Clonal evaluation of early onset prostate cancer by expression profiling of ERG, SPINK1, ETV1, and ETV4 on whole-mount radical prostatectomy tissue.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Next-generation sequencing implicates oncogenic roles for p53 and JAK/STAT signaling in microcystic adnexal carcinomas.

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 (<8) prioritized somatic mutations, and lacked an ultraviolet (UV) signature. The most recurrent mutation was TP53 inactivation in four (22%) tumors. Frame-preserving insertions affecting the kinase domain of JAK1 were detected in three (17%) cases, and were nonoverlapping with TP53 mutations. Seven (39%) cases demonstrated copy number gain of at least one oncogene. By immunohistochemistry, p53 expression was significantly higher in microcystic adnexal carcinomas with TP53 mutations compared with those without such mutations and syringomas. Similarly, phospho-STAT3 expression was significantly higher in microcystic adnexal carcinomas harboring JAK1 kinase insertions compared with those with wild-type JAK1 and syringomas. In conclusion, microcystic adnexal carcinomas are molecularly heterogeneous tumors, with inactivated p53 or activated JAK/STAT signaling in a subset. Unlike most other nonmelanoma skin cancers involving sun-exposed areas, most microcystic adnexal carcinomas lack evidence of UV damage, and hence likely originate from a relatively photo-protected progenitor population in the dermis. These findings have implications for the biology, diagnosis, and treatment of microcystic adnexal carcinomas, including potential for therapeutic targeting of p53 or the JAK/STAT pathway in advanced tumors.

Clonal evaluation of prostate cancer molecular heterogeneity in biopsy samples by dual immunohistochemistry and dual RNA in situ hybridization.

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 with Caucasian American patients. About 47% of the biopsy cores with discontinuous tumor foci showed clonal differences with distinct molecular aberrations. ERG positivity occurred in low-grade cancer, whereas ETV4 expression was observed mostly in high-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.

Gene fusion characterisation of rare aggressive prostate cancer variants-adenosquamous carcinoma, pleomorphic giant-cell carcinoma, and sarcomatoid carcinoma: an analysis of 19 cases.

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.

A pediatric case of pigmented epithelioid melanocytoma with chromosomal copy number alterations in 15q and 17q and a novel NTRK3-SCAPER gene fusion.

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.

Prostate cancer with comedonecrosis is frequently, but not exclusively, intraductal carcinoma: a need for reappraisal of grading criteria.

AIMS: Comedonecrosis in prostate cancer has always been Gleason pattern 5. However, we aimed to evaluate how intraductal carcinoma (not graded) with comedonecrosis should be considered. METHODS AND RESULTS: From 52 radical prostatectomy patients, 40 were informative and evaluated with immunohistochemistry for basal cells. Clinical outcome was assessed for biochemical recurrence, metastatic disease and the need for adjuvant therapy. Comedonecrosis was predominantly located in intraductal carcinoma (24, 60%). However, nine (23%) had comedonecrosis within invasive cancer and seven (18%) within both invasive and intraductal carcinoma. Extraprostatic extension rarely showed comedonecrosis (5, 13%), but rather perineural invasion within cribriform glands. Tumours were largely high-stage (15, 38% pT3a and 19, 48% pT3b), with 15 (37%) having positive lymph nodes and four distant metastases. Most cases (25, 63%) had other patterns of Gleason pattern 5 (single cells, solid), although 10 were reclassified as containing no invasive pattern 5. Of these, most were pT3 (eight of 10), but none had positive lymph nodes. Lymph node metastases were more common in patients with invasive cancer containing comedonecrosis (P = 0.02), and the need for androgen deprivation was near significance (P = 0.07), but biochemical recurrence was not significantly different (P = 0.58). CONCLUSIONS: Prostate cancer with comedonecrosis is often intraductal; however, these tumours are largely high-stage, showing a higher rate of positive lymph nodes with invasive comedonecrosis. Immunohistochemistry may be considered when comedonecrosis may significantly change the tumour grade. However, it is not clear at present that excluding intraductal carcinoma from the grade is superior to including it in grading when it is associated with high-grade invasive cancer.

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma.

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.

Wnt receptor Frizzled 8 is a target of ERG in prostate cancer.

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.

Pseudosarcomatous myofibroblastic proliferations of the genitourinary tract are genetically different from nodular fasciitis and lack USP6, ROS1 and ETV6 gene rearrangements.

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.

