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2.  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.
PMCID: PMC3524342  PMID: 23211287
Familiar renal cancer syndrome; Fumarate hydratase mutation; HLRCC; Uterine Leiomyoma
3.  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.
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.
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.
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.
PMCID: PMC3856891  PMID: 23083876
renal cell cancer (RCC); hereditary kidney cancer; Krebs cycle; Succinate dehydrogenase
4.  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.
PMCID: PMC3677622  PMID: 23781281
microRNA; Prostate Cancer; biomarkers.
5.  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.
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
6.  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.
PMCID: PMC3515204  PMID: 23104009
8.  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.
9.  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
10.  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.
PMCID: PMC3527899  PMID: 22446944
translocation renal cell carcinoma; TFE3; TFEB
11.  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.
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.
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.
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.
PMCID: PMC3526004  PMID: 22343504
Prostate cancer; 11C-Acetate PET; Multi-parametric prostate MRI
12.  Ectopic growth hormone-releasing hormone secretion by a bronchial carcinoid tumor: clinical experience following tumor resection and long-acting octreotide therapy 
Pituitary  2012;15(2):260-265.
Acromegaly resulting from the ectopic secretion of growth hormone-releasing hormone (GHRH) is rare. We present a case of acromegaly secondary to proven GHRH-secretion by a bronchial carcinoid tumor in a type 1 diabetic subject and document the clinical course pre- and post-resection of the tumor and of subsequent octreotide therapy. A 54-year-old Caucasian man was referred for evaluation of acromegalic symptoms and significantly increased insulin requirements. He had a history of left lung surgery 20 years prior for hemoptysis. Initial laboratory results indicated acromegaly. Fasting serum growth hormone (GH): 26.1 ng/mL (0–5 ng/mL), insulin-like growth factor 1 (IGF-1): 635 ng/mL (87–283 ng/mL), GH at 60 min post-ingestion of 75 grams of oral glucose during a glucose tolerance test: 8.3 ng/mL (normal<1 ng/mL). Pituitary magnetic resonance imaging (MRI) revealed diffuse pituitary enlargement without adenoma. A 4.4 cm left hilar mass was noted on chest computed tomography (CT) scan. Further evaluation for a suspected GHRH-secreting neuroendocrine tumor was pursued. Plasma GHRH level was elevated: 198 pg/mL (<50 pg/mL). Octreoscan showed radiolabelled-octreotide uptake in the left lung mass and pituitary gland. Surgical resection of the lung mass was performed. Immunohisto-chemical study of the tumor tissue indicated a neuroendocrine tumor secreting GHRH. Postoperatively, serum GHRH, GH and IGF-1 levels fell precipitously. At 10 months, IGF-1 levels were mildly elevated and 7 months of 10 mg long-acting octreotide therapy (Sandostatin® LAR®) was trialed. At 20 months, off octreotide, serum IGF-1 levels had normalized, acromegalic features were receding, and the patient's daily insulin requirements had decreased by 57%.
PMCID: PMC3470463  PMID: 20379782
Acromegaly; Ectopic acromegaly; Growth hormone-releasing hormone; Neuroendocrine tumor; Carcinoid; Somatostatin analogue; Octreotide
13.  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
14.  Metastatic pheochromocytoma: clinical, genetic, and histopathologic characteristics 
Pheochromocytomas are tumors arising from chromaffin tissue located in the adrenal medulla associated with typical symptoms and signs which may occasionally develop metastases, which are defined as the presence of tumor cells at sites where these cells are not found. This retrospective analysis was focused on clinical, genetic, and histopathologic characteristics of primary metastatic versus primary benign pheochromocytomas.
Materials and methods
We identified 41 subjects with metastatic pheochromocytoma and 108 subjects with apparently benign pheochromocytoma. We assessed dimension and biochemical profile of the primary tumor, age at presentation, and time to develop metastases.
Subjects with metastatic pheochromocytoma presented at a significantly younger age (41.4±14.7 vs. 50.2±13.7 years; P<0.001), with larger primary tumors (8.38±3.27 cm vs. 6.18±2.75 cm; P<0.001) and secreted more frequently norepinephrine (95.1% vs. 83.3 %; P=0.046) compared to subjects with apparently benign pheochromocytomas. No significant differences were found in the incidence of genetic mutations in both groups of subjects (25.7 % in the metastatic group and 14.7 % in the benign group; P=0.13). From available histopathologic markers of potential malignancy, only necrosis occurred more frequently in subjects with metastatic pheochromocytoma (27.6 % vs. 0 %; P<0.001). The median time to develop metastases was 3.6 years with the longest interval 24 years.
