Search

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 ETV4expression 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.

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 ETV4expression 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.

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 TP53inactivation in four (22%) tumors. Frame-preserving insertions affecting the kinase domain of JAK1were detected in three (17%) cases, and were nonoverlapping with TP53mutations. Seven (39%) cases demonstrated copy number gain of at least one oncogene. By immunohistochemistry, p53 expression was significantly higher in microcystic adnexal carcinomas with TP53mutations compared with those without such mutations and syringomas. Similarly, phospho-STAT3 expression was significantly higher in microcystic adnexal carcinomas harboring JAK1kinase insertions compared with those with wild-type JAK1and 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.

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 TP53inactivation in four (22%) tumors. Frame-preserving insertions affecting the kinase domain of JAK1were detected in three (17%) cases, and were nonoverlapping with TP53mutations. Seven (39%) cases demonstrated copy number gain of at least one oncogene. By immunohistochemistry, p53 expression was significantly higher in microcystic adnexal carcinomas with TP53mutations compared with those without such mutations and syringomas. Similarly, phospho-STAT3 expression was significantly higher in microcystic adnexal carcinomas harboring JAK1kinase insertions compared with those with wild-type JAK1and 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.

Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting tumor heterogeneity. We have performed multi-omic profiling of the kinome in bladder cancer patients with the goal of identify therapeutic targets. Our analyses revealed amplification, overexpression, and elevated kinase activity of P21 (RAC1) activated kinase 4 (PAK4) in a subset of Bladder cancer (BLCA). Using bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, we observed 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 and RNA interference of PAK4. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared with 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.

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.

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 VHLgene 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 VHLmutation, 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 VHLpromoter hypermethylation. Nine could be reclassified as distinct or emerging entities: translocation renal cell carcinoma (n=3), TCEB1mutant 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 TCEB1mutant). 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 TFE3gene fusions also exhibited VHLmutation, 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 VHLmutation, 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.

Supplemental Digital Content is available in the text.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 TFEBgene, 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 TFEBfusions 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 VHLdeletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHLmutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FHmutation and SMARCB1mutation. Chromosome 6p amplification including TFEBis 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-ALKgene 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 ALKgene). 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.

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.

Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2and ERGgenes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERGgene 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-ERGgene 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.

Supplemental Digital Content is Available in the Text.

Recent cancer research has demonstrated the existence of circulating tumor cells (CTCs) in cancer patient's blood. Once identified, CTC biomarkers will be invaluable tools for clinical diagnosis, prognosis and treatment. In this review, we propose ex vivoculture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample, to derive enough CTCs for accurate and reproducible characterization of the biophysical, biochemical, gene expressional and behavioral properties of the harvested cells. Because of tumor cell heterogeneity, it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis. By analyzing critical steps and technical issues in ex vivoCTC culture, we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells, relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.

The presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well-recognized finding on prostate biopsy. Cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area. Importantly, this parameter is a common enrollment criterion for active surveillance protocols. We hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones, impacting cancer involvement assessment. Hence, we used dual ERG/SPINK1 immunohistochemistry to determine the frequency of separate clones—when separate tumor foci showed discordant ERG and/or SPINK1 status—in discontinuously involved prostate biopsy cores from two academic institutions. In our cohort of 97 prostate biopsy cores with spatially discrete tumor foci (from 80 patients), discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and Gleason scores ranged from 6 to 9. Twenty-four (25%) of 97 discontinuously involved cores harbored clonally distinct cancer foci by discordant ERG and/or SPINK1 expression status: 58% (14/24) had one ERG+focus, and one ERG−/SPINK1−focus; 29% (7/24) had one SPINK1+focus and one ERG−/SPINK1−focus; and 13% (3/24) had one ERG+focus and one SPINK1+focus. ERG and SPINK1 overexpression were mutually exclusive in all tumor foci. In summary, our results show that ~25% of discontinuously involved prostate biopsy cores showed tumor foci with discordant ERG/SPINK1 status, consistent with multiclonal disease. The relatively frequent presence of multiclonality in discontinuously involved prostate biopsy cores warrants studies on the potential clinical impact of clonality assessment, particularly in cases where tumor volume in a discontinuous core may impact active surveillance eligibility.

The presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well-recognized finding on prostate biopsy. Cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area. Importantly, this parameter is a common enrollment criterion for active surveillance protocols. We hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones, impacting cancer involvement assessment. Hence, we used dual ERG/SPINK1 immunohistochemistry to determine the frequency of separate clones—when separate tumor foci showed discordant ERG and/or SPINK1 status—in discontinuously involved prostate biopsy cores from two academic institutions. In our cohort of 97 prostate biopsy cores with spatially discrete tumor foci (from 80 patients), discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and Gleason scores ranged from 6 to 9. Twenty-four (25%) of 97 discontinuously involved cores harbored clonally distinct cancer foci by discordant ERG and/or SPINK1 expression status: 58% (14/24) had one ERG+focus, and one ERG−/SPINK1−focus; 29% (7/24) had one SPINK1+focus and one ERG−/SPINK1−focus; and 13% (3/24) had one ERG+focus and one SPINK1+focus. ERG and SPINK1 overexpression were mutually exclusive in all tumor foci. In summary, our results show that ~25% of discontinuously involved prostate biopsy cores showed tumor foci with discordant ERG/SPINK1 status, consistent with multiclonal disease. The relatively frequent presence of multiclonality in discontinuously involved prostate biopsy cores warrants studies on the potential clinical impact of clonality assessment, particularly in cases where tumor volume in a discontinuous core may impact active surveillance eligibility.

To examine the impact of common somatic mutations in prostate cancer (PCa) on androgen receptor (AR) signaling, mouse models were designed to perturb sequentially the AR, ETV1, and PTEN pathways. Mice with “humanized” AR (hAR) alleles that modified AR transcriptional strength by varying polyglutamine tract (Q-tract) length were crossed with mice expressing a prostate-specific, AR-responsive ETV1 transgene (ETV1Tg). While hARallele did not grossly affect ETV1-induced neoplasia, ETV1 strongly antagonized global AR regulation and repressed critical androgen-induced differentiation and tumor suppressor genes, such as Nkx3-1and Hoxb13. When Ptenwas varied to determine its impact on disease progression, mice lacking one Ptenallele (Pten+/−) developed more frequent prostatic intraepithelial neoplasia (PIN). Yet, only those with the ETV1 transgene progressed to invasive adenocarcinoma. Furthermore, progression was more frequent with the short Q-tract (stronger) AR, suggesting that the AR, ETV1, and PTEN pathways cooperate in aggressive disease. On the Pten+/−background, ETV1 had markedly less effect on AR target genes. However, a strong inflammatory gene expression signature, notably upregulation of Cxcl16, was induced by ETV1. Comparison of mouse and human patient data stratified by the presence of E26 transformation-specific ETS fusion genes highlighted additional factors, some not previously associated with prostate cancer but for which targeted therapies are in development for other diseases. In sum, concerted use of these mouse models illuminates the complex interplay of AR, ETV1, and PTEN pathways in pre-cancerous neoplasia and early tumorigenesis, disease stages difficult to analyze in man.

Prostate biopsy is the key clinical specimen for disease diagnosis. However, various conditions used during biopsy processing for histologic analysis may affect the performance of diagnostic tests, such as hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), or in situ hybridization (ISH). One such condition that may affect diagnostic test performance is fixation duration in 10 neutral buffered formalin (NBF). For example, prostate needle biopsies are often <1 mm in diameter and thus overfixed. It is important to understand the impact of tissue fixation duration on diagnostic test performance to enable optimized assay procedures. This study was designed to study the effect of 10 NBF fixation duration of prostate needle biopsy on multiplexed quantum dot (QD) ISH assay of ERGand PTEN, 2 genes commonly altered in prostate cancer. The samples were also evaluated for H&E staining and ERG and PTEN IHC. H&E staining and ERG and PTEN IHC were acceptable for all the durations of fixation tested. For QD ISH, we observed good signals with biopsy samples fixed from 4 to 120 hours. Biopsy specimens fixed between 8 and 72 hours gave the best signal as scored by the study pathologist. In a separate cohort of 18 routinely processed prostate biopsy cores, all cores were stained successfully with the QD ISH assay, and results were 100 concordant to ERG and PTEN IHC. We conclude that 8 to 72 hours duration of fixation for prostate needle biopsies in 10 NBF results in optimal QD ISH assay performance.