Morpheaform Basal Cell Carcinomas With Areas of Predominantly Single-Cell Pattern of Infiltration: Diagnostic Utility of p63 and Cytokeratin.

BACKGROUND: 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. DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

Molecular profiling of ETS and non-ETS aberrations in prostate cancer patients from northern India.

BACKGROUND: Molecular stratification of prostate cancer (PCa) based on genetic aberrations including ETS or RAF gene-rearrangements, PTEN deletion, and SPINK1 over-expression show clear prognostic and diagnostic utility. Gene rearrangements involving ETS transcription factors are frequent pathogenetic somatic events observed in PCa. Incidence of ETS rearrangements in Caucasian PCa patients has been reported, however, occurrence in Indian population is largely unknown. The aim of this study was to determine the prevalence of the ETS and RAF kinase gene rearrangements, SPINK1 over-expression, and PTEN deletion in this cohort. METHODS: In this multi-center study, formalin-fixed paraffin embedded (FFPE) PCa specimens (n = 121) were procured from four major medical institutions in India. The tissues were sectioned and molecular profiling was done using immunohistochemistry (IHC), RNA in situ hybridization (RNA-ISH) and/or fluorescence in situ hybridization (FISH). RESULTS: ERG over-expression was detected in 48.9% (46/94) PCa specimens by IHC, which was confirmed in a subset of cases by FISH. Among other ETS family members, while ETV1 transcript was detected in one case by RNA-ISH, no alteration in ETV4 was observed. SPINK1 over-expression was observed in 12.5% (12/96) and PTEN deletion in 21.52% (17/79) of the total PCa cases. Interestingly, PTEN deletion was found in 30% of the ERG-positive cases (P = 0.017) but in only one case with SPINK1 over-expression (P = 0.67). BRAF and RAF1 gene rearrangements were detected in ∼1% and ∼4.5% of the PCa cases, respectively. CONCLUSIONS: This is the first report on comprehensive molecular profiling of the major spectrum of the causal aberrations in Indian men with PCa. Our findings suggest that ETS gene rearrangement and SPINK1 over-expression patterns in North Indian population largely resembled those observed in Caucasian population but differed from Japanese and Chinese PCa patients. The molecular profiling data presented in this study could help in clinical decision-making for the pursuit of surgery, diagnosis, and in selection of therapeutic intervention.

Evaluation of tissue PCA3 expression in prostate cancer by RNA in situ hybridization--a correlative study with urine PCA3 and TMPRSS2-ERG.

PCA3 is a prostate-specific non-coding RNA, with utility as a urine-based early detection biomarker. Here, we report the evaluation of tissue PCA3 expression by RNA in situ hybridization in a cohort of 41 mapped prostatectomy specimens. We compared tissue PCA3 expression with tissue level ERG expression and matched pre-prostatectomy urine PCA3 and TMPRSS2-ERG levels. Across 136 slides containing 138 foci of prostate cancer, PCA3 was expressed in 55% of cancer foci and 71% of high-grade prostatic intraepithelial neoplasia foci. Overall, the specificity of tissue PCA3 was >90% for prostate cancer and high-grade prostatic intraepithelial neoplasia combined. Tissue PCA3 cancer expression was not significantly associated with urine PCA3 expression. PCA3 and ERG positivity in cancer foci was positively associated (P<0.01). We report the first comprehensive assessment of PCA3 expression in prostatectomy specimens, and find limited correlation between tissue PCA3 and matched urine in prostate cancer.

Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.

Cutaneous spindle cell malignancies such as sarcomatoid squamous cell carcinoma (SCC), leiomyosarcoma, desmoplastic melanoma (DM) and atypical fibroxanthoma (AFX) may be morphologically indistinguishable, yet accurate diagnosis is important for appropriate clinical management. The distinction among these entities relies on immunohistochemical evaluation for epidermal, muscle or melanocytic differentiation. Epidermal differentiation markers include cytokeratins and p63. p63 is expressed as two distinct isoforms, ΔNp63 (p40) and TAp63. p40 positivity is highly specific for pulmonary SCC and head and neck sarcomatoid SCC. We examined the utility of p40 vs. p63 immunostaining in the differentiation of a variety of cutaneous spindle cell malignancies, including sarcomatoid SCC (n = 27), AFX (n = 34) and DM (n = 10). p40 was less sensitive than p63 for detecting sarcomatoid SCC (56% and 81%, respectively). p63 and p40 were comparably specific for sarcomatoid SCC relative to AFX, with only rare weak staining of tumor cells for p63 and/or p40 in a minority of AFX cases, including one case with approximately 10% of cells staining weakly for p40. All cases of DM were negative for p40 and p63. Our results support continued use of p63 for diagnosis of cutaneous sarcomatoid SCC because of greater sensitivity relative to p40.

