Overexpression of PPP1R3B or knockdown of LOC157273 increased glycogen but did not result in decreased LOC157273 or increased PPP1R3B, respectively, suggesting that the effects may not all occur via affecting RNA levels. Based on electronic health record (EHR) data, rs4841132-A associates with all components of the metabolic syndrome (MetS). However, rs4841132-A associated with decreased low-density lipoprotein (LDL) cholesterol and risk for myocardial infarction (MI). A metabolic signature for rs4841132-A includes increased glycine, lactate, triglycerides, and decreased acetoacetate and beta-hydroxybutyrate. Conclusions These results show that rs4841132-A promotes a hepatic glycogen storage disease by increasing PPP1R3B and decreasing LOC157273. rs4841132-A promotes glycogen accumulation and development of MetS but lowers LDL cholesterol and risk for MI. These results suggest that elevated hepatic glycogen is one cause of MetS that does not invariably promote MI.

Atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and spindle cell lipoma are lipomatous tumors with distinct clinical, molecular, and prognostic features. Although histological and immunophenotypic features can overlap between ALT/WDL and spindle cell lipoma, the oncogenesis and clinical behavior are markedly different. In borderline cases, molecular analysis for MDM2 or CDK4 amplification can aid in distinguishing ALT/WDL from spindle cell lipoma. Although dedifferentiated liposarcoma has been reported to harbor both MDM2 amplification and loss of the RB1 region, we are not aware of a reported RB1 loss in well-differentiated ALT/WDL. In this article, we present a 69-year-old woman with a lipomatous tumor in the gluteal region that histologically, immunohistochemically, and molecularly mimicked spindle cell lipoma (with positive immunohistochemical staining for CD34 and loss of the RB1 gene region), yet harbored amplification of MDM2 and CDK4 confirmed by fluorescence in situ hybridization, supporting classification as ALT/WDL. This case strengthens the argument that in atypical clinical contexts, molecular studies for MDM2/CDK4 should be considered in tumors resembling spindle cell lipoma.

Prostate cancer (PCa) is one of the most frequently diagnosed cancers among men. Many molecular changes have been detailed during PCa progression. The gene encoding the transcription factor ERG shows recurrent rearrangement, resulting in the overexpression of ERG in the majority of prostate cancers. Overexpression of ERG plays a critical role in prostate oncogenesis and development of metastatic disease. Among the downstream effectors of ERG, Frizzled family member FZD4 has been shown to be a target of ERG. Frizzled-8 (FZD8) has been shown to be involved in PCa bone metastasis. In the present study, we show that the expression of FZD8 is directly correlated with ERG expression in PCa. Furthermore, we show that ERG directly targets and activates FZD8 by binding to its promoter. This activation is specific to ETS transcription factor ERG and not ETV1. We propose that ERG overexpression in PCa leads to induction of Frizzled family member FZD8, which is known to activate the Wnt pathway. Taken together, these findings uncover a novel mechanism for PCa metastasis, and indicate that FZD8 may represent a potential therapeutic target for PCa.

Aims Pseudosarcomatous myofibroblastic proliferations of the genitourinary tract have a debatable relationship with inflammatory myofibroblastic tumour (generally lacking ALK rearrangement); however, they share several overlapping features with nodular fasciitis of soft tissue. As rearrangement of the USP6 gene has been recently recognised as a recurrent alteration in soft tissue nodular fasciitis, and several other alternative gene fusions have been recently recognised in inflammatory myofibroblastic tumour, the aim of this study was to investigate whether USP6, ROS1 or ETV6 rearrangements were present in these lesions (12 cases). Methods and results Fluorescence in-situ hybridisation analysis was performed by the use of bacterial artificial chromosome-derived break-apart probes against USP6, ROS1, and ETV6. Two cases with adequate genetic material from recent paraffin tissue blocks were also tested by use of a solid tumour gene fusion detection assay via next-generation sequencing, targeting >50 known genes involved in recurrent fusions. None of the genitourinary pseudosarcomatous myofibroblastic proliferations was found to harbour USP6 (0/12), ROS1 (0/8) or ETV6 (0/7) rearrangements, and no gene fusions were detected in two cases studied by sequencing. Conclusions Despite overlap in histological and immunohistochemical features between pseudosarcomatous myofibroblastic proliferation and nodular fasciitis, these tumours lack the recently recognised USP6 rearrangements that occur in nodular fasciitis, as well as alternative fusions found in ALK-negative inflammatory myofibroblastic tumours. At present, this diagnosis remains based primarily on clinical, histological and immunohistochemical features.

Clear cell renal cell carcinoma is by far the most common form of kidney cancer; however, a number of histologically similar tumors are now recognized and considered distinct entities. The Cancer Genome Atlas published data set was queried (http://cbioportal.org) for clear cell renal cell carcinoma tumors lacking VHL gene mutation and chromosome 3p loss, for which whole-slide images were reviewed. Of the 418 tumors in the published Cancer Genome Atlas clear cell renal cell carcinoma database, 387 had VHL mutation, copy number loss for chromosome 3p, or both (93%). Of the remaining, 27/31 had whole-slide images for review. One had 3p loss based on karyotype but not sequencing, and three demonstrated VHL promoter hypermethylation. Nine could be reclassified as distinct or emerging entities: translocation renal cell carcinoma (n=3), TCEB1 mutant renal cell carcinoma (n=3), papillary renal cell carcinoma (n=2), and clear cell papillary renal cell carcinoma (n=1). Of the remaining, 6 had other clear cell renal cell carcinoma-associated gene alterations (PBRM1, SMARCA4, BAP1, SETD2), leaving 11 specimens, including 2 high-grade or sarcomatoid renal cell carcinomas and 2 with prominent fibromuscular stroma (not TCEB1 mutant). One of the remaining tumors exhibited gain of chromosome 7 but lacked histological features of papillary renal cell carcinoma. Two tumors previously reported to harbor TFE3 gene fusions also exhibited VHL mutation, chromosome 3p loss, and morphology indistinguishable from clear cell renal cell carcinoma, the significance of which is uncertain. In summary, almost all clear cell renal cell carcinomas harbor VHL mutation, 3p copy number loss, or both. Of tumors with clear cell histology that lack these alterations, a subset can now be reclassified as other entities. Further study will determine whether additional entities exist, based on distinct genetic pathways that may have implications for treatment.