The genetic basis of 50 to 60 of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific(ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERGrearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.

Recent genetic epidemiologic studies identified a germline mutation in the homeobox transcription factor, HOXB13G84E, which is associated with markedly increased risk for prostate cancer, particularly early-onset hereditary prostate cancer. The histomorphologic and molecular features of cancers arising in such carriers have not been studied. Here, we reviewed prostatectomy specimens from 23 HOXB13G84E mutation carriers, mapping the total cancer burden by anatomically distinct cancer focus and evaluating morphologic features. We also assessed basic molecular subtypes for all cancer foci (ERGSPINK1 status) by dual immunohistochemistry staining on full sections. The cohort showed a median age of 58 years, a median serum PSA level of 5.7 ngmL, and a median of 6 cancer foci (range, 1 to 14) per case. Of evaluable cases, dominant foci were Gleason score 6 in 23, 34=7 in 41, 43=7 in 23, and ≥8 in 14; biochemical recurrence was observed in 1 case over a median of 36 months follow-up. Histologic review found a high prevalence of cases showing cancers with a spectrum of features previously described with pseudohyperplastic carcinomas, with 45 of cases showing a dominant focus with such features. Molecular subtyping revealed a strikingly low prevalence of ERGcancer with increased prevalence of SPINK1cancer (dominant focus ERG17, SPINK126, ERG−SPINK1−52, single ERGSPINK1focus 4). One ERG−SPINK1−dominant focus showed aberrant p63immunophenotype. In summary, HOXB13G84E variant–related prostate cancers show frequent pseudohyperplastic-type features and markedly low prevalence of ERGcancers relative to unselected cases and, especially, to early-onset cohorts. These findings suggest that novel molecular pathways may drive disease in HOXB13G84E carriers.

PCA3is a prostate-specific non-coding RNA, with utility as a urine-based early detection biomarker. Here, we report the evaluation of tissue PCA3expression by RNA in situhybridization in a cohort of 41 mapped prostatectomy specimens. We compared tissue PCA3expression with tissue level ERG expression and matched pre-prostatectomy urine PCA3and TMPRSS2-ERGlevels. Across 136 slides containing 138 foci of prostate cancer, PCA3was expressed in 55% of cancer foci and 71% of high-grade prostatic intraepithelial neoplasia foci. Overall, the specificity of tissue PCA3was >90% for prostate cancer and high-grade prostatic intraepithelial neoplasia combined. Tissue PCA3cancer expression was not significantly associated with urine PCA3expression. PCA3and ERG positivity in cancer foci was positively associated (P<0.01). We report the first comprehensive assessment of PCA3expression in prostatectomy specimens, and find limited correlation between tissue PCA3and matched urine in prostate cancer.

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.

