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1.  Defining Early-Onset Kidney Cancer: Implications for Germline and Somatic Mutation Testing and Clinical Management 
Journal of Clinical Oncology  2013;32(5):431-437.
Purpose
Approximately 5% to 8% of renal cell carcinoma (RCC) is hereditary. No guidelines exist for patient selection for RCC germline mutation testing. We evaluate how age of onset could indicate the need for germline mutation testing for detection of inherited forms of kidney cancer.
Patients and Methods
We analyzed the age distribution of RCC cases in the SEER-17 program and in our institutional hereditary kidney cancer population. The age distributions were compared by sex, race, histology, and hereditary cancer syndrome. Models were established to evaluate the specific age thresholds for genetic testing.
Results
The median age of patients with RCC in SEER-17 was 64 years, with the distribution closely approaching normalcy. Statistical differences were observed by race, sex, and subtype (P < .05). The bottom decile cutoff was ≤ 46 years of age and slightly differed by sex, race, and histology. The mean and median ages at presentation of 608 patients with hereditary kidney cancer were 39.3 years and 37 years, respectively. Although age varied by specific syndrome, 70% of these cases were found to lie at or below the bottom age decile. Modeling age-based genetic testing thresholds demonstrated that the 10th percentile maximized sensitivity and specificity.
Conclusion
Early age of onset might be a sign of hereditary RCC. Even in the absence of clinical manifestations and personal/family history, an age of onset of 46 years or younger should trigger consideration for genetic counseling/germline mutation testing and may serve as a useful cutoff when establishing genetic testing guidelines.
doi:10.1200/JCO.2013.50.8192
PMCID: PMC3912328  PMID: 24378414
2.  Imaging and pathology findings after an initial negative MRI-US fusion-guided and 12-core extended sextant prostate biopsy session 
PURPOSE
A magnetic resonance imaging-ultrasonography (MRI-US) fusion-guided prostate biopsy increases detection rates compared to an extended sextant biopsy. The imaging characteristics and pathology outcomes of subsequent biopsies in patients with initially negative MRI-US fusion biopsies are described in this study.
MATERIALS AND METHODS
We reviewed 855 biopsy sessions of 751 patients (June 2007 to March 2013). The fusion biopsy consisted of two cores per lesion identified on multiparametric MRI (mpMRI) and a 12-core extended sextant transrectal US (TRUS) biopsy. Inclusion criteria were at least two fusion biopsy sessions, with a negative first biopsy and mpMRI before each.
RESULTS
The detection rate on the initial fusion biopsy was 55.3%; 336 patients had negative findings. Forty-one patients had follow-up fusion biopsies, but only 34 of these were preceded by a repeat mpMRI. The median interval between biopsies was 15 months. Fourteen patients (41%) were positive for cancer on the repeat MRI-US fusion biopsy. Age, prostate- specific antigen (PSA), prostate volume, PSA density, digital rectal exam findings, lesion diameter, and changes on imaging were comparable between patients with negative and positive rebiopsies. Of the patients with positive rebiopsies, 79% had a positive TRUS biopsy before referral (P = 0.004). Ten patients had Gleason 3+3 disease, three had 3+4 disease, and one had 4+4 disease.
CONCLUSION
In patients with a negative MRI-US fusion prostate biopsy and indications for repeat biopsy, the detection rate of the follow-up sessions was lower than the initial detection rate. Of the prostate cancers subsequently found, 93% were low grade (≤3+4). In this low risk group of patients, increasing the follow-up time interval should be considered in the appropriate clinical setting.
doi:10.5152/dir.2014.13345
PMCID: PMC4289157  PMID: 24509182
3.  Vandetanib in Children and Adolescents with Multiple Endocrine Neoplasia Type 2B Associated Medullary Thyroid Carcinoma 
Purpose
Medullary thyroid carcinoma (MTC) is a manifestation of multiple endocrine neoplasia type 2 (MEN2) syndromes caused by germline, activating mutations in the RET proto-oncogene. Vandetanib, a VEGF and EGF receptor inhibitor, blocks RET tyrosine kinase activity and is active in adults with hereditary MTC.
Experimental Design
We conducted a phase I/II trial of vandetanib for children (5–12 years) and adolescents (13–18 years) with MTC to define a recommended dose and assess anti-tumor activity. The starting dose was 100 mg/m2 administered orally, once daily, continuously for 28 day treatment cycles. The dose could be escalated to 150 mg/m2/d after 2 cycles. Radiographic response to vandetanib was quantified using RECIST(v1.0), biomarker response was measured by comparing post-treatment serum calcitonin and carcinoembryonic antigen (CEA) levels to baseline, and a patient reported outcome was used to assess clinical benefit.
Results
Sixteen patients with locally advanced or metastatic MTC received vandetanib for a median (range) 27 (2–52) cycles. Eleven patients remain on protocol therapy. Diarrhea was the primary dose-limiting toxicity. In subjects with M918T RET germline mutations (n=15) the confirmed objective partial response rate was 47% (exact 95%CI, 21%, 75%). Biomarker partial response was confirmed for calcitonin in twelve subjects and for CEA in eight subjects.