Purpose: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCCs. Detection of MCPyV in MCCs has diagnostic utility and prognostic potential. We investigated whether RNAscope, an RNA in situ hybridization (ISH) assay for detection of RNA transcripts in tissues, is useful for MCPyV detection. Experimental Design: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMA) and whole-tissue sections encompassing 87 MCCs from 75 patients, 14 carcinomas of other types, and benign tissues. For comparison, qPCR was performed on 57 cases of MCC from 52 patients. Results: RNA-ISH demonstrated the presence of MCPyV in 37 of 75 cases (49.3%). Notably, tumors from younger patients ( Conclusions: RNA-ISH is comparably sensitive to qPCR for detection of MCPyV and allows for correlation with tissue morphology. This study also reveals a significant association between age, gender, and MCPyV positivity. Clin Cancer Res; 23(18); 5622–30. ©2017 AACR.

Amplification of chromosome 6p has been implicated in aggressive behavior in several cancers, but has not been characterized in renal cell carcinoma (RCC). We identified 9 renal tumors with amplification of chromosome 6p including the TFEB gene, 3 by fluorescence in situ hybridization, and 6 from the Cancer Genome Atlas (TCGA) databases. Patients' ages were 28 to 78 years (median, 61 y). Most tumors were high stage (7/9 pT3a, 2/9 pN1). Using immunohistochemistry, 2/4 were positive for melanocytic markers and cathepsin K. Novel TFEB fusions were reported by TCGA in 2; however, due to a small composition of fusion transcripts compared with full-length transcripts (0.5/174 and 3.3/132 FPKM), we hypothesize that these represent secondary fusions due to amplification. Five specimens (4 TCGA, 1 fluorescence in situ hybridization) had concurrent chromosome 3p copy number loss or VHL deletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHL mutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FH mutation and SMARCB1 mutation. Chromosome 6p amplification including TFEB is a previously unrecognized cytogenetic alteration in RCC, associated with heterogenous tubulopapillary eosinophilic and clear cell histology. The combined constellation of features does not fit cleanly into an existing tumor category (unclassified), most closely resembling papillary or translocation RCC. The tendency for high tumor stage, varied tubulopapillary morphology, and a subset with melanocytic marker positivity suggests the possibility of a unique tumor type, despite some variation in appearance and genetics.

The emergence of liquid biopsy using circulating tumor cells (CTCs) as a resource to identify genomic alterations in cancer presents new opportunities for diagnosis, therapy, and surveillance. We identified EML4-ALK gene rearrangement in expanded CTCs from a patient with ALK-positive lung adenocarcinoma. At the time of radiographic progression, CTCs obtained from the patient revealed a drug resistance mutation (i.e., L1196M on the ALK gene). CTCs were expanded ex vivo and drug sensitivity testing was performed using two ALK inhibitors, crizotinib and ceritinib. The half maximal inhibitory concentration of ceritinib was 1664 nM compared with crizotinib (2268 nM), showing that ceritinib was a more potent ALK inhibitor. We show that it is feasible to detect serial genetic alterations in expanded CTCs and perform in vitro drug screening. These findings support the clinical utility of CTCs not only for diagnosis, but also a potential tool for drug sensitivity testing in distinct subsets of lung cancer and for personalized precision medicine.

Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2 to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2 and ERG genes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERG gene fusions. However, the origins of DNA breaks that underlie gene fusion formation in prostate cancers are far from clear. We demonstrate a role for inflammation-induced oxidative stress in the formation of DNA breaks leading to recurrent TMPRSS2-ERG gene fusions. The transcriptional status and epigenetic features of the target genes influence this effect. Importantly, inflammation-induced de novo genomic rearrangements are blocked by homologous recombination (HR) and promoted by non-homologous end-joining (NHEJ) pathways. In conjunction with the association of proliferative inflammatory atrophy (PIA) with human prostate cancer, our results support a working model in which recurrent genomic rearrangements induced by inflammatory stimuli lead to the development of prostate cancer.

(1) Background:One of the most common cancers that affect North American men and men worldwide is prostate cancer. The Gleason score is a pathological grading system to examine the potential aggressiveness of the disease in the prostate tissue. Advancements in computing and next-generation sequencing technology now allow us to study the genomic profiles of patients in association with their different Gleason scores more accurately and effectively. (2) Methods: In this study, we used a novel machine learning method to analyse gene expression of prostate tumours with different Gleason scores, and identify potential genetic biomarkers for each Gleason group. We obtained a publicly-available RNA-Seq dataset of a cohort of 104 prostate cancer patients from the National Center for Biotechnology Information&rsquo, s (NCBI) Gene Expression Omnibus (GEO) repository, and categorised patients based on their Gleason scores to create a hierarchy of disease progression. A hierarchical model with standard classifiers in different Gleason groups, also known as nodes, was developed to identify and predict nodes based on their mRNA or gene expression. In each node, patient samples were analysed via class imbalance and hybrid feature selection techniques to build the prediction model. The outcome from analysis of each node was a set of genes that could differentiate each Gleason group from the remaining groups. To validate the proposed method, the set of identified genes were used to classify a second dataset of 499 prostate cancer patients collected from cBioportal. (3) Results: The overall accuracy of applying this novel method to the first dataset was 93.3%, the method was further validated to have 87% accuracy using the second dataset. This method also identified genes that were not previously reported as potential biomarkers for specific Gleason groups. In particular, PIAS3 was identified as a potential biomarker for Gleason score 4 + 3 = 7, and UBE2V2 for Gleason score 6. (4) Insight: Previous reports show that the genes predicted by this newly proposed method strongly correlate with prostate cancer development and progression. Furthermore, pathway analysis shows that both PIAS3 and UBE2V2 share similar protein interaction pathways, the JAK/STAT signaling process.