Parathyroid cancer is a rare, clinically aggressive cause of primary hyperparathyroidism, and whether these malignancies generally evolve from pre-existing benign adenomas or arise de novo is unclear. Furthermore, while inactivation of the CDC73(HRPT2) tumor suppressor gene, encoding parafibromin, is a major contributor, other genes essential to parathyroid carcinogenesis remain unknown. We sought to identify genomic regions potentially harboring such oncogenes or tumor suppressor genes, and to gain insight into the origins and molecular relationship of malignant versus benign parathyroid tumors. We performed genome-wide copy-number and loss of heterozygosity analysis using Affymetrix 50K SNP mapping arrays and/or comparative genomic hybridization on 16 primary parathyroid carcinomas, local recurrences or distant metastases, and matched normal controls, from 10 individuals. Recurrent regions of allelic loss were observed on chromosomes 1p, 3, and 13q suggesting that key parathyroid tumor suppressor genes are located in these chromosomal locations. Recurrent allelic gains were seen on chromosomes 1q and 16, suggesting the presence of parathyroid oncogenes on these chromosomes. Importantly, the most common alteration in benign parathyroid adenomas, loss of 11q, was not found as a recurrent change in the malignant parathyroid tissues. Molecular allelotyping using highly polymorphic microsatellite markers provided further confirmation that the prevalence of 11q loss is markedly and significantly lower in carcinomas as compared with adenomas. Our observations provide molecular support for the concept that sporadic parathyroid cancer usually arises de novo, rather than evolving from a pre-existing typical benign adenoma. Furthermore, these results help direct future investigation to ultimately determine which of the candidate genes in these chromosomal locations make significant contributions to the molecular pathogenesis of parathyroid cancer.

E26 transformation-specific (ETS) transcription factors are known to be involved in gene aberrations in various malignancies including prostate cancer; however, their role in melanoma oncogenesis has yet to be fully explored. We have completed a comprehensive fluorescence in situhybridization (FISH)-based screen for all 27 members of the ETS transcription factor family on two melanoma tissue microarrays, representing 223 melanomas, 10 nevi, and 5 normal skin tissues. None of the melanoma cases demonstrated ETS fusions; however, 6 of 114 (5.3%) melanomas were amplified for ETV1 using a break-apart FISH probe. For the six positive cases, locus-controlled FISH probes revealed that two of six cases were amplified for the ETV1 region, whereas four cases showed copy gains of the entire chromosome 7. The remaining 26 ETS family members showed no chromosomal aberrations by FISH. Quantitative polymerase chain reaction showed an average 3.4-fold (Pvalue = .00218) increased expression of ETV1 in melanomas, including the FISH ETV1-amplified cases, when compared to other malignancies (prostate, breast, and bladder carcinomas). These data suggest that a subset of melanomas overexpresses ETV1 and amplification of ETV1 may be one mechanism for achieving high gene expression.

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.

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 situhybridization (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.

Histologic variants of prostate carcinoma account for 5–10% of the disease and are typically seen in association with conventional acinar carcinoma. These variants often differ from the latter in clinical, immunophenotypic, and biologic potential. Recently, recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS transcription factors ERG, ETV1, ETV4, or ETV5 have been identified in a majority of conventional prostate carcinomas. However, the frequency and significance of this critical molecular event is unknown in the histologic variants of prostate carcinoma. Here, we used break-apart fluorescence in situ hybridization to assess TMPRSS2 and ETS aberrations in a series of select histologic variants: foamy gland carcinoma (N=17), ductal adenocarcinoma (N=18), mucinous carcinoma (N=18), and small cell carcinoma (N=7). A histologic variation of acinar adenocarcinoma, demonstrating glomeruloid morphology (N=9), was also investigated. Overall, 55% of histologic variant or variation morphologies demonstrated ETS aberrations (ERG in 54% and ETV1 in 1%). TMPRSS2:ERG fusion was identified in 83% (15/18), 71% (5/7), 50% (9/18), 33% (3/9), and 29% (5/17) of mucinous, small cell, ductal, glomeruloid, and foamy gland prostate carcinomas, respectively. Previously, we reported that 100% of androgen-independent metastatic prostate carcinomas harboring TMPRSS2:ERG gene fusion were associated with interstitial deletion (Edel). Interestingly, ERG rearrangement in small cell carcinomas occurred exclusively through Edel, supporting the notion that TMPRSS2:ERG with Edel is an aggressive molecular subtype. SPINK1, a biomarker expressed exclusively in a subset of ETS negative prostate carcinomas, was expressed in 6% of ETS negative histologic variants, specifically in ductal adenocarcinoma. Notably, 88% (43/49) variant morphologies in this cohort showed concordance of TMPRSS2:ERG fusion with associated conventional acinar type, suggesting that variant morphology is clonally related to the latter. Overall, our data provide insight into the origin, molecular mechanism, and phenotypic association of ETS fusions in histologic variants of prostate carcinoma.