An integrative proteomics approach identifies tyrosine kinase KIT as a therapeutic target for SPINK1-positive prostate cancer.

Elevated serine peptidase inhibitor, Kazal type 1 (SPINK1) levels in ∼10%-25% of prostate cancer (PCa) patients associate with aggressive phenotype, for which there are limited treatment choices and dismal clinical outcomes. Using an integrative proteomics approach involving label-free phosphoproteome and proteome profiling, we delineated the downstream signaling pathways involved in SPINK1-mediated tumorigenesis and identified tyrosine kinase KIT as highly enriched. Furthermore, high to moderate levels of KIT expression were detected in ∼85% of SPINK1-positive PCa specimens. We show KIT signaling orchestrates SPINK1-mediated oncogenesis, and treatment with KIT inhibitor reduces tumor growth and metastases in preclinical mice models. Mechanistically, KIT signaling modulates WNT/ß-catenin pathway and confers stemness-related features in PCa. Notably, inhibiting KIT signaling led to restoration of AR/REST levels, forming a feedback loop enabling SPINK1 repression. Overall, we uncover the role of KIT signaling downstream of SPINK1 in maintaining lineage plasticity and provide distinct treatment modalities for advanced-stage SPINK1-positive patients.

Novel dual-color immunohistochemical methods for detecting ERG-PTEN and ERG-SPINK1 status in prostate carcinoma.

Identification of new molecular markers has led to the molecular classification of prostate cancer based on driving genetic lesions. The translation of these discoveries for clinical use necessitates the development of simple, reliable and rapid detection systems to screen patients for specific molecular aberrations. We developed two dual-color immunohistochemistry-based assays for the simultaneous assessment of ERG-PTEN and ERG-SPINK1 in prostate cancer. A total of 232 cases from 184 localized and 48 metastatic prostate cancers were evaluated for ERG-PTEN and 284 cases from 228 localized and 56 metastatic prostate cancers were evaluated for ERG-SPINK1. Of the 232 cases evaluated for ERG-PTEN, 81 (35%) ERG-positive and 77 (33%) PTEN-deleted cases were identified. Of the 81 ERG-positive cases, PTEN loss was confirmed in 35 (15%) cases by fluorescence in situ hybridization (FISH). PTEN status was concordant in 203 cases (sensitivity 90% and specificity 87%; P<0.0001) by both immunohistochemisty and FISH; however, immunohistochemisty could not distinguish between heterozygous and homozygous deletion status of PTEN. Of the 284 cases evaluated for ERG-SPINK1, 111 (39%) cases were positive for ERG. In the remaining 173 ERG-negative cases, SPINK1 was positive in 26 (9%) cases. SPINK1 expression was found to be mutually exclusive with ERG expression; however, we identified two cases, of which one showed concomitant expression of ERG and SPINK1 in the same tumor foci, and in the second case ERG and SPINK1 were seen in two independent foci of the same tumor nodule. Unlike the homogenous ERG staining in cancer tissues, heterogeneous SPINK1 staining was observed in the majority of the cases. Further studies are required to understand the molecular heterogeneity of cases with concomitant ERG-SPINK1 expression. Automated dual ERG-PTEN and ERG-SPINK1 immunohistochemisty assays are simple, reliable and portable across study sites for the simultaneous assessment of these proteins in prostate cancer.

Loss of KLK4::KLKP1 pseudogene expression by RNA chromogenic in-situ hybridization is associated with PTEN loss and increased risk of biochemical recurrence in a cohort of middle eastern men with prostate cancer.