Background Cap-dependent mRNA translation is essential for the translation of key oncogenic proteins at optimal levels and is highly regulated by the rate limiting, initiation step in protein synthesis. Eukaryotic Translation Initiation Factor 4 Gamma 1 (EIF4G1) serves as a scaffold for assembly of cap-dependent translation components in EIF4F complex formation. In the current study, we analyzed the role and expression of EIF4G1 in Pan human cancer panels through various approaches. Methods Immunohistochemistry analysis of EIF4G1 protein was done on high-density multi-organ Human Cancer tissue microarray (TMA) derived from the patient samples from different cancers. We used multiple clinical cohorts to analyze the EIF4G1 mRNA expression across human cancers. TCGA data analysis of EIF4G1 was done through Ualcan and c-bioportal web servers. Western blots for EIF4G1 protein was done for different cell lines in representing the multiple cancer types. Dependency score was calculated through Cancer Dependency Map. Clonogenic, tumorosphere assay and cell invasion assay were done with EIF4G complex inhibitor. Association of EIF4G1 mRNA and Kaplan–Meier survival analysis was done on available TCGA datasets. Results We observed an increase in EIF4G1 protein levels in tissue sections from different cancers as compared to their respective normal tissue. Our analysis of the TCGA data revealed that EIF4G1 mRNA expression is significantly increased in tumor tissues compared to respective control tissues across human cancers and variable expression was observed among different datasets. We discovered that alteration frequency in EIF4G1 is prevalent in human cancers e.g. prostate cancer (~ 25%), ovarian cancer (~ 15%), Head and Neck cancer (~ 13%) and cervical cancer (~ 12.5%). EIF4G1 mRNA and protein levels were high across cancer cell lines from multiple organs. Our analysis of DepMap datasets utilizing depletion assays revealed that EIF4G1 is critical for cancer cell survival. Treatment with EIF4G complex inhibitor impaired clonogenic, tumorosphere formation potential and inhibited cell invasion. Moreover, higher EIF4G1 mRNA level was associated with a lower median survival of patients in multiple tumor types. Conclusions These studies show that EIF4G1 is amplified/over-expressed in multiple cancers and plays an essential role in cancer cell survival, as such EIF4G1 could serve as a novel potential target for therapeutic intervention across many cancers.

Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Disease incidence and survival rates vary based on aggressiveness and treatment options. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. For a minority (∼20%) of patients, T-cell checkpoint inhibitors provide durable benefits following prior platinum therapy. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting heterogeneity in these tumors and pointing to the importance of molecular characterization of MIBCs to provide effective treatment. We have performed multi-omic profiling of the kinome to identify therapeutic targets that are overexpressed in a subset of BLCAs. Our analyses revealed amplification and overexpression of P21 (RAC1) activated kinase 4 (PAK4) in a subset of BLCAs. For these tumors, multiplex kinase assay profiling identified corresponding PAK4 target substrates. By performing experiments using cultured bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, our studies showed that a PAK4 inhibitor was effective in curtailing growth of BLCA cells. Transcriptomic analyses identified elevated expression of another kinase, Protein Tyrosine Kinase 6 (PTK6), upon treatment with a PAK4 inhibitor. Similarly, RNA interference of PAK4 led to elevated expression of PTK6. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared to either drug alone. Thus, PAK4 may be therapeutically actionable for a subset of MIBC patients with amplified and/or overexpressed PAK4 in their tumors. Our results also indicate that combined inhibition of PAK4 and PTK6 may overcome resistance to PAK4. These observations warrant clinical investigations with selected BLCA patients.

Expression profiles of erythroblast transformation-specific (ETS)-related gene fusions and serine protease inhibitor Kazal-type 1 (SPINK1) in early onset prostate cancer have not been thoroughly explored.We retrieved 151 radical prostatectomy specimens from young men with prostate cancer (55 years) and characterized the expression of ETS-related gene (ERG), SPINK1, ETS Variant 1 (ETV1), and ETV4 by dual immunohistochemistry and dual RNA in situ hybridization. Age, race, family history, preoperative prostate-specific antigen, biochemical recurrence, and pathological variables using whole-mount radical prostatectomy tissue were collected.A total of 313 tumor nodules from 151 men including 68 (45%) Caucasians and 61 (40%) African Americans were included in the analysis. Positive family history of prostate cancer was seen in 65 (43%) patients. Preoperative prostate-specific antigen ranged from 0.3 to 52.7 ng/mL (mean = 7.04). The follow-up period ranged from 1 to 123.7 months (mean = 30.3). Biochemical recurrence was encountered in 8 of 151 (5%). ERG overexpression was observed in 85 of 151 (56%) cases, followed by SPINK1 in 61 of 151 (40%), ETV1 in 9 of 149 (6%), and ETV4 in 4 of 141 (3%). There were 25 of 151 (17%) cases showing both ERG and SPINK1 overexpression within different regions of either the same tumor focus or different foci. Higher frequency of ERG overexpression was seen in younger patients (≤45 years old; 76% vs 49%, P = .002), Caucasian men (71% vs 41% P = .0007), organ-confined tumors (64% vs 33%, P = .0008), and tumors of Gleason Grade groups 1 and 2 (62% vs 26%, P = .009). SPINK1 overexpression was more in African American men (68% vs 26%, P = .00008), in tumors with high tumor volume (20%) and with anterior located tumors. ETV1 and ETV4 demonstrated rare overexpression in these tumors, particularly in the higher-grade tumors.This study expands the knowledge of the clonal evolution of multifocal cancer in young patients and support differences in relation to racial background and genetics of prostate cancer.

Expression profiles of ETS related genes and SPINK1 in early onset prostate cancer have not been thoroughly explored. We retrieved 151 radical prostatectomy specimens from young men with prostate cancer (ETV1andETV4by dual immunohistochemistry and dual RNAin-situhybridization. Age, race, family history, preoperative prostate-specific antigen, biochemical recurrence and pathological variables using whole mount radical prostatectomy tissue were collected. 313 tumor nodules from 151 men including 68 (45%) Caucasians and 61 (40%) African Americans. Positive family history of prostate cancer was observed in 65 (43%) patients. Preoperative prostate-specific antigen ranged from 0.3 to 52.7 ng/ml (mean 7.04). Follow-up period ranged from 1 to 123.7 months (Mean 30.3). Biochemical recurrence was encountered in 8/151 (5%). ERG overexpression was observed in 85/151 (56%) cases, followed by SPINK1 in 61/151 (40%),ETV1in 9/149 (6%), andETV4in 4/141 (3%). There were 25/151 (17%) cases showing both ERG and SPINK1 overexpression within different regions of either the same tumor focus or different foci. Higher frequency of ERG overexpression was seen in younger patients (≤ 45 years old) (76% vs. 49%,p= 0.002213), Caucasian men (71% vs. 41%p= 0.000707), organ-confined tumors (64% vs. 33%,p= 0.00079), and tumors of Grade Groups 1 and 2 (62% vs. 26%,p= 0.008794). SPINK1 overexpression was more in African American men (68% vs. 26%,p= 0.00008), in tumors with high tumor volume (> 20%) and with anterior located tumors.ETV1andETV4demonstrated rare overexpression in these tumors, particularly in the higher-grade tumors. This study expands the knowledge of the clonal evolution of multifocal cancer in young patients and support differences in relation to racial background and genetics of prostate cancer.