In conclusion, regardless of a genetic background, the size of a primary pheochromocytoma and age of its first presentation are two independent risk factors associated with the development of metastatic disease.
PMCID: PMC3170415  PMID: 21692797
malignant pheochromocytoma; norepinephrine; epinephrine
15.  Documenting the location of systematic transrectal ultrasound-guided prostate biopsies: correlation with multi-parametric MRI 
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.
PMCID: PMC3080122  PMID: 21450548
Prostate cancer; multi-parametric MR imaging; TRUS/MRI fusion tracking
16.  Intrauterine growth retardation associated with precocious puberty and Sertoli cell hyperplasia 
The original description of patients with Russell-Silver syndrome included precocious puberty, the mechanism of which was unclear. We describe a child with a Russell-Silver syndrome-like phenotype who presented with precocious puberty that was associated with hyperplasia of the Sertoli cells. The patient was found to have an immature cryptorchid testicle; hyperplastic Sertoli cells were also aneuploid carrying trisomy 8. This chromosomal abnormality was present in Sertoli cells only and could not be detected in peripheral lymphocytes, tunica vaginalis, or other, normal, testicular tissue. Sertoli cells in culture showed excess aromatization providing an explanation for the rapid advancement of the patient’s bone age. We conclude that in a patient with a Russell-Silver syndrome-like phenotype, Sertoli cell hyperplasia was associated with somatic trisomy 8, increased aromatization and gonadotropin-independent precocious puberty.
PMCID: PMC3412584  PMID: 20411478
17.  A unique spectrum of somatic PIK3CA (p110α) mutations within primary endometrial carcinomas 
The goal of this study was to comprehensively define the incidence of mutations in all exons of PIK3CA in both endometrioid endometrial cancer (EEC) and non-endometrioid endometrial cancer (NEEC).
Experimental design
We resequenced all coding exons of PIK3CA and PTEN, and exons 1 and 2 of KRAS, from 108 primary endometrial tumors. Somatic mutations were confirmed by sequencing matched normal DNAs. The biochemical properties of a subset of novel PIK3CA mutations were determined by exogenously expressing wildtype and mutant constructs in U2OS cells and measuring levels of AKTSer473 phosphorylation.
Somatic PIK3CA mutations were detected in 52.4% of 42 EECs and 33.3% of 66 NEECs. Half (29 of 58) of all nonsynonymous PIK3CA mutations were in exons 1–7 and half were in exons 9 and 20. The exons 1–7 mutations localized to the ABD, ABD-RBD linker and C2 domains of p110α. Within these regions, Arg88, Arg93, Gly106, Lys111, Glu365, and Glu453, were recurrently mutated; Arg88, Arg93 and Lys111 formed mutation hotspots. The p110α-R93W, -G106R, -G106V, -K111E, -delP449-L455, and -E453K mutants led to increased levels of phospho-AKTSer473 compared to wild-type p110α. Overall, 62% of exons 1–7 PIK3CA mutants and 64% of exon 9–20 PIK3CA mutants were activating; 72% of exon 1–7 mutations have not previously been reported in endometrial cancer.
Our study identified a new subgroup of endometrial cancer patients with activating mutations in the amino-terminal domains of p110α; these patients might be appropriate for consideration in clinical trials of targeted therapies directed against the PI3K pathway.
PMCID: PMC3060282  PMID: 21266528
Endometrial; cancer; PIK3CA; mutation; spectrum
18.  Documenting the location of prostate biopsies with image fusion 
Bju International  2011;107(1):53-57.
To develop a system that documents the location of transrectal ultrasonography (TRUS)-guided prostate biopsies by fusing them to MRI scans obtained prior to biopsy, as the actual location of prostate biopsies is rarely known.
Fifty patients (median age 61) with a median prostate-specific antigen (PSA) of 5.8 ng/ml underwent 3T endorectal coil MRI prior to biopsy. 3D TRUS images were obtained just prior to standard TRUS-guided 12-core sextant biopsies wherein an electromagnetic positioning device was attached to the needle guide and TRUS probe in order to track the position of each needle pass. The 3D-TRUS image documenting the location of each biopsy was fused electronically to the T2-weighted MRI. Each biopsy needle track was marked on the TRUS images and these were then transposed onto the MRI. Each biopsy site was classified pathologically as positive or negative for cancer and the Gleason score was determined.