Conclusion
Using an innovative trial design and selecting patients based on target gene expression, we conclude that vandetanib 100 mg/m2/d is a well tolerated and highly active new treatment for children and adolescents with MEN2B and locally advanced or metastatic MTC.
doi:10.1158/1078-0432.CCR-13-0071
PMCID: PMC4274128  PMID: 23766359
MEN2B; Medullary Thyroid Carcinoma; Phase 1/2 Trial; Rare Disease; Vandetanib
4.  New Syndrome of Paraganglioma and Somatostatinoma Associated With Polycythemia 
Journal of Clinical Oncology  2013;31(13):1690-1698.
Purpose
The occurrence of ≥ two distinct types of tumors, one of them paraganglioma (PGL), is unusual in an individual patient, except in hereditary cancer syndromes.
Patients and Methods
Four unrelated patients were investigated, with thorough clinical evaluation. Plasma and tissue catecholamines and metanephrines were measured by high-performance liquid chromatography. Anatomic and functional imaging were performed for tumor visualization. Germline and tumor tissue DNA were analyzed for hypoxia-inducible factor 2 alpha (HIF2A) mutations. The prolyl hydroxylation and stability of the mutant HIF2α protein, transcriptional activity of mutant HIF2A, and expression of hypoxia-related genes were also investigated. Immunohistochemical staining for HIF1/2α was performed on formalin-fixed, paraffin-embedded tumor tissue.
Results
Patients were found to have polycythemia, multiple PGLs, and duodenal somatostatinomas by imaging or biochemistry with somatic gain-of-function HIF2A mutations. Each patient carried an identical unique mutation in both types of tumors but not in germline DNA. The HIF2A mutations in these patients were clustered adjacent to an oxygen-sensing proline residue, affecting HIF2α interaction with the prolyl hydroxylase domain 2–containing protein, decreasing the hydroxylation of HIF2α, and reducing HIF2α affinity for the von Hippel–Lindau protein and its degradation. An increase in the half-life of HIF2α was associated with upregulation of the hypoxia-related genes EPO, VEGFA, GLUT1, and END1 in tumors.
Conclusion
Our findings indicate the existence of a new syndrome with multiple PGLs and somatostatinomas associated with polycythemia. This new syndrome results from somatic gain-of-function HIF2A mutations, which cause an upregulation of hypoxia-related genes, including EPO and genes important in cancer biology.
doi:10.1200/JCO.2012.47.1912
PMCID: PMC3807138  PMID: 23509317
5.  Prospective Screening in Familial Non-medullary Thyroid Cancer 
Surgery  2013;154(6):10.1016/j.surg.2013.06.019.
Background
Approximately 8% of non-medullary thyroid cancers are familial. The optimal age for screening in familial non-medullary thyroid cancer (FNMTC) is unknown.
Method
Kindreds with FNMTC (2 or more first-degree relatives affected) were prospectively screened by thyroid ultrasound.
Results
Fifteen kindreds showed an overall prevalence of thyroid nodule(s) ≥ 5 mm of 44.4% at screening; 19.2% in the second generation, and 90% in the generation anterior to the index case. The youngest age of detection was 10 years for thyroid nodules and 18 years for thyroid cancer. Thyroid nodule microcalcification at screening was associated with a higher risk of cancer (p < 0.05). Family members diagnosed with thyroid cancer by ultrasound screening were diagnosed at a younger age and had a lower rate of extrathyroidal invasion (p < 0.05).
Conclusions
In FNMTC, first-degree relatives 10 years or older, including the generation anterior to the index case, should have thyroid ultrasound screening. This may result in earlier diagnosis.
doi:10.1016/j.surg.2013.06.019
PMCID: PMC3857607  PMID: 23978593
Familial non-medullary thyroid cancer; screening; thyroid nodule
6.  Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancer uncovers rare somatic mutations in TNK2 and DDR1 
BMC Cancer  2014;14(1):884.
Background
Endometrial cancer (EC) is the 8th leading cause of cancer death amongst American women. Most ECs are endometrioid, serous, or clear cell carcinomas, or an admixture of histologies. Serous and clear ECs are clinically aggressive tumors for which alternative therapeutic approaches are needed. The purpose of this study was to search for somatic mutations in the tyrosine kinome of serous and clear cell ECs, because mutated kinases can point to potential therapeutic targets.
Methods
In a mutation discovery screen, we PCR amplified and Sanger sequenced the exons encoding the catalytic domains of 86 tyrosine kinases from 24 serous, 11 clear cell, and 5 mixed histology ECs. For somatically mutated genes, we next sequenced the remaining coding exons from the 40 discovery screen tumors and sequenced all coding exons from another 72 ECs (10 clear cell, 21 serous, 41 endometrioid). We assessed the copy number of mutated kinases in this cohort of 112 tumors using quantitative real time PCR, and we used immunoblotting to measure expression of these kinases in endometrial cancer cell lines.
Results
Overall, we identified somatic mutations in TNK2 (tyrosine kinase non-receptor, 2) and DDR1 (discoidin domain receptor tyrosine kinase 1) in 5.3% (6 of 112) and 2.7% (3 of 112) of ECs. Copy number gains of TNK2 and DDR1 were identified in another 4.5% and 0.9% of 112 cases respectively. Immunoblotting confirmed TNK2 and DDR1 expression in endometrial cancer cell lines. Three of five missense mutations in TNK2 and one of two missense mutations in DDR1 are predicted to impact protein function by two or more in silico algorithms. The TNK2P761Rfs*72 frameshift mutation was recurrent in EC, and the DDR1R570Q missense mutation was recurrent across tumor types.