Histologic variants of prostate carcinoma account for 5–10% of the disease and are typically seen in association with conventional acinar carcinoma. These variants often differ from the latter in clinical, immunophenotypic, and biologic potential. Recently, recurrent gene fusions between the androgen-regulated gene TMPRSS2and the ETStranscription factors ERG, ETV1, ETV4,or ETV5have been identified in a majority of conventional prostate carcinomas. However, the frequency and significance of this critical molecular event is unknown in the histologic variants of prostate carcinoma. Here, we used break-apart fluorescence in situhybridization to assess TMPRSS2and ETSaberrations in a series of select histologic variants: foamy gland carcinoma (N=17), ductal adenocarcinoma (N=18), mucinous carcinoma (N=18), and small cell carcinoma (N=7). A histologic variation of acinar adenocarcinoma, demonstrating glomeruloid morphology (N=9), was also investigated. Overall, 55% of histologic variant or variation morphologies demonstrated ETSaberrations (ERGin 54% and ETV1in 1%). TMPRSS2:ERGfusion was identified in 83% (15/18), 71% (5/7), 50% (9/18), 33% (3/9), and 29% (5/17) of mucinous, small cell, ductal, glomeruloid, and foamy gland prostate carcinomas, respectively. Previously, we reported that 100% of androgen-independent metastatic prostate carcinomas harboring TMPRSS2:ERGgene fusion were associated with interstitial deletion (Edel). Interestingly, ERGrearrangement in small cell carcinomas occurred exclusively through Edel, supporting the notion that TMPRSS2:ERGwith Edel is an aggressive molecular subtype. SPINK1, a biomarker expressed exclusively in a subset of ETSnegative prostate carcinomas, was expressed in 6% of ETSnegative histologic variants, specifically in ductal adenocarcinoma. Notably, 88% (43/49) variant morphologies in this cohort showed concordance of TMPRSS2:ERGfusion with associated conventional acinar type, suggesting that variant morphology is clonally related to the latter. Overall, our data provide insight into the origin, molecular mechanism, and phenotypic association of ETSfusions in histologic variants of prostate carcinoma.

The link between ERGrearrangement and PTEN(phosphatase and tensin homolog deleted on chromosome 10) deletion is unclear in prostate cancer progression. Using fluorescence in situhybridization, we systematically validated the frequency and distribution of ERGand PTENaberrations in a cohort of 73 benign prostate tissues, 59 high-grade prostatic intraepithelial neoplasia (HGPIN) foci, 281 localized prostate cancer and 47 androgen-independent metastatic prostate cancer patients. Overall, ERGrearrangement was present in 15% (5/33) of HGPIN, 45% (121/267) of localized cancers and 35% (15/43) of metastases. By contrast, PTENdeletion was identified in 9% (3/33) of HGPIN, 17% (42/251) of localized cancers and 54% (22/41) of metastases, of which 0%, 40% (17/42) and 45% (10/22) were homozygous, respectively. Concomitance of ERGrearrangement and PTENdeletion was observed in a subset of HGPIN. Significantly, association between PTENdeletion and ERGrearrangement was present both in localized cancers (P=0.0008) and metastases (P=0.02). Further, immunohistochemistry revealed significant correlation of decreased PTEN protein expression with PTENgenomic deletion both in localized and metastatic cancer. Of note, ERGaberration, but not PTENdeletion, was consistently identical both in localized cancer and adjacent HGPIN. Similarly, whereas all metastases (41/41, 100%) shared the same ERGstatus across multiple sites from the same patient, 5% (2/41) of cases showed discordance for PTENdeletion status across multiple sites. Collectively, our data support PTENdeletion as a late genetic event in human prostate cancer, presumably a ‘second hit’ after ERGrearrangement. PTENdeletion and ERGrearrangement may cooperate, but contribute at different stages, in prostate cancer progression.