Analysis of next generation transcriptome sequencing data of prostate cancer identified a novel gene fusion formed by the fusion of a protein coding gene (KLK4) with a non-coding pseudogene (KLKP1) and expression of its cognate protein. Screening of 659 prostate cancer TMA showed about 32% of positive cases predominantly expressed in higher Gleason grade tumors. Concomitant expression with ERG but not with SPINK1 and other ETS fusion positive tumors. Fusion gene expression potentially regulated by AR and ERG. Antibody specific to the KLK4-KLKP1 fusion protein was validated by immunohistochemistry and western blot methods. Oncogenic properties were validated by in vitro and in vivo functional studies. Clinical data analysis shows significant association with prostate cancer in young men and overall survival analysis indicate favorable prognosis. Non-invasive detection in urine samples has been confirmed. Taken together, we present a novel biomarker for routine screening of high Gleason grade prostate cancer at diagnosis.SIGNIFICANCEWe discovered and validated a novel prostate cancer (PCa) specific fusion gene involving a protein coding (KLK4) and a pseudogene (KLKP1) and its cognate protein. The unique feature of this fusion gene is the conversion of the noncoding pseudogene into a protein coding gene and its unique expression only in about 30% of high Gleason grade PCa. Expression of this gene is found to be concomitant in ERG fusion positive prostate cancer but mutually exclusive with SPINK1, ETV1, ETV4 and ETV5 positive tumors. Like other ETS family gene fusions, KLK4-KLKP1 can be detected in the urine samples of patients with prostate cancer enabling non-invasive detection of high Gleason grade prostate cancer. Given the unique feature of this fusion oncogenic potential, high Gleason grade specific expression and noninvasive detection, this novel gene fusion has a potential to be used as a biomarker for early detection of high-grade prostate cancer and a therapeutic target.

INTRODUCTION AND OBJECTIVES:Comedonecrosis has always been assigned Gleason pattern 5. Recent recommendations suggest against the grading of intraductal carcinoma. We hypothesized that prostatic adenocarcinoma with comedonecrosis may be frequently intraductal and therefore prostate cancer grading system may need to be revised.METHODS:We searched our pathology database from 2014-2018 for radical prostatectomy cases with documented comedonecrosis (specific grade patterns accounting for pattern 4 and 5 have been documented over this period). From an initial cohort of 52 cases with reported comedonecrosis, 40 cases were retrieved and had identifiable areas of comedonecrosis on additional whole mount slides prepared for immunohistochemistry. Immunohistochemical staining was performed for prostatic basal cell markers p63 and high molecular weight cytokeratin on whole-mount tumor blocks with the largest areas of comedonecrosis.RESULTS:In 24 cases (60%), the comedonecrosis was located only in intraductal carcinom...

The Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) overexpression represents ~10-25% of the prostate cancer (PCa) cases associated with shorter recurrence-free survival and poor prognosis. Nonetheless, androgen-deprivation therapy (ADT) remains the mainstay treatment for locally advanced and metastatic PCa patients. However, majority of these individuals eventually progress to castration-resistant stage, and a subset of these patients develop ADT-induced neuroendocrine PCa. Despite adverse effects of ADT, possible role of androgen signaling in SPINK1-mediated prostate oncogenesis remains unexplored. Here, we show that androgen receptor (AR) and its corepressor, the RE1-silencing transcription factor (REST), occupySPINK1promoter and functions as a direct transcriptional repressor ofSPINK1, thus blocking AR signaling via ADT relieves its repression, leading to SPINK1 upregulation. In agreement, an inverse association between SPINK1 levels and AR expression was observed across multiple PCa cohorts, and in neuroendocrine differentiated cells. While, lineage reprogramming factor SOX2 in turn binds toSPINK1promoter leading to its transactivation in androgen-deprived conditions with concomitant increase in neuroendocrine markers. Additionally, we also confirm the role ofSPINK1in epithelial-mesenchymal transition, drug resistance, stemness and cellular plasticity. Moreover, we show that Casein Kinase 1 inhibitor stabilizes the REST levels, which in cooperation with AR, conjures transcriptional repression ofSPINK1expression, and impedes SPINK1-mediated oncogenesis. Collectively, our findings provide a plausible explanation to the paradoxical clinical outcomes of ADT, possibly due to increased SPINK1 levels. This study highlights the need to take a well-informed decision prior to ADT and develop alternative therapeutic strategies for castrate-resistant PCa patients.

Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by androgen receptor (AR). First-line CRPC treatments typically target AR signaling, but are rapidly bypassed, resulting in only a modest survival benefit with antiandrogens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional coactivator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at superenhancer sites, and is essential for AR-mediated transcription. In addition, a CDK7-specific inhibitor, THZ1, blunts AR-dependent neoplastic growth by blocking AR/MED1 corecruitment genome-wide, as well as reverses the hyperphosphorylated MED1-associated enzalutamide-resistant phenotype. In vivo, THZ1 induces tumor regression of AR-amplified human CRPC in a xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC. Significance: Potent inhibition of AR signaling is critical to treat CRPC. This study uncovers a driver role for CDK7 in regulating AR-mediated transcription through phosphorylation of MED1, thus revealing a therapeutically targetable potential vulnerability in AR-addicted CRPC. See related commentary by Russo et al., p. 1490. This article is highlighted in the In This Issue feature, p. 1469

Morpheaform basal cell carcinoma (BCC) is a variant of BCC characterized by narrow strands and nests of basaloid cells with dense sclerotic stroma. The histologic extent often exceeds the clinical impression, leading to high recurrence rates after standard excision. The authors encountered a case with single-cell invasion distant from the main tumor. To date a systematic review of single-cell infiltration in morpheaform BCC has yet to be performed.Ten morpheaform BCCs, 10 nonmorpheaform aggressive BCCs, 5 desmoplastic trichoepitheliomas, and 2 microcystic adnexal carcinomas were identified by database search and confirmed on hematoxylin and eosin. Cases were evaluated by hematoxylin and eosin, immunohistochemical staining for p63, and (in a subset) broad-spectrum cytokeratin. Single-cell pattern was defined as individual cells, 2-cell clusters, or single-file invasion.Three types of single-cell pattern were identified: intratumoral (single cells within the main tumor mass), peripheral, and distant. Single cells were typically a minor component relative to larger tumor nodules and strands. Eight of the 10 cases of morpheaform BCC demonstrated areas of single-cell pattern: 3 intratumoral, 3 peripheral, and 2 with distant spread (0.75 and 1.0 mm from the main tumor). Eight of the 10 aggressive BCC demonstrated a peripheral single-cell pattern. Rare intratumoral single cells were identified in 3/5 desmoplastic trichoepitheliomas and 1/2 microcystic adnexal carcinomas.Single-cell pattern is frequently a component of morpheaform BCC. Tumor cells at a significant distance from the main component were unique to morpheaform BCC. Thus, when evaluating margins for morpheaform BCC, increased caution is recommended, and immunohistochemical stains for p63 or cytokeratins may be helpful.