Conclusions
This is the first study to systematically search for mutations in the tyrosine kinome in clear cell endometrial tumors. Our findings indicate that high-frequency somatic mutations in the catalytic domains of the tyrosine kinome are rare in clear cell ECs. We uncovered ten new mutations in TNK2 and DDR1 within serous and endometrioid ECs, thus providing novel insights into the mutation spectrum of each gene in EC.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-884) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-884
PMCID: PMC4258955  PMID: 25427824
Endometrial; Cancer; Mutation; TNK2; ACK1; DDR1; Copy number; Tyrosine kinase; Tyrosine kinome
7.  A Novel Germline Mutation in BAP1 Predisposes to Familial Clear-Cell Renal Cell Carcinoma 
Molecular cancer research : MCR  2013;11(9):1061-1071.
Renal cell carcinoma (RCC) clusters in some families. Familial RCC arises from mutations in several genes, including VHL, which is also mutated in sporadic RCC. However, a significant percentage of familial RCC remains unexplained. Recently, we discovered that the BAP1 gene is mutated in sporadic RCC. BAP1, which encodes a nuclear deubiquitinase, is a two-hit tumor suppressor gene. Somatic BAP1 mutations are associated with high-grade ccRCC and poor patient outcomes. To determine whether BAP1 predisposes to familial RCC, we sequenced the BAP1 gene in 83 unrelated probands with unexplained familial RCC. We identified a novel variant (c.41T>A; p.L14H), which cosegregated with the RCC phenotype. The p.L14H variant disrupts a highly conserved residue in the catalytic domain, a domain frequently targeted by missense mutations. The family with the BAP1 variant was characterized by early-onset clear cell RCC, occasionally of high Fuhrman grade, and lacked other features that characterize von Hippel-Lindau syndrome. These findings suggest that BAP1 is a familial RCC predisposing gene.
doi:10.1158/1541-7786.MCR-13-0111
PMCID: PMC4211292  PMID: 23709298
renal cell carcinoma; BAP1; cancer; inherited RCC; predisposition; tumor suppressor
8.  Phase II and Biomarker Study of the Dual MET/VEGFR2 Inhibitor Foretinib in Patients With Papillary Renal Cell Carcinoma 
Journal of Clinical Oncology  2012;31(2):181-186.
Purpose
Foretinib is an oral multikinase inhibitor targeting MET, VEGF, RON, AXL, and TIE-2 receptors. Activating mutations or amplifications in MET have been described in patients with papillary renal cell carcinoma (PRCC). We aimed to evaluate the efficacy and safety of foretinib in patients with PRCC.
Patients and Methods
Patients were enrolled onto the study in two cohorts with different dosing schedules of foretinib: cohort A, 240 mg once per day on days 1 through 5 every 14 days (intermittent arm); cohort B, 80 mg daily (daily dosing arm). Patients were stratified on the basis of MET pathway activation (germline or somatic MET mutation, MET [7q31] amplification, or gain of chromosome 7). The primary end point was overall response rate (ORR).
Results
Overall, 74 patients were enrolled, with 37 in each dosing cohort. ORR by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0 was 13.5%, median progression-free survival was 9.3 months, and median overall survival was not reached. The presence of a germline MET mutation was highly predictive of a response (five of 10 v five of 57 patients with and without germline MET mutations, respectively). The most frequent adverse events of any grade associated with foretinib were fatigue, hypertension, gastrointestinal toxicities, and nonfatal pulmonary emboli.
Conclusion
Foretinib demonstrated activity in patients with advanced PRCC with a manageable toxicity profile and a high response rate in patients with germline MET mutations.
doi:10.1200/JCO.2012.43.3383
PMCID: PMC3532390  PMID: 23213094
9.  Germline PTEN Mutation Cowden Syndrome: An Under-Appreciated Form of Hereditary Kidney Cancer 
The Journal of urology  2013;190(6):1990-1998.
Introduction
Cowden syndrome (CS) is a hereditary cancer syndrome associated with a germline mutation in PTEN. Patients are predisposed to multiple malignancies including renal cell carcinoma (RCC).
Methods
Patients with CS were evaluated as part of a clinical protocol. Those with a history of RCC underwent review of clinical features, tumor characteristics, and family history. Renal tumors were evaluated for loss of heterozygosity (LOH).
Results
Among 24 CS patients, 4 were identified with RCC (16.7%). Three patients had solitary tumors, two with papillary type I histology and one with clear cell histology. The fourth patient had bilateral, synchronous chromophobe tumors. No patients had a prior family history of RCC. All RCC patients had dermatologic manifestations of CS and had macrocephaly. LOH at the PTEN mutation was identified in 4 tumors (80%). No genotype-phenotype association was found, as the same mutation was identified in different RCC histologies.