The link between ERG rearrangement and PTEN (phosphatase and tensin homolog deleted on chromosome 10) deletion is unclear in prostate cancer progression. Using fluorescence in situ hybridization, we systematically validated the frequency and distribution of ERG and PTEN aberrations in a cohort of 73 benign prostate tissues, 59 high-grade prostatic intraepithelial neoplasia (HGPIN) foci, 281 localized prostate cancer and 47 androgen-independent metastatic prostate cancer patients. Overall, ERG rearrangement was present in 15% (5/33) of HGPIN, 45% (121/267) of localized cancers and 35% (15/43) of metastases. By contrast, PTEN deletion was identified in 9% (3/33) of HGPIN, 17% (42/251) of localized cancers and 54% (22/41) of metastases, of which 0%, 40% (17/42) and 45% (10/22) were homozygous, respectively. Concomitance of ERG rearrangement and PTEN deletion was observed in a subset of HGPIN. Significantly, association between PTEN deletion and ERG rearrangement was present both in localized cancers (P=0.0008) and metastases (P=0.02). Further, immunohistochemistry revealed significant correlation of decreased PTEN protein expression with PTEN genomic deletion both in localized and metastatic cancer. Of note, ERG aberration, but not PTEN deletion, was consistently identical both in localized cancer and adjacent HGPIN. Similarly, whereas all metastases (41/41, 100%) shared the same ERG status across multiple sites from the same patient, 5% (2/41) of cases showed discordance for PTEN deletion status across multiple sites. Collectively, our data support PTEN deletion as a late genetic event in human prostate cancer, presumably a ‘second hit’ after ERG rearrangement. PTEN deletion and ERG rearrangement may cooperate, but contribute at different stages, in prostate cancer progression.Modern Pathology (2009) 22, 1083–1093; doi:10.1038/modpathol.2009.69; published online 1 May 2009

Invasive cervical carcinomas almost invariably carry extra copies of chromosome arm 3q, resulting in a gain of the human telomerase gene (TERC). This provided the rationale for the development of a multicolor fluorescence in situhybridization (FISH) probe set as a diagnostic tool for the direct detection of TERCgains in Pap smears. We previously used this probe set to show that cervical intraepithelial neoplasia (CIN) 2 and CIN3 lesions could be distinguished from normal samples, atypical squamous cell of undetermined significance (ASCUS) and CIN1, with a sensitivity and specificity exceeding 90%, independent of the cytomorphological assessment. In the current study, we explored whether gain of 3q and amplification of TERCcould predict progression from CIN1/CIN2 to CIN3 and invasive carcinoma. We applied our probe set to a series of 59 previously stained Pap smears for which repeat Pap smears and clinical follow-up were available. The samples included CIN1/CIN2 lesions that progressed to CIN3 (progressors), CIN1/CIN2 lesions that regressed spontaneously (regressors), and normal Pap smears from women who subsequently developed CIN3 or cervical cancer. Here, we show that progressors displayed a gain of 3q whereas none of the regressors showed this genetic aberration. These data suggest that 3q gain is required for the transition from CIN1/CIN2 to CIN3 and that it predicts progression. Of note, 3q gain was found in 33% of cytologically normal Pap smears from women who were diagnosed with CIN3 or invasive cervical carcinoma after a short latency. The sensitivity of our test for predicting progression from CIN1/CIN2 to CIN3 was 100% and the specificity, ie, the prediction of regression, was 70%. We conclude that the detection of 3q gain and amplification of TERCin routinely collected Pap smears can assist in identifying low-grade lesions with a high progression risk and in decreasing false-negative cytological screenings.

Although it has been suggested that REL is the critical target gene of 2p12-16 amplification in diffuse large B-cell lymphoma (DLBCL), little experimental evidence supports this notion. In the present study, we sought to evaluate the relationship between REL amplification and REL function in a panel of 46 newly diagnosed DLBCLs and to correlate with DLBCL subgroups as identified by gene expression profiles and clinical features. The results indicate that amplification of the REL locus is not associated with accumulation of the active form of REL, as evaluated by immunofluorescence analysis. Upon subgrouping of the DLBCL cases based on gene expression signatures, REL amplification was detected in all subgroups, while high levels of nuclear-located REL were more frequently detected in activated B-cell–like DLBCL. Correlative analyses of REL copy number and REL nuclear accumulation with clinical parameters did not reveal any significant associations. Together these results indicate that 2p12-16 amplification does not lead to abnormal REL activation, suggesting that REL may not be the functional target of the amplification event. Nonetheless, these data indicate that DLBCLs are heterogeneous with respect to REL and thus nuclear factor–κB (NF-κB) activity.