BACKGROUND: KLK4::KLKP1 fusion is a recently described pseudogene that is enriched in prostate cancer (PCa). This new biomarker has not been characterized in the Middle Eastern population. OBJECTIVE: To establish the incidence and prognostic value of KLK4::KLKP1 fusion in a cohort of Middle Eastern men with PCa and explore the relationship of this marker to other relevant biomarkers (PTEN, ERG, SPINK1). DESIGN, SETTING, AND PARTICIPANTS: We interrogated a cohort of 340 Middle Eastern men with localized PCa treated by radical prostatectomy between 2005 and 2015. KLK4::KLKP1 fusion status was assessed by RNA Chromogenic in situ hybridization (CISH) and correlated to pathological and clinical parameters. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: RNA-CISH expression of KLK4::KLKP1 was correlated with prognostic factors, ERG, PTEN, and SPINK1 expression, and biochemical recurrence (BCR) following prostatectomy. RESULTS AND LIMITATIONS: 51.7% of patient samples showed positive KLK4::KLKP1 expression; more commonly in cores of PCa (38%) versus non-cancer (20.6%) (p < 0.0001) and in lower Gleason Grade Group tumors (1-3) vs (4-5). KLK4::KLKP1 expression positively correlated with ERG positivity and inversely associated with PTEN loss. No significant association was found with SPINK1 expression, seminal vesicle invasion, positive surgical margin, pathological stage, or patient age (< 50 or ≥ 50). The association between PTEN loss and BCR increased when combined with KLK4::KLKP1 negativity (HR 2.31, CI 1.03-5.20, p = 0.042). CONCLUSIONS: KLK4::KLKP1 expression is more common in this cohort of Middle Eastern men than has been reported in North American men. It is associated with ERG positivity and inversely correlated with PTEN loss. In isolation, KLK4::KLKP1 expression was not significantly associated with clinical outcome or pathological parameters. However, its expression is associated with certain molecular subtypes (ERG-positive, PTEN-intact) and as we demonstrate may help further stratify the risk of recurrence within these groups.

Brief Report: Prognostic Relevance of 3q Amplification in Squamous Cell Carcinoma of the Lung.

Introduction: Amplification of 3q is the most common genetic alteration identified in squamous cell carcinoma of the lung (LUSC), with the most frequent amplified region being 3q26 to 3q28. Methods: In this analysis, we aim to describe the prognostic relevance of 3q amplification by focusing on a minimal common region (MCR) of amplification constituted of 25 genes. We analyzed 511 cases of LUSC from The Cancer Genome Atlas and included 476 in the final analysis. Results: We identified a 25-gene MCR that was amplified in 221 (44.3%) cases and was associated with better disease-specific survival (not reported [NR] versus 9.25 y, 95% confidence interval [CI]: 5.24-NR, log-rank p = 0.011) and a progression-free interval of 8 years (95% CI: 5.1-NR) versus 4.9 years (95% CI: 3.5-NR, log-rank p = 0.020). Multivariable analysis revealed that MCR amplification was associated with improved disease-specific survival and progression-free interval. Conclusions: Amplification of the 25-gene MCR within 3q was present in 44% of this cohort, consisting mainly of Caucasian patients with early stage LUSC. This analysis strongly indicates the prognostic relevance of the 25-gene MCR within 3q. We are further evaluating its prognostic and predictive relevance in a racially diverse patient population with advanced LUSC.

Macrophage migration inhibitory factor promotes tumor growth in the context of lung injury and repair.

RATIONALE: Tissue injury and repair involve highly conserved processes governed by mechanisms that can be co-opted in tumors. We hypothesized that soluble factors released during the repair response to lung injury would promote orthotopic tumor growth. OBJECTIVES: To determine whether lung injury promoted growth of orthotopic lung tumors and to study the molecular mechanisms. METHODS: We initiated lung injury in C57Bl6 mice using different stimuli, then injected Lewis lung carcinoma cells during the repair phase. We assessed tumor growth 14 days later. We measured tumor angiogenesis, cytokine expression, proliferation, and apoptosis. MEASUREMENTS AND MAIN RESULTS: Regardless of the mechanism, injured lungs contained more numerous and larger tumors than sham-injured lungs. Tumors from injured lungs were no more vascular, but had higher levels of proliferation and reduced rates of apoptosis. The cytokine macrophage migration inhibitory factor (MIF) was highly expressed in both models of tissue injury. We observed no increase in tumor growth after lung injury in MIF knockout mice. We induced lung-specific overexpression of MIF in a double-transgenic mouse, and observed that MIF overexpression by itself was sufficient to accelerate the growth of orthotopic Lewis lung carcinoma tumors. CONCLUSIONS: Lung injury leads to increased expression of the cytokine MIF, which results in protection from apoptosis and increased proliferation in orthotopic tumors injected after the acute phase of injury.

Transcriptional network involving ERG and AR orchestrates Distal-less homeobox-1 mediated prostate cancer progression.

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.

Virus-positive Merkel Cell Carcinoma Is an Independent Prognostic Group with Distinct Predictive Biomarkers.

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.