Conclusion
RCC is an underappreciated feature of CS. As most patients lack a prior family history or a distinctive RCC histology, recognition of the associated non-renal features should target referral for genetic counseling. PTEN LOH is common in CS renal tumors. Because loss of PTEN can activate mTOR and mTOR inhibitors are FDA-approved to treat RCC, these agents have clinical potential in RCC associated with CS.
doi:10.1016/j.juro.2013.06.012
PMCID: PMC4193347  PMID: 23764071
PTEN; RCC; Cowden syndrome; hereditary; mTOR
10.  An investigation of risk factors for renal cell carcinoma by histologic subtype in two case-control studies 
To investigate whether renal cell carcinoma (RCC) histologic subtypes possess different etiologies, we conducted analyses of established RCC risk factors by subtype (clear cell, papillary, chromophobe) in two case-control studies conducted in the United States (1,217 cases, 1,235 controls) and Europe (1,097 cases, 1,476 controls). Histology was ascertained for 706 U.S. cases (58% of total) and 917 European cases (84%) through a central slide review conducted by a single pathologist. For the remaining cases, histology was abstracted from the original diagnostic pathology report. Case-only analyses were performed to compute odds ratios (ORs) and 95% confidence intervals (CI) summarizing subtype differences by age, sex, and race. Case-control analyses were performed to compute subtype-specific ORs for other risk factors using polytomous regression. In case-only analyses, papillary cases (N=237) were older (OR=1.2, 95% CI=1.1–1.4 per 10-year increase), less likely to be female (OR=0.5, 95 % CI=0.4- 0.8) and more likely to be black (OR=2.6, 95% CI=1.8–3.9) compared to clear cell cases (N=1,524). In case-control analyses, BMI was associated with clear cell (OR=1.2, 95% CI=1.1–1.3 per 5kg/m2 increase) and chromophobe RCC (N=80; OR=1.2, 95% CI=1.1- 1.4), but not papillary RCC (OR=1.1, 95% CI=1.0–1.2; test vs. clear cell, P=0.006). No subtype differences were observed for associations with smoking, hypertension or family history of kidney cancer. Our findings support the existence of distinct age, sex and racial distributions for RCC subtypes, and suggest that the obesity-RCC association differs by histology.
doi:10.1002/ijc.27934
PMCID: PMC3717609  PMID: 23150424
Renal cell carcinoma; histology; case-control studies; body mass index
11.  Comprehensive microRNA Profiling of Prostate Cancer 
Journal of Cancer  2013;4(5):350-357.
MicroRNAs are small non-coding RNA molecules that have been shown to regulate the expression of genes linked to cancer. The relevance of microRNAs in the development, progression and prognosis of prostate cancer is not fully understood. It is also possible that these specific molecules may assist in the recognition of aggressive tumors and the development of new molecular targets. Our study investigated the importance of several microRNAs in cases of prostate cancer from 37 patients that were manually microdissected to obtain pure populations of tumor cells, normal epithelium and adjacent stroma. MicroRNA was extracted for PCR array profiling. Differentially expressed miRNAs for each case were used to compare tumor vs. normal epithelium and tumor-adjacent stroma samples.
Loss of 18 miRNAs (e.g.miR-34c, miR-29b, miR-212 and miR-10b) and upregulation of miR-143 and miR-146b were significantly found in all the tumors in comparison with normal epithelium and/or stroma (p≤ 0.001). A different signature was found in the high grade tumors (Gleason score ≥ 8) when compared with tumors Gleason score 6. Upregulation of miR-122, miR-335, miR-184, miR-193, miR-34, miR-138, miR-373, miR-9, miR-198, miR-144 and miR-215 and downregulation of miR-96, miR-222, miR-148, miR-92, miR-27, miR-125, miR-126, miR-27 were found in the high grade tumors.
MicroRNA profiling in prostate cancer appears to have unique expression patterns in comparison with normal tissue. These differential expressed miRNAs may provide novel diagnostic and prognostic tools that will assist in the recognition of prostate cancers with aggressive behavior.
doi:10.7150/jca.6394
PMCID: PMC3677622  PMID: 23781281
microRNA; Prostate Cancer; biomarkers.
13.  Morphologic and molecular characteristics of uterine leiomyomas in hereditary leiomyomatosis and renal cancer (HLRCC) syndrome 
Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome where affected individuals are predisposed to the development of multiple leiomyomas of the skin and uterus and aggressive forms of kidney cancer. Affected individuals harbor a germline heterozygous loss-of-function mutation of fumarate hydratase (FH) gene. Uterine leiomyomas are present in up to 77 percent of women with this syndrome. Previous studies have shown that inactivation of the FH gene is unusual for non-syndromic leiomyomas. Therefore, it might be possible to distinguish two genetic groups of smooth muscle tumors: the most common group of sporadic uterine leiomyomas without FH gene inactivation and the more unusual group of HLRCC leiomyomas in patients that harbor a germline mutation of FH, although the exact prevalence of hereditary HLRCC is unknown. We reviewed the clinical, morphological and genotypical features of uterine leiomyomas in 19 HLRCC patients with FH germline mutations. Patients with HLRCC syndrome were younger in age compared with regular leiomyomata. DNA was extracted by microdissection and analysis of LOH at 1q43 was performed. Uterine leiomyomas in HLRCC have young age onset and are multiple with size ranging from 1 to 8 cm. Histopathologically, HLRCC leiomyomas frequently had increased cellularity, multinucleated cells and atypia. All cases showed tumor nuclei with large orangiophillic, nucleoi surrounded by a perinucleolar halo similar to the changes found in HLRCC renal cell cancer. Occasional mitoses were found in three cases; however the tumors did not fulfill the criteria for malignancy. Our study also showed that LOH at 1q43 was frequent in HLRCC leiomyomas (8/10 cases), similarly to what it has been previously found in renal cell carcinomas from HLRCC patients. LOH is considered to be the second hit that inactivates the FH gene. We conclude that uterine leiomyomas associated with HLRCC syndrome have characteristic morphologic features. Both, uterine leiomyomas and renal cell carcinoma share some morphological nuclear changes and genotypical features in HLRCC patients. The specific morphological features of the uterine leiomyomas that we describe, may help to identify patients that may be part of the hereditary syndrome.