The band 1q21 is among the most common sites affected by chromosomal translocations in lymphoid, myeloid, epithelial, and sarcomatous lesions. In non-Hodgkin’s lymphoma (NHL), translocations and duplications affecting this chromosomal site are frequently, but not exclusively, seen in association with primary abnormalities such as the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, suggesting a role for 1q21 rearrangements in tumor progression. We report here the characterization and cloning of breakpoints in a case of extranodal ascitic B-cell lymphoma with a t(1;14)(q21;q32) translocation. The breakpoints on the der(1) and der(14) chromosomes were mapped by fluorescence in situ hybridization and Southern blot analysis and cloned using an IGHG(Cγ) probe. The translocation linked theIGHG4switch (Sγ4) sequences of the productively rearranged allele to chromosome 1 sequences downstream of MUC1, leaving the MUC1transcriptional unit intact. MUC1was markedly overexpressed in the tumor at the mRNA and protein levels relative to lymphoma cell lines lacking a 1q21 rearrangement. Presumably,MUC1transcription is aberrantly regulated by the IGHA(Cα) 3′ enhancer element retained on the same chromosome. Screening of a panel of B-cell lymphomas by Southern blot analysis identified a subset with a 3′ MUC1breakpoint and another with low-level amplification of MUC1. MUC-1 mucin has previously been shown to be frequently overexpressed in human epithelial cancers and to be associated with tumor progression and poor clinical outcome. Thus, MUC1activation by chromosomal translocation, rearrangement, and amplification, identified here for the first time in NHL, is consistent with its suggested role in tumorigenesis.

The band 1q21 is among the most common sites affected by chromosomal translocations in lymphoid, myeloid, epithelial, and sarcomatous lesions. In non-Hodgkin's lymphoma (NHL), translocations and duplications affecting this chromosomal site are frequently, but not exclusively, seen in association with primary abnormalities such as the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, suggesting a role for 1q21 rearrangements in tumor progression. We report here the characterization and cloning of breakpoints in a case of extranodal ascitic B-cell lymphoma with a t(1;14)(q21;q32) translocation. The breakpoints on the der(1) and der(14) chromosomes were mapped by fluorescence in situ hybridization and Southern blot analysis and cloned using an IGHG (C?) probe. The translocation linked theIGHG4 switch (S?4) sequences of the productively rearranged allele to chromosome 1 sequences downstream of MUC1, leaving the MUC1 transcriptional unit intact. MUC1 was markedly overexpressed in the tumor at the mRNA and protein levels relative to lymphoma cell lines lacking a 1q21 rearrangement. Presumably,MUC1 transcription is aberrantly regulated by the IGHA(C?) 3' enhancer element retained on the same chromosome. Screening of a panel of B-cell lymphomas by Southern blot analysis identified a subset with a 3' MUC1 breakpoint and another with low-level amplification of MUC1. MUC-1 mucin has previously been shown to be frequently overexpressed in human epithelial cancers and to be associated with tumor progression and poor clinical outcome. Thus, MUC1 activation by chromosomal translocation, rearrangement, and amplification, identified here for the first time in NHL, is consistent with its suggested role in tumorigenesis.

Dovitinib is therapeutically active in a subset of patients with prostate cancer bone metastases, partly due to blockade of FGFR-mediated stromal-epithelial interactions in the bone microenvironment.

Urine TMPRSS2:ERGgene fusion could be used for stratification of patients at higher risk for prostate cancer.

One partner of a fusion gene found in gastric cancer, CD44-SLC1A2, may contribute to the tumor’s abnormal metabolism.