Background Targeted sequencing of patients with epidemiologically low-risk (ELR) head and neck squamous cell carcinoma (HNSCC) could help identify novel drivers or lost suppressors leading to precision medicine protocols and improved survival rates. Methods A patient with ELR-HNSCC was selected for targeted sequencing. We then assessed next generation sequencing cohorts from the Oncomine Powertool Database, which contains pan-cancer data from The Cancer Genome Atlas (TCGA). Results Targeted sequencing revealed fibroblast growth factor receptor-1 (FGFR1) amplifications as a putative driver of the patient's tumor. Patients with HNSCC from TCGA data demonstrated fibroblast growth factor (FGF) family mutations, rearrangements, or amplifications in over 35% of HNSCC cases, with a statistically significant higher frequency in African American populations. FGF alterations were unique from activating phosphatidylinositol 3-kinase (PIK3CA) mutations. Conclusion Together, these data suggest that FGF signaling may be critical for a subset of patients with HNSCC independent of other known pathways and provides rationale for leveraging patients with ELR-HNSCC to define molecular subsets of high-risk HNSCC. © 2016 Wiley Periodicals, Inc. Head Neck, 2016

Objective: Molecular subtypes have been found to be associated with prognosis and treatment response for prostate cancer (PCa) patients and have the potential to aid personalized treatment planning. We aim to stratify molecular subtypes by lesion characterization from multiparametric magnetic resonance images (mp-MRI) using convolutional neural networks (CNN) and knowledge transferred from lesion malignancy classification task. Methods: We identified 23 PCa patients with available mpMRI and molecular subtype information. Each patient may harbor multi-focal lesions of different molecular subtypes. Automated antibody based dual-color immunohistochemistry assays were developed for the simultaneous assessment of ERG-PTEN and ERG-SPINK1 status in PCa on the whole mount microscopic sections. In 7 patients 11 intraprostatic lesions (ILs) were identified as ERG+, 3 patients 3 ILs as SPINK1+, and 10 patients 11 ILs as triple negative (ERG-, SPINK1- and ETS-). We fed a CNN using intratumoral region of interest defined in a slice-by-slice manner from T2WI, ADC and DWIb50. The feature maps from the third convolutional layer were flattened into 7,744 dimensional vectors to represent each IL slice. To compensate for the small sample size, we utilized transfer learning, from the task of tumor malignancy stratification. The CNN model was pre-trained on mpMRI with 320 ILs from 201 patients for malignancy stratification using a different cohort we published previously, which was later fined tuned on this cohort on malignancy classification for domain adaptation purpose. Results: The clustering accuracy (Top 3 estimates) using cosine similarity metrics was shown in Table 1 for each molecular subtype category respectively. The preliminary results supported our hypothesis that task of lesion malignancy and molecular sub-types stratification were correlated in the imaging features derived from mpMRI. Table 1:Clustering accuracy for each molecular category. Each ILs input had 3 consecutive slices in a sequence, know as 2.5D, to incorporate lesion growth pattern.Accuracy (# of 2.5D image slices)Top 1 predictionsTop 2 predictionsTop 3 predictionsERG+ (21)0.670.900.95SPINK1+ (4)0.50.750.75Negative (17)0.650.760.76 Conclusions: This work showed the potential to classify the molecular subtypes of PCa from mp-MRI. The small sample size problem was tackled using transfer learning. Acknowledgement: The work was supported by a Research Scholar Grant: RSG-15-137-01-CCE from the American Cancer Society. Citation Format: Weiwei Zong, Eric N. Carver, Aharon Feldman, Nallasivam Palanisamy, Ning Wen. Molecular subtype stratification for prostate cancer from mpMRI and histopathology images using convolutional neural networks and transfer learning [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5302.

Prostate cancer is a heterogeneous disease with unique molecular aberrations present in patient sub-groups. Distinct prostate cancer molecular changes have been shown to associate with specific clinical outcomes, suggesting the potential of molecular markers as diagnostic and prognostic biomarkers for prostate cancer. Recurrent ETS family gene fusions, BRAF and SPINK1 overexpression account for about 50-60% of the prostate cancer cases. Genetic aberrations in the remaining 40-50% of the cases is not known. Our attempt to identify new molecular markers in prostate cancer led to the identification of NAALADL2 gene shown to be associated with aggressive prostate cancer. In this study, we carried out a comprehensive analysis of genomic changes of NAALADL2 in prostate and other solid cancers by analyzing copy number changes and gene expression using TCGA next generation RNA sequencing data. We observed recurrent amplification, rearrangement, deletion, mutations and over-expression of NAALADL2 in prostate cancer cases. Notably, we observed mutually exclusive aberrations in NAALADL2 when compared to other cases with known prostate cancer aberrations including ETS gene fusions, indicating NAALADL2 as a distinct molecular sub-set of prostate cancer. Independent validation by fluorescent in situ hybridization (FISH) analysis using break apart probe for NAALADL2 on 874 prostate cancer revealed recurrent amplification and rearrangements in about 8% (71/874) [PN1] of the cases with higher prevalence in Caucasian American than African American cases. Based on these results, we explored prostate cancer TGCA gene fusion database and identified additional cases with gene fusions involving NAALADL2. We selected one of the gene fusions identified in the TCGA database involving NAALADL2-PIK3CA and conducted in vitro functional characterization studies and showed its oncogenic properties. Gene expression microarray analysis of RWPE1 cells transfected with NAALADL2-PIK3CA showed dysregulation of genes involved in cancer related pathways, further suggesting a role for NAALADL2-PIK3CA in prostate cancer development. Based on these studies, we explored the incidence of NAALADL2 gene fusion in other solid cancers including lung squamous cell cancer (LUSC), ovarian cancer, head and neck cancer, cervical cancer and breast cancer shown to have recurrent gene fusions identified in the TCGA gene fusion database. Notably, recurrent rearrangements and amplification are seen in a large subset (45%) of LUSC patients, but not in lung adenocarcinoma, suggesting that NAALADL2 could be developed as a novel biomarker in LUSC. Further validation studies using FISH in our independent cohort of LUSC and breast cancer patients including 79 patients from Ghana confirmed recurrent rearrangements and amplification in a subset of cases. In conclusion, similar to ERG, BRAF and FGFR genes, we show recurrent gene fusions of NAALADL2 across multiple solid cancer with potential applications as a pan cancer molecular marker for cancer diagnosis and a potential target for drug development. Citation Format: Pavithra D. Arachchige, Shannon Carskadon, James Hu, Justin Fernando, Darshan S. Chandrashekar, Nilesh S. Gupta, Sean R. Williamson, Dhananjay A. Chitale, Craig G. Rogers, James O. Peabody, Mani Menon, Tarek A. Bismar, Evelyn Jiagge, Sooryanarayana Varambally, Nallasivam Palanisamy. Recurrent rearrangements of NAALADL2 in prostate, breast, cervical, head and neck and lung squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2012.