doi:10.1097/PAS.0b013e31825ec16f
PMCID: PMC3524342  PMID: 23211287
Familiar renal cancer syndrome; Fumarate hydratase mutation; HLRCC; Uterine Leiomyoma
14.  Succinate Dehydrogenase Kidney Cancer (SDH-RCC): An Aggressive Example of the Warburg Effect in Cancer 
The Journal of urology  2012;188(6):10.1016/j.juro.2012.08.030.
Purpose
Recently, a new renal cell cancer (RCC) syndrome has been linked to germline mutation of multiple subunits (SDHB/C/D) of the Krebs cycle enzyme, succinate dehydrogenase. We report our experience with diagnosis, evaluation and treatment of this novel form of hereditary kidney cancer.
Materials and Methods
Patients with suspected hereditary kidney cancer were enrolled on an NCI-IRB approved protocol to study inherited forms of kidney cancer. Individuals from families with germline SDHB, SDHC and SDHD mutations and kidney cancer underwent comprehensive clinical and genetic evaluation.
Results
Fourteen patients from twelve SDHB mutation families were evaluated. Patients presented with RCC at an early age, 33 yrs (range 15–62 yrs), four developed metastatic kidney cancer and some families were found to have no manifestations other than kidney tumors. An additional family with six individuals found to have clear cell RCC that presented at a young average age, 47 yrs (range 40–53yrs), was identified with a germline SDHC mutation (R133X), two of which developed metastatic disease. A patient with a history of carotid body paragangliomas and a very aggressive form of kidney cancer was evaluated from a family with germline SDHD mutation.
Conclusions
SDH-RCC can be an aggressive type of kidney cancer, especially in younger individuals. Although detection and management of early tumors is most often associated with good outcome, based on our initial experience with these patients and our long term experience with HLRCC, we recommend careful surveillance of patients at risk for SDH-RCC and wide surgical excision of renal tumors.
doi:10.1016/j.juro.2012.08.030
PMCID: PMC3856891  PMID: 23083876
renal cell cancer (RCC); hereditary kidney cancer; Krebs cycle; Succinate dehydrogenase
15.  miR-21 Expression in Pregnancy-Associated Breast Cancer: A Possible Marker of Poor Prognosis 
Journal of Cancer  2011;2:67-75.
Aims: microRNAs (miRNAs) are a class of small noncoding RNAs that can act as key modulators in tumorigenesis-related genes. Specifically, it has been suggested that miR-21 overexpression plays a role in the development and progression of breast cancer. So far, the role of miRNAs in pregnancy-associated breast cancer (PABC) has not been investigated.
Methods and Results: We evaluated miR-21 expression by quantitative RT-PCR in 35 patients, 25 with PABC and 10 control breast cancer cases not pregnancy-associated with similar clinicopathological features. We then analyzed protein expression for PTEN, BCL2 and PDCD4 as miR-21 target genes by IHC, and finally correlated the results with patients' clinicopathological features.
Significant overexpression of miR-21 in PABC tumors compared to normal adjacent tissue was found. Overexpression of miR-21 was frequently found in high grade tumors with loss of hormone receptor expression and was significantly associated with positive lymph nodes (p=0.025). In PABC patients, PTEN, BCL2 and PDCD4 target protein expression was decreased in 80%, 76% and 40% respectively.
Conclusion: Our study supports the involvement of miR-21 in breast cancer progression and metastasis formation in PABC implying a role of this miRNA as a marker for poor prognosis in PABC patients.
PMCID: PMC3039223  PMID: 21326627
Pregnancy-associated breast cancer; breast cancer; microRNA; miR-21; PTEN.
16.  Regulatory Effects of microRNA-92 (miR-92) on VHL Gene Expression and the Hypoxic Activation of miR-210 in Clear Cell Renal Cell Carcinoma 
Journal of Cancer  2011;2:515-526.
Background & Aims: In order to understand the role of miRNAs in renal tumorigenesis, we undertook a stepwise approach that included a comprehensive differential miRNA expression analysis for the most common histological subtypes of human renal neoplasms appearing in either sporadic or hereditary forms. We also aimed to test the hypothesis that microRNAs can act as an alternative mechanism of VHL gene inactivation and therefore might be correlated with tumorigenesis in ccRCC. Finally, we wanted to explore whether the well-known hypoxic activation of ccRCC is followed by a specific pattern of miRNA expression.
Methods: Tumor and normal adjacent kidney parenchyma from patients with RCC were tested for microRNA expression. Twenty cases of different histologies were used for profiling by PCR miRNA arrays. For validation, a separate cohort of samples used to test specifically miR92a expression and its involvement in VHL gene mRNA silencing. Finally, miR210 as a marker of hypoxia was evaluated. Expression values were correlated with important clinicopathologic features from the patients.