The location of all (n = 605) needle biopsy tracks was successfully documented on the T2-weighted (T2W) MRI. Among 50 patients, 20 had 56 positive cores. At the sites of biopsy, T2W signal was considered ‘positive’ for cancer (i.e. low in signal intensity) in 34 of 56 sites.
It is feasible to document the location of TRUS-guided prostate biopsies on pre-procedure MRI by fusing the pre-procedure TRUS to an endorectal coil MRI using electromagnetic needle tracking. This procedure may be useful in documenting the location of prior biopsies, improving quality control and thereby avoiding under-sampling of the prostate as well as directing subsequent biopsies to regions of the prostate not previously sampled.
PMCID: PMC3272674  PMID: 20590543
prostate cancer; MRI; TRUS-guided sextant biopsy; MRI–TRUS fusion; biopsy mapping
19.  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.
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).
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.
Foretinib demonstrated activity in patients with advanced PRCC with a manageable toxicity profile and a high response rate in patients with germline MET mutations.
PMCID: PMC3532390  PMID: 23213094
20.  Expression of Benign and Malignant Thyroid Tissue in Ovarian Teratomas and the Importance of Multimodal Management as Illustrated by a BRAF-Positive Follicular Variant of Papillary Thyroid Cancer 
Thyroid  2010;20(9):981-987.
The most common type of ovarian germ cell tumor is the teratoma. Thyroid tissue, both benign and malignant, may be a component of an ovarian teratoma. Here we review this topic and illustrate major features by presenting multimodal management of a patient with BRAF-positive disseminated follicular thyroid cancer arising in an ovarian teratoma.
Malignant thyroid tissue is often difficult to distinguish from benign thyroid tissue arising in ovarian teratomas. Preoperatively, an elevated thyroglobulin (Tg) level, laboratory or clinical evidence of hyperthyroidism, or ultrasonography appearance of “struma pearl” should prompt referral to oncologist for surgical management of a possibly malignant ovarian teratoma. Postoperatively, tumor tissue should be referred to pathologists experienced with differentiating benign from malignant struma ovarii. Once diagnosed, treatment of this rare condition should be handled by a team of specialists with combined treatment modalities. We cared for woman with disseminated thyroid cancer arising in an ovarian teratoma whose history illustrates the complexity of managing ovarian teratomas with malignant thyroid tissue. At age 33 she had an intraoperative rupture of an ovarian cyst, thought to be struma ovarii. During her next pregnancy, pelvic masses were noted; biopsies revealed well-differentiated papillary thyroid carcinoma, follicular variant. She was euthyroid, but had elevated serum Tg levels. Surgical staging demonstrated widely metastatic intraabdominal dissemination. A thyroidectomy revealed no malignancy. A post-131I treatment scan revealed diffuse uptake throughout the abdomen. She then developed abdominal pain and, on computed tomography, was found to have multiple intraabdominal foci of disease. Serum Tg was 264 ng/mL while on L-thyroxine for hypothyroidism and to obtain thyrotropin suppression. A 18 fluorodeoxyglucose positron emission tomography scan showed no pathological uptake. The tumor was found to be BRAF mutation positive (K601E). She underwent extensive secondary debulking and a second course of 131I with lithium pretreatment. Posttreatment scan revealed diffuse abdominal uptake. Six months posttherapy, the patient is asymptomatic with a serum Tg of 18.1 ng/mL.
Aggressive multimodal management appears to be the most promising approach for malignant thyroid tissue arising in ovarian teratomas.
PMCID: PMC2964358  PMID: 20718682
21.  UOK 262: Fumarate Hydratase (-/-) Hereditary Leiomyomatosis Renal Cell Carcinoma: In Vitro and In Vivo Model of an Aberrant Energy Metabolic Pathway in Human Cancer 
Cancer genetics and cytogenetics  2010;196(1):45-55.