Prostate cancer (PCa) is a heterogeneous disease and the diverse clinical outcomes may be associated with hitherto unidentified tumor molecular heterogeneity. In the era of precision medicine, new approaches to identify distinct molecular subsets of tumors may lead to the development of targeted treatment options. Given the disparity in the disease progression between African American (AA) and Caucasian American (CA) patients, understanding the existence of distinct molecular subtypes between the two racial groups may facilitate effective cancer management. Therefore, we carried out a comprehensive molecular analysis using prostate cancer-specific molecular markers, including ERG, ETV1, ETV4, ETV4, ETV5 and SPINK1, to study tumor molecular heterogeneity in a large cohort of AA and CA PCa patients. A total of 1,117 PCa cases comprising 575 (52%) CA and 453 (41%) AA patients were included in this study. Dual IHC for ERG/SPINK1 and RNA ISH for ETV1, ETV4 and ETV5 were carried out on whole mount prostatectomy specimens. Incidence of markers in each tumor foci, difference between the two racial groups, and the association of the markers with the clinical and pathologic findings in each racial group were analyzed. Independent clonal origin of tumor foci in patients with multifocal disease were observed with the presence of dual and triple marker positivity in dominant and/or secondary tumor foci. ERG was more frequently overexpressed among CA patients (P=0.0000) while SPINK1 was more prevalent among AA patients (P=0.0000). The incidence of dual marker positive cases for ERG+/SPINK1+ and SPINK1+/ETV1+ were more prevalent among AA patients than CA patients (P=0.0124 and P=0.0417, respectively). In both CA and AA patients, ERG overexpression is associated with young men age lower than 50 and a positive perineural invasion,(CA; P=0.0037 and P=0.0199, respectively; AA; P= 0.0338 and P=0.0309, respectively). Among CA patients, ERG overexpression also associated with a lower Gleason grade group (group 1 and 2, P=0.0483). SPINK1 overexpression associated with grade group lower than 5 and a negative family history among AA patients (P=0.0003 and P=0.0063). In CA patients, SPINK1 overexpression associated with grade group higher than 1, pT3 stage and tumor volume larger than 10% (P= 0.0036, P=0.0426 and P=0.0377, respectively). ETV1 overexpression is associated with positive family history (P=0.0043) and ETV4 overexpression associated with a tumor volume of 1-10% (P=0.0385) in CA patients. In AA patients, ETV4 expression associated with an age higher than 70 (P=0.0212). ERG/SPINK1 overexpression associated with grade group 2, tumor volume of 10-20%, and an age lower than 70 among CA patients (P=0.0013, P=0.0316, and P=0.0191, respectively). AA patients, on the other hand, showed an association between ERG/SPINK1 and age group lower than 40 (P=0.0092). We will discuss the details on the identification of new molecular subsets of PCa with clonal molecular heterogeneity in racial disparity perspective. Citation Format: Shannon Carskadon, Mireya Diaz-Insua, Nilesh Gupta, Sean Williamson, Craig Rogers, James Peabody, Mani Menon, Nallasivam Palanisamy. Comprehensive molecular mapping of prostate cancer- Approaching health disparities in molecular tumor heterogeneity perspective [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A113.

Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers, and current methods for isolation of clusters may fall short. Methods: We have applied the inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs and CTC clusters from patients with metastatic non-small cell lung cancer (NSCLC). Results: Using the Labyrinth device, CTCs (PanCK+/DAPI+/CD45-) were isolated from metastatic NSCLC patients (n=25). Heterogeneous CTC populations were detected, including CTCs expressing epithelial (EpCAM), mesenchymal (vimentin), or both markers. CTCs were isolated from 100% of patients (417±1023 CTCs/mL), and CTCs that were EpCAM negative were significantly higher in numbers than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients, of which 75% were negative for EpCAM. Patients with higher number of CTC clusters than single CTCs had worse progression-free survival (PFS) (p=0.05). Recovered CTCs from patients with RET, ROS1, and ALK-rearranged tumors revealed identical genetic aberrations as the primary tumor for each gene using FISH analysis. We have successfully expanded the recovered CTCs from 2 patients and screened for therapeutic targeting. We have found that TPX-0005 might be effective in these patients and would direct them to a clinical trial using this compound. Conclusions: The label-free Labyrinth device demonstrated the capability of collecting recovered CTCs from the device using a continuous processing technique while in a suspension state. This advantage opens the opportunities not only for CTC expansion off-chip, but also for ex vivo drug testing to direct patient-specific therapies. Citation Format: Mina Zeinali, Wei Huang, Maggie Lee, Arthi Nadhan, Anvya Mathur, Casey Hedman, Eric Lin, Ramdane Harouaka, Max S. Wicha, Lili Zhao, Nallasivam Palanisamy, Mathias Hafner, Rishindra Reddy, Gregory P. Kalemkerian, Bryan J. Schneider, Khaled A. Hassan, Nithya Ramnath, Sunitha Nagrath. High-throughput label-free isolation and expansion of circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) patients for personalized treatments [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B45.