Results: We identified unique miRNA expression signatures for each histologic subtype of kidney tumors. Expression values for downregulated miRNAs ranged from 0.3-fold (in VHL-clear cell RCC) up to 0.393 fold (in papillary type II (HLRCC) tumors). For the upregulated miRNAs, fold-changes ranged from 2.1 up to 290-fold. Specific patterns together with type-specific profiles were observed. Twenty-three miRNAs were found to be differentially expressed in both sporadic and VHL-dependent ccRCC. Sporadic clear cell tumors showed a unique pattern of 14-miRNA that were absent from the VHL-dependent tumors. These also showed 15 miRNAs specific to the hereditary type. Common miRNAs to both sporadic and hereditary forms included miR-92a and miR-210. For miR-92a, and a striking inverse correlation with VHL mRNA levels was found. For the hypoxia-regulated miR-210, clear cell tumors showed significantly higher expression levels when compared to tumor of non-clear cell histology (9.90-fold vs. 1.36, p<0.001).
Conclusions: microRNA expression seems to be involved in every step of RCC pathogenesis: both as an element for tumor development as well as a consequence of or in response to the initial malignant transformation and part of tumor progression. Our data show consistent disregulation of miRNAs in human kidney cancer, some of which are potentially involved in critical gene silencing in RCC and others that are activated as part of the pathophysiological response in these tumors.
PMCID: PMC3204400  PMID: 22043236
miRNA; VHL; RCC; ccRCC; BHD; HLRCC; TSC; BHD.
17.  Protein Expression Profiling in the Spectrum of Renal Cell Carcinomas 
Journal of Cancer  2010;1:184-196.
In this study, we aimed to evaluate the protein expression profile of a spectrum of renal cell carcinomas (RCC) to find potential biomarkers for disease onset and progression and therefore, prospective therapeutic targets. A 2D-gel based proteomic analysis was used to outline differences in protein levels among different subtypes of renal cell carcinomas, including clear cell carcinomas, papillary lesions, chromophobe tumors and renal oncocytomas. Spot pattern was compared to the corresponding normal kidney from the same patients and distinctive, differentially expressed proteins were characterized by mass spectrometry. Twenty-one protein spots were found differentially expressed between clear cell RCC and normal tissue and 38 spots were found expressed in chromophobe tumors. Eleven proteins were identified, with most differentially expressed -by fold change- between clear cell tumors and the corresponding normal tissue. Two of the identified proteins, Triosephosphate isomerase 1 (TPI-1) and Heat Shock protein 27 (Hsp27), were further validated in a separate set of tumors by immunohistochemistry and expression levels were correlated with clinicopathologic features of the patients. Hsp27 was highly expressed in 82% of the tumors used for validation, and all cases showed strong immunoreactivity for TPI-1. In both Hsp27 and TPI-1, protein expression positively correlated with histologic features of the disease. Our results suggest that the subjacent cytogenetic abnormalities seen in different histological types of RCC are followed by specific changes in protein expression. From these changes, Hsp27 and TPI-1 emerged as potential candidates for the differentiation and prognosis in RCC.
PMCID: PMC2962428  PMID: 20975849
Renal cell carcinoma; proteomics; protein profiling; biomarker; Hsp27; TPI-1
18.  Sequencing of Candidate Chromosome Instability Genes in Endometrial Cancers Reveals Somatic Mutations in ESCO1, CHTF18, and MRE11A 
PLoS ONE  2013;8(6):e63313.
Most endometrial cancers can be classified histologically as endometrioid, serous, or clear cell. Non-endometrioid endometrial cancers (NEECs; serous and clear cell) are the most clinically aggressive of the three major histotypes and are characterized by aneuploidy, a feature of chromosome instability. The genetic alterations that underlie chromosome instability in endometrial cancer are poorly understood. In the present study, we used Sanger sequencing to search for nucleotide variants in the coding exons and splice junctions of 21 candidate chromosome instability genes, including 19 genes implicated in sister chromatid cohesion, from 24 primary, microsatellite-stable NEECs. Somatic mutations were verified by sequencing matched normal DNAs. We subsequently resequenced mutated genes from 41 additional NEECs as well as 42 endometrioid ECs (EECs). We uncovered nonsynonymous somatic mutations in ESCO1, CHTF18, and MRE11A in, respectively, 3.7% (4 of 107), 1.9% (2 of 107), and 1.9% (2 of 107) of endometrial tumors. Overall, 7.7% (5 of 65) of NEECs and 2.4% (1 of 42) of EECs had somatically mutated one or more of the three genes. A subset of mutations are predicted to impact protein function. The co-occurrence of somatic mutations in ESCO1 and CHTF18 was statistically significant (P = 0.0011, two-tailed Fisher's exact test). This is the first report of somatic mutations within ESCO1 and CHTF18 in endometrial tumors and of MRE11A mutations in microsatellite-stable endometrial tumors. Our findings warrant future studies to determine whether these mutations are driver events that contribute to the pathogenesis of endometrial cancer.
doi:10.1371/journal.pone.0063313
PMCID: PMC3670891  PMID: 23755103
19.  Enhanced homing permeability and retention of bone marrow stromal cells (BMSC) by non-invasive pulsed focused ultrasound 
Stem Cells (Dayton, Ohio)  2012;30(6):1216-1227.