Energy deregulation and abnormalities of tumor cell metabolism are critical issues in our understanding of cancer. Hereditary leiomyomatosis renal cell carcinoma (HLRCC) is an aggressive form of RCC characterized by germline mutation of the Krebs cycle enzyme, fumarate hydratase (FH), and is known to be highly metastatic and unusually lethal. There is significant utility in establishing preclinical cell and xenograft models for study of disorder of energy metabolism as well as development of new therapeutic approaches targeting of tricarboxylic acid (TCA) cycle deficient human cancers. Here we report the first immortal cell line derived from a patient having aggressive HLRCC-associated recurring kidney cancer, designated as UOK 262. We investigated gene expression, chromosome profiles, efflux bioenergetic analysis, mitochondrial ultrastructure, FH catabolic activity, invasiveness, and optimal glucose requirements for in vitro growth. UOK 262 cells have isochromosome 1q [i(1)(q10)] as recurring chromosome abnormality; demonstrate compromised oxidative phosphorylation and in vitro dependence on anaerobic glycolysis consistent with the clinical manifestation of HLRCC. Furthermore the cells display glucose-dependent growth, an elevated rate of lactate efflux, over-expression of the glucose transporter Glut 1 and lactate dehydrogenase (LDH) 5. Mutant FH protein was primarily present in edematous mitochondria, but its catalytic activity was nearly undetectable. UOK 262 xenografts retain the characteristics of HLRCC histopathology. Our findings indicate that the severe compromise of oxidative phosphorylation and rapid glycolytic flux in UOK 262 are an essential feature of this TCA cycle enzyme deficient form of kidney cancer. This tumor model is the embodiment of the “Warburg effect”. UOK 262 provides a unique in vitro and in vivo preclinical mode to study the bioenergetics of the Warburg effect in human cancer.
PMCID: PMC2827193  PMID: 19963135
papillary kidney cancer; oxidative phosphorylation; glycolysis; FH gene; HLRCC: Hereditary Leiomyomatosis Renal Cell Carcinoma
22.  Decrease in CD8+ lymphocyte number and altered cytokine profile in human prostate cancer 
The tumor microenvironment is comprised of multiple cell types arranged in a three-dimensional structure. Interactions amongst the various cell components play an important role in neoplasia, including the inflammatory reaction that occurs as part of the host response. In this study, the regional lymphocyte subpopulations and cytokine profiles associated with prostate cancer were examined using a quantitative imaging approach and expression microarray analysis. Lymphocytes were measured in four different epithelial phenotypes in prostate cancer specimens: carcinoma; prostatic intraepithelial neoplasia (PIN); benign prostate hyperplasia (BPH); and normal epithelium. The data indicate that CD8 positive, cytotoxic T lymphocytes are significantly decreased in regions adjacent to hyperplasia and carcinoma as compared to normal epithelium and PIN. In contrast the relative number of CD4 positive and CD20 positive lymphocytes did not change markedly. Parallel mRNA expression array analysis of the normal and tumor microenvironments identified a distinct cytokine profile in cancer, with 24 dysregulated genes in tumor epithelium and nine altered in tumor-associated stroma. Overall, these data indicate that the spatial distribution of CD8 positive, cytotoxic T lymphocytes is dysregulated in human prostate glands that contain cancer, and cytokine profiles are altered at the mRNA level.
PMCID: PMC3180108  PMID: 21969236
Prostate cancer; lymphocytes; cytokines; histomathematics; histopathology
23.  Association of Germline Mutations in the Fumarate Hydratase Gene and Uterine Fibroids in Women With Hereditary Leiomyomatosis and Renal Cell Cancer 
Archives of dermatology  2008;144(12):1584-1592.
To investigate the risk of uterine fibroids and other reproductive risk factors in women with hereditary leiomyomatosis and renal cell cancer (HLRCC).
Case-control study.
National Institutes of Health, Rockville, Maryland.
A family-based case-control study was conducted between July 1, 2004, and June 30, 2006, including 105 women from families with HLRCC ascertained throughout North America. A telephone interview was conducted with all participants using a standardized questionnaire that elicited information about their menstrual, pregnancy, uterine fibroid, and hormonal contraceptive use history. Diagnosis of uterine fibroids was confirmed by pathologic diagnosis and by medical record review. DNA was extracted from blood samples and was screened for germline mutations in the fumarate hydratase (FH) gene.
Main Outcome Measures
FH germline mutation status, presence of uterine fibroids, age at diagnosis, and symptoms and treatment of uterine fibroids.
Of 105 women, 77 reported a history of uterine fibroids. Regardless of uterine fibroid status, 75 of 105 women had a germline mutation in FH (FHmut positive). The risk of uterine fibroids in FHmut-positive women was statistically significantly increased compared with that in FHmut-negative women (odds ratio [OR], 7.6; 95% confidence interval [CI], 2.9–20.0), as it was among women clinically affected with HLRCC compared with those clinically unaffected with HLRCC (8.6; 3.1–24.0). The median age at uterine fibroid diagnosis for FHmut-positive women (28 years) was significantly younger than that for FHmut-negative women (38 years) (P=.03). Women with a germline mutation in FH or clinically affected with HLRCC reported younger age at menarche (P < .004) compared with FHmut-negative women (P =.02) or women who were clinically unaffected with HLRCC. Women with HLRCC were more likely to have had treatment for uterine fibroids (OR, 4.6; 95% CI, 1.4–15.8), including hysterectomy (P=.02) at an earlier age compared with women who were clinically unaffected with HLRCC.