BACKGROUND AND OBJECTIVE: We have previously demonstrated a significant correlative relationship between PTEN deletion and ERG rearrangement both in development of clinically localized prostate cancers and metastases. Herein, we evaluate the cooperative role of ERG and PTEN in oncological outcome after radical prostatectomy for clinically localized prostate cancer. METHODS: We evaluated ERG and PTEN status using three previously described cohorts. The first cohort included 235 clinically localized prostate cancer cases represented on tissue microarrays (TMA), evaluated using previously validated FISH assays for ERG and PTEN. The second cohort included 167 cases of clinically localized prostate cancer on TMAs evaluated for PTEN by FISH, and for PTEN and ERG by dual IHC. The third cohort comprised 59 clinically localized prostate cancer cases assessed by array comparative genomic hybridization (aCGH). Kaplan Meir plots and long rank tests were used to assess the association of ERG and PTEN status with biochemical recurrence after radical prostatectomy for clinically localized prostate cancer. RESULTS: Of the 317 cases eligible for analyses with evaluable ERG and PTEN status, 88 (27.8%) patients developed biochemical recurrence over a median follow-up of 5.7 years. Overall, 45% (142/317) of cases demonstrated ERG rearrangement and 20% (62/317) of cases demonstrated PTEN loss. Hemizygous and homozygous deletion of PTEN was seen in 10% (18/175) and 3% (5/175) of ERG negative cases, respectively. In contrast, hemizygous and homozygous deletion of PTEN was seen in 11% (15/142) and 17% (24/123) of ERG positive cases, respectively. PTEN loss (heterozygous or homozygous) was significantly associated with shorter time to biochemical recurrence compared to no PTEN loss (p

PurposeSerine Peptidase Inhibitor, Kazal type-1 (SPINK1) overexpression defines the second most recurrent and aggressive prostate cancer (PCa) subtype. However, the underlying molecular mechanism and pathobiology of SPINK1 in PCa remains largely unknown.Experimental DesignMicroRNA-prediction tools were employed to examine theSPINK1-3’UTR for miRNAs binding. Luciferase reporter assays were performed to confirm theSPINK1-3’UTR binding of shortlisted miR-338-5p/miR-421. Further, miR-338-5p/-421 overexpressing cancer cells (SPINK1-positive) were evaluated for oncogenic properties using cell-based functional assays and mice xenograft model. Global gene expression profiling was performed to unravel the biological pathways altered by miR-338-5p/-421. Immunohistochemistry and RNAin-situhybridization was carried-out on PCa patients’ tissue microarray for SPINK1 andEZH2expression respectively. Chromatin immunoprecipitation assay was performed to examine EZH2 occupancy on the miR-338-5p/-421 regulatory regions. Bisulfite sequencing and methylated DNA-immunoprecipitation was performed on PCa cell lines and patients’ specimens.ResultsWe established a critical role of miRNA-338-5p/-421 in post-transcriptional regulation ofSPINK1. Ectopic expression of miRNA-338-5p/-421 in SPINK1-positive PCa cells abrogate oncogenic properties including cell-cycle progression, stemness and drug resistance, and show reduced tumor burden and distant metastases in mice model. Importantly, we show SPINK1-positive PCa patients exhibit increased EZH2 expression, suggesting its role in miRNA-338-5p/-421 epigenetic silencing. Furthermore, presence of CpG dinucleotide DNA methylation marks on the regulatory regions of miR-338-5p/-421 in SPINK1-positive PCa cells and patients’ specimens confirms epigenetic silencing.ConclusionOur findings revealed that miRNA-338-5p/-421 are epigenetically silenced in SPINK1-positive PCa, while restoring the expression of these miRNAs using epigenetic drugs or synthetic mimics could abrogate SPINK1-mediated oncogenesis.TRANSLATIONAL IMPACTWe establish a regulatory model involving the functional interplay between SPINK1, miRNA-338-5p/miRNA-421 and EZH2, thereby, revealing hitherto unknown mechanism of SPINK1 up-regulation in SPINK1-positive subtype. Our findings provide a strong rationale for the development of potential therapeutic strategies for SPINK1-positive malignancies. We demonstrate that restoring miRNA-338-5p/miRNA-421 expression using epigenetic drugs including DNMTs inhibitors in combination with HDACs or HKMTs inhibitors or miRNA synthetic mimics in SPINK1-positive prostate cancer abrogate SPINK1-mediated oncogenicity. The major findings of this study will not only advance the prostate cancer field, but will also be valuable for treatment and disease management of other SPINK1-positive malignancies.

Metastatic castration-resistant prostate cancer (CRPC) is an aggressive disease with high mortality rate, primarily resulting from the transcriptional addiction driven by Androgen Receptor (AR). First-line CRPC treatments typically target AR-signaling, but are rapidly bypassed, resulting in only a modest survival benefit with the FDA approved anti-androgen therapies such abiraterone or enzalutamide. Therefore, molecular approaches that more effectively block the AR-transcriptional program are urgently needed. Here, we present evidence demonstrating that AR transcriptional signaling is activated through a “phosphoswitch” catalyzed by cyclin-dependent-kinase 7 (CDK7). Specifically, CDK7 phosphorylates (in vitro and in vivo) the transcriptional co-activator MED1, a key subunit of Mediator complex, at Threonine 1457 to promote the formation of the AR-transcriptional complex at enhancers and super-enhancer (SE) sites. Furthermore, knockdown or inhibition of CDK7 with the recently developed covalent inhibitor THZ1 abolished T1457 phosphorylation that led to MED1 decruitment from the chromatin, attenuated AR-signaling and eliminated AR-addicted naïve or enzalutamide refractory prostate cancer cells. Interestingly, the reduced AR transcriptional output and cellular phenotypes associated with CDK7 knockdown or inhibition was reversed by T1457D phosphomimic suggesting MED1 as a major effector substrate of CDK7 transcription kinase. Finally, THZ1 demonstrated tumor regression in CRPC xenograft models in vivo. In summary, our identification of MED1 T1457 as a novel CDK7 substrate, which is essential for driving AR-mediated transcription, makes CDK7 a potential “non-oncogene dependency” in AR addicted advanced prostate cancer. Taken together, we will present data that strongly support the clinical evaluation of CDK7 specific inhibitors as a monotherapy or in combination with second generation anti-androgens in refractory castration-resistant prostate cancer. Conflict of Interest: None (P.S. This work is undergoing final revision for publication in Cancer Discovery) Citation Format: Reyaz Rasool, Ramakrishnan Natesan, qu deng, Shweta Aras, Priti Lal, Jessica Posimo, Nallasivam Palanisamy, Goutham Narla, Robert Den, Matthew Freedman, donita brady, irfan a asangani. CDK7 inhibition suppresses AR addicted Castration-Resistant Prostate Cancer through MED1 inactivation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B016. doi:10.1158/1535-7163.TARG-19-B016