Bone marrow stromal cells (BMSC) have shown significant promise in the treatment of disease, but their therapeutic efficacy is often limited by inefficient homing of systemically-administered cells, which results in low numbers of cells accumulating at sites of pathology. BMSC home to areas of inflammation where local expression of integrins and chemokine gradients are present. We demonstrated that non-destructive pulsed focused ultrasound (pFUS) exposures that emphasize the mechanical effects of ultrasound-tissue interactions induced local and transient elevations of chemoattractants (i.e., cytokines, integrins, and growth factors) in the murine kidney. pFUS-induced upregulation of cytokines occurred through approximately 1 day post-treatment and returned to contralateral kidney levels by day 3. This window of significant increases in cytokine expression was accompanied by local increases of other trophic factors and integrins that have been shown to promote BMSC homing. When BMSC were administered intravenously following pFUS treatment to a single kidney, enhanced homing, permeability, and retention of BMSC was observed in the treated kidney versus the contralateral kidney. Histological analysis revealed up to 8 times more BMSC in the peritubular regions of the treated kidneys on days 1 and 3 post-treatment. Furthermore, cytokine levels in pFUS-treated kidneys following BMSC administration were found to be similar to controls, suggesting modulation of cytokine levels by BMSC. pFUS could potentially improve cell-based therapies as a noninvasive modality to target BMSC homing by establishing local chemoattractant gradients and increasing expression of integrins to enhance tropism of BMSC toward treated tissues.
doi:10.1002/stem.1099
PMCID: PMC3356926  PMID: 22593018
mesenchymal stem cells; pulsed focused ultrasound; high intensity focused ultrasound; mechanotransduction; stem cell migration; cytokines; enhanced homing permeability and retention (EHPR); ICAM; VCAM; integrins; ferumoxides; protamine
20.  Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes 
Nature genetics  2012;44(12):1310-1315.
Endometrial cancer is the 6th most commonly diagnosed cancer among women worldwide, causing ~74,000 deaths annually 1. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology 2-4. We used whole exome sequencing (WES) to comprehensively search for somatic mutations within ~22,000 protein-encoding genes among 13 primary serous endometrial tumors. We subsequently resequenced 18 genes that were mutated in more than one tumor, and/or were genes that formed an enriched functional grouping, from 40 additional serous tumors. We identified high frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%). Overall, 36.5% of serous tumors had mutated a chromatin-remodeling gene and 35% had mutated a ubiquitin ligase complex gene, implicating the frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.
doi:10.1038/ng.2455
PMCID: PMC3515204  PMID: 23104009
21.  Documenting the location of systematic transrectal ultrasound-guided prostate biopsies: correlation with multi-parametric MRI 
Cancer Imaging  2011;11(1):31-36.
Abstract
During transrectal ultrasound (TRUS)-guided prostate biopsies, the actual location of the biopsy site is rarely documented. Here, we demonstrate the capability of TRUS-magnetic resonance imaging (MRI) image fusion to document the biopsy site and correlate biopsy results with multi-parametric MRI findings. Fifty consecutive patients (median age 61 years) with a median prostate-specific antigen (PSA) level of 5.8 ng/ml underwent 12-core TRUS-guided biopsy of the prostate. Pre-procedural T2-weighted magnetic resonance images were fused to TRUS. A disposable needle guide with miniature tracking sensors was attached to the TRUS probe to enable fusion with MRI. Real-time TRUS images during biopsy and the corresponding tracking information were recorded. Each biopsy site was superimposed onto the MRI. Each biopsy site was classified as positive or negative for cancer based on the results of each MRI sequence. Sensitivity, specificity, and receiver operating curve (ROC) area under the curve (AUC) values were calculated for multi-parametric MRI. Gleason scores for each multi-parametric MRI pattern were also evaluated. Six hundred and 5 systemic biopsy cores were analyzed in 50 patients, of whom 20 patients had 56 positive cores. MRI identified 34 of 56 positive cores. Overall, sensitivity, specificity, and ROC area values for multi-parametric MRI were 0.607, 0.727, 0.667, respectively. TRUS-MRI fusion after biopsy can be used to document the location of each biopsy site, which can then be correlated with MRI findings. Based on correlation with tracked biopsies, T2-weighted MRI and apparent diffusion coefficient maps derived from diffusion-weighted MRI are the most sensitive sequences, whereas the addition of delayed contrast enhancement MRI and three-dimensional magnetic resonance spectroscopy demonstrated higher specificity consistent with results obtained using radical prostatectomy specimens.
doi:10.1102/1470-7330.2011.0007
PMCID: PMC3080122  PMID: 21450548
Prostate cancer; multi-parametric MR imaging; TRUS/MRI fusion tracking
23.  Translocation Renal Cell Carcinomas in Adults: A Single Institution Experience 
Translocation renal cell carcinoma is a newly recognized subtype of renal cell carcinoma (RCC) with chromosomal translocations involving TFE3 (Xp11.2) or, less frequently, TFEB (6p21). Xp11 translocation RCC was originally described as a pediatric neoplasm representing 20–40% of pediatric RCCs with a much lower frequency in the adult population. TFEB translocation RCC is very rare, with approximately 10 cases reported in the literature. Here, we describe the clinicopathological features of adult translocation RCC from a single institution. Utilizing tissue microarray (TMA), immunohistochemistry, cytogenetic examination, and FISH, we identified 6 (~5%) cases of TFE3 translocation RCC and 1 (<1%) case of TFEB translocation RCC in 121 consecutive adult renal cell carcinoma cases between 2001 and 2009. Our results suggest that weak TFE3 staining of a significant proportion of RCC cases may due to expression of the full length TFE3 protein rather than the chimeric fusion protein resulting from chromosomal translocation.