This study provides the first evidence (to our knowledge) that women with germline mutations in FH and with clinical HLRCC have an increased risk of developing uterine fibroids. These women also have a younger age at uterine fibroid diagnosis and are more likely to have treatment for uterine fibroids at a younger age than women without HLRCC in their families.
PMCID: PMC2937541  PMID: 19075141
24.  Analyses of Resected Human Brain Metastases of Breast Cancer Reveal the Association between Up-regulation of Hexokinase 2 and Poor Prognosis 
Molecular cancer research : MCR  2009;7(9):1438-1445.
Brain metastases of breast cancer appear to be increasing in incidence as systemic therapy improves. Metastatic disease in the brain is associated with high morbidity and mortality. We present the first gene expression analysis of laser captured epithelial cells from resected human brain metastases of breast cancer compared to unlinked primary breast tumors. The tumors were matched for histology, TNM stage and hormone receptor status. Most differentially expressed genes were down-regulated in the brain metastases which included, surprisingly, many genes associated with metastasis. Q-PCR analysis confirmed statistically significant differences or strong trends in the expression of six genes: BMP1, PEDF, LAMγ3, SIAH, STHMN3 and TSPD2. Hexokinase 2 (HK2) was also of interest because of its increased expression in brain metastases. HK2 is important in glucose metabolism and apoptosis. In agreement with our microarray results, HK2 levels (both mRNA and protein) were elevated in a brain metastatic derivative (231-BR) of the human breast carcinoma cell line MDA-MB-231 relative to the parental cell line (231-P), in vitro. Knockdown of HK2 expression in 231-BR cells using shRNA reduced cell proliferation when cultures were maintained in glucose limiting conditions. Finally, HK2 expression was analyzed in a cohort of 123 resected brain metastases of breast cancer. High HK2 expression was significantly associated with poor patient survival post-craniotomy (P=0.028). The data suggest that HK2 overexpression is associated with metastasis to the brain in breast cancer and it may be a therapeutic target.
PMCID: PMC2746883  PMID: 19723875
25.  Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-β signaling 
Molecular Cancer  2010;9:160.
Germline mutations in the FLCN gene are responsible for the development of fibrofolliculomas, lung cysts and renal neoplasia in Birt-Hogg-Dube' (BHD) syndrome. The encoded protein folliculin (FLCN) is conserved across species but contains no classic motifs or domains and its function remains unknown. Somatic mutations or loss of heterozygosity in the remaining wild type copy of the FLCN gene have been found in renal tumors from BHD patients suggesting that FLCN is a classic tumor suppressor gene.
To examine the tumor suppressor function of FLCN, wild-type or mutant FLCN (H255R) was stably expressed in a FLCN-null renal tumor cell line, UOK257, derived from a BHD patient. When these cells were injected into nude mice, tumor development was inversely dependent upon the level of wild-type FLCN expression. We identified genes that were differentially expressed in the cell lines with or without wild-type FLCN, many of which are involved in TGF-β signaling, including TGF-β2 (TGFB2), inhibin β A chain (INHBA), thrombospondin 1 (THBS1), gremlin (GREM1), and SMAD3. In support of the in vitro data, TGFB2, INHBA, THBS1 and SMAD3 expression levels were significantly lower in BHD-associated renal tumors compared with normal kidney tissue. Although receptor mediated SMAD phosphorylation was not affected, basal and maximal TGF-β-induced levels of TGFB2, INHBA and SMAD7 were dramatically reduced in FLCN-null cells compared with FLCN-restored cells. Secreted TGF-β2 and activin A (homo-dimer of INHBA) protein levels were also lower in FLCN-null cells compared with FLCN-restored cells. Consistent with a growth suppressive function, activin A (but not TGF-β2) completely suppressed anchorage-independent growth of FLCN-null UOK257 cells.
Our data demonstrate a role for FLCN in the regulation of key molecules in TGF-β signaling and confirm deregulation of their expression in BHD-associated renal tumors. Thus, deregulation of genes involved in TGF-β signaling by FLCN inactivation is likely to be an important step for tumorigenesis in BHD syndrome.
PMCID: PMC2907329  PMID: 20573232

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