Homeobox genes are the master regulatory transcription factors involved in the embryonic development and their deregulation in homeostatic biological processes can lead to cancer initiation and progression. A member of Distal less homeobox gene family (DLX), namely DLX1 plays pivotal role in the craniofacial, jaw and GABAergic (gamma aminobutyric acid) interneuron development. Higher expression levels of DLX1 has been associated with primary and metastatic prostate tumor. Moreover, DLX1 and HOXC6 are well-established diagnostic biomarkers for the early detection of prostate cancer (PCa). However, the mechanism involved in DLX1 up-regulation and functional significance in metastatic castration-resistant prostate cancer (mCRPC) progression remains unexplored. Here, we show that benign prostate epithelial cells acquire several oncogenic properties upon ectopic overexpression of DLX1. Similarly, CRISPR/Cas9 mediated DLX1 knockout in castrate-resistant 22RV1 cells show reduced proliferation, invasion, foci formation in cell-based assays, and a remarkable reduction (~80%) in tumor burden in mouse xenograft model, confirming its role in tumor progression. Moreover, our global gene expression profiling data established the role of DLX1 in regulating critical cancer hallmarks such as epithelial-to-mesenchymal transition (EMT), apoptosis and cell cycle regulation. We also demonstrate positive association between DLX1 and the most recurrent TMPRSS2-ERG gene fusion in many independent PCa cohorts, wherein, ~96% of the TMPRSS2-ERG fusion positive patients also harbour higher level of DLX1. Our ChIP-qPCR and luciferase-reporter assay data show that ERG occupies the DLX1 promoter region, and orchestrate its transcriptional control. Nevertheless, a subset of ERG-fusion negative patients also exhibits a higher DLX1 expression, where androgen receptor (AR) and AR variant 7 (AR-V7) plays critical role in transcriptional regulation of DLX1 in an ERG-independent manner. Since, Bromodomain and extra-terminal domain inhibitors (BETi) are known to inhibit the transcriptional activity of AR, AR-V7 and ERG, we examined the effect of BETi on DLX1. Interestingly, a significant decrease in the expression of DLX1 in BETi treated PCa cell lines with a concomitant loss of their oncogenic potential was observed. Taken together, our findings reveal that DLX1 serves as an oncogene thereby regulating several oncogenic properties associated with PCa progression. We discovered a novel transcriptional regulatory network involving ERG dependent up-regulation of DLX1 in TMPRSS2-ERG fusion positive PCa, while AR and AR-V7 governed expression in ERG-independent manner in CRPC cells. Conclusively, our findings suggest that BET domain inhibitors can be used as an adjuvant therapy for DLX1-positive subset of metastatic castration resistant prostate cancer patients. Citation Format: Sakshi Goel, Vipul Bhatia, Mahendra Shyamlal Palecha, Shannon Carskadon, Nallasivam Palanisamy, Bushra Ateeq. ERG mediated transcriptional regulation of DLX1 homeobox gene represents a novel mechanism underlying prostate cancer progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B124. doi:10.1158/1535-7163.TARG-19-B124

Androgen deprivation therapy (ADT) remains the clinical paradigm for the management of prostate cancer (PCa) patients. However, most of the cases inevitably become resistant to ADT, leading to a more aggressive, hormone-refractory stage known as castrate-resistant prostate cancer (CRPC). A subset of CRPC patients undergoing ADT develop tumors with low AR-signaling dependence where the luminal prostate cancer cells consequently acquire alternative lineage programs, resulting in the development of neuroendocrine prostate cancer (NEPC). In the past decade, several molecular features have been associated with progression of the adenocarcinoma to NEPC, including loss of RE1-silencing transcription factor (REST), N-MYC proto-oncogene amplification, upregulation of Aurora kinase A, neural transcription factor BRN2, and reprogramming factor SRY (sex determining region Y)-box 2 (SOX2). Nonetheless, the underlying molecular mechanism involved in lineage plasticity in neuroendocrine (NE) transdifferentaition is poorly understood and remains a matter of speculation. Overexpression of Serine Peptidase Inhibitor, Kazal type 1 (SPINK1) represents the second major molecular PCa subtype (~15-20% of the cases) associated with aggressive stage and poor patient outcome. Oncogenic role of SPINK1 in PCa has already been established, and recently SPINK1 produced in the tumor stroma was found to act as a senescence-associated secretory factor and contributes to chemoresistance in a paracrine manner. Here, we show that androgen receptor (AR) along with its corepressor REST functions as a direct transcriptional repressor of SPINK1, and blocking AR signaling using anti-androgens relieve this repression, leading to increase in SPINK1 expression. We also show that the lineage reprogramming factor SOX2 binds to the SPINK1 promoter and positively regulate its expression in androgen deprivation induced NE-transdifferentiated PCa cells. Conversely, silencing SPINK1 in the NE-transdifferentiated PCa cells result in reduced expression of epithelial-mesenchymal transition (EMT) and neuroendocrine markers accompanied with a concomitant decrease in the neurite-like projections. Moreover, castration-resistant mice xenografts treated with anti-androgens show increase in SPINK1 levels as well as EMT and neuroendocrine markers. Likewise, higher SPINK1 expression was observed in NE patient-derived organoids and NEPC clinical specimens, indicating its plausible role in cellular plasticity and NE progression. Since, REST along with AR negatively regulates SPINK1 expression, and Casein kinase 1 elicits ubiquitin-mediated proteasomal degradation of REST. Therefore, we treated SPINK1-positive PCa cells with Casein kinase 1 inhibitor (iCK1), which results in restoration of the REST expression, leading to transcriptional repression of SPINK1 as well as decrease in SPINK1-mediated oncogenesis. Taken together, we highlight the clinical complications associated with high SPINK1 levels and discovered its possible role in maintaining cellular plasticity in prostate cancer cells. Notably, stabilization of REST levels using iCK1 suggests a novel therapeutic strategy for the management of SPINK1-positive subtype and also open new avenues for the treatment modalities for CRPC patients. Citation Format: Nishat Manzar, Ritika Tiwari, Vipul Bhatia, Anjali Yadav, Shannon Carskadon, Nilesh Gupta, Amina Zoubeidi, Matti Poutanen, Himisha Beltran, Nallasivam Palanisamy, Bushra Ateeq. Reprogramming transcription factors SOX2 and REST modulates SPINK1 expression in governing cellular plasticity in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C128. doi:10.1158/1535-7163.TARG-19-C128

To assess the prognostic value of ERG and PTEN protein expression as two of the most common genetic aberration in men with prostate cancer managed non-surgically by androgen deprivation therapy (ADT). 463 tumor samples were assessed by double immunohistochemistry stains for ERG and PTEN and data correlated with clinical pathological features including, Gleason score, patients’ outcome and ADT. ERG expression and PTEN protein loss were present in 28.2% and 38% of total patients respectively. There was a significant interplay between ERG and PTEN expression with 21.8% PTEN negative tumors being ERG positive (p