doi:10.1097/PAS.0b013e31824f24a6
PMCID: PMC3527899  PMID: 22446944
translocation renal cell carcinoma; TFE3; TFEB
24.  11C-Acetate PET/CT Imaging in Localized Prostate Cancer: A study with MRI and Histopathologic Correlation 
This work characterizes the uptake of 11C-Acetate in prostate cancer (PCa), benign prostate hyperplasia (BPH) and normal prostate tissue in comparison with multi-parametric MRI, whole mount histopathology and clinical markers, to evaluate its potential utility for delineating intra-prostatic tumors in a population of patients with localized PCa.
METHODS
39 men with presumed localized PCa underwent dynamic/static abdomen-pelvic 11C-Acetate PET/CT for 30-minutes and 3T multi-parametric (MP) MRI prior to prostatectomy. PET/CT images were registered to MRI using pelvic bones for initial rotation-translation, followed by manual adjustments to account for prostate motion and deformation from the MRI endorectal coil. Whole-mount pathology specimens were sectioned using an MRI-based patient specific mold resulting in improved registration between the MRI, PET and pathology. 11C-Acetate PET standardized uptake values were compared with MP-MRI and pathology.
RESULTS
11C-Acetate uptake was rapid but reversible, peaking at 3–5 minutes post-injection and reaching a relative plateau at ~10 minutes. The average SUVmax(10–12min) of tumors was significantly higher than that of normal prostate tissue (4.4±2.05, range 1.8–9.2 vs. 2.1±0.94, range 0.7–3.4; p<0.001); however it was not significantly different from benign prostatic hyperplasia (4.8±2.01; range 1.8–8.8). A sector-based comparison with histopathology, including all tumors > 0.5 cm, revealed a sensitivity and specificity of 61.6 % and 80.0 % for 11C-Acetate PET/CT, and 82.3% and 95.1% for MRI, respectively. Considering only tumors >0.9 cm the 11C-Acetate accuracy was comparable to that of MRI. In a small cohort (n=9), 11C-Acetate uptake was independent of fatty acid synthase expression based on immunohistochemistry.
CONCLUSION
11C-Acetate PET/CT demonstrates higher uptake in tumor foci than normal prostate tissue; however 11C-Acetate uptake in tumors is similar to BPH nodules. While 11C-Acetate PET/CT is not likely to have utility as an independent modality for evaluation of localized PCa, the high uptake in tumors may make it useful for monitoring focal therapy, where tissue damage after therapy may limit anatomic imaging methods.
doi:10.2967/jnumed.111.096032
PMCID: PMC3526004  PMID: 22343504
Prostate cancer; 11C-Acetate PET; Multi-parametric prostate MRI
25.  gp100 Peptide Vaccine and Interleukin-2 in Patients with Advanced Melanoma 
The New England journal of medicine  2011;364(22):2119-2127.
Background
Stimulating an immune response against cancer with the use of vaccines remains a challenge. We hypothesized that combining a melanoma vaccine with interleukin-2, an immune activating agent, could improve outcomes. In a previous phase 2 study, patients with metastatic melanoma receiving high-dose interleukin-2 plus the gp100:209–217(210M) peptide vaccine had a higher rate of response than the rate that is expected among patients who are treated with interleukin-2 alone.
Methods
We conducted a randomized, phase 3 trial involving 185 patients at 21 centers. Eligibility criteria included stage IV or locally advanced stage III cutaneous melanoma, expression of HLA⋆A0201, an absence of brain metastases, and suitability for high-dose interleukin-2 therapy. Patients were randomly assigned to receive interleukin-2 alone (720,000 IU per kilogram of body weight per dose) or gp100:209–217(210M) plus incomplete Freund’s adjuvant (Montanide ISA-51) once per cycle, followed by interleukin-2. The primary end point was clinical response. Secondary end points included toxic effects and progression-free survival.
Results
The treatment groups were well balanced with respect to baseline characteristics and received a similar amount of interleukin-2 per cycle. The toxic effects were consistent with those expected with interleukin-2 therapy. The vaccine–interleukin-2 group, as compared with the interleukin-2–only group, had a significant improvement in centrally verified overall clinical response (16% vs. 6%, P = 0.03), as well as longer progression-free survival (2.2 months; 95% confidence interval [CI], 1.7 to 3.9 vs. 1.6 months; 95% CI, 1.5 to 1.8; P = 0.008). The median overall survival was also longer in the vaccine–interleukin-2 group than in the interleukin-2–only group (17.8 months; 95% CI, 11.9 to 25.8 vs. 11.1 months; 95% CI, 8.7 to 16.3; P = 0.06).
Conclusions
In patients with advanced melanoma, the response rate was higher and progression-free survival longer with vaccine and interleukin-2 than with interleukin-2 alone. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00019682.)
doi:10.1056/NEJMoa1012863
PMCID: PMC3517182  PMID: 21631324

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