Elwira Bakuła-Zalewska

microRNAs in uterine sarcomas and mixed epithelial-mesenchymal uterine tumors: a preliminary report.

Uterine sarcomas and mixed epithelial–mesenchymal uterine tumors are a heterogeneous group of rare tumors for which there are very few diagnostic markers available. As aberrant microRNA (miRNA) expression patterns represent putative diagnostic cancer markers, we aimed to identify miRNA expression profiles of the major uterine sarcoma subtypes and mixed epithelial–mesenchymal tumors of the uterus. Eighty-eight miRNAs were assessed by quantitative RT-PCR in cancerous and non-cancerous tissue samples collected from 29 patients with endometrial sarcoma, leiomyosarcoma, and mixed epithelial–mesenchymal tumors. Tumor and control samples significantly (Pu2009<u20090.05) differed in the expression of miR-23b, miR-1, let-7f, and let-7c in endometrial sarcomas, and miR-1, let-7c, miR-133b, let-7b, miR-143, let-7a, let-7d, let-7e, let-7g, miR-222, let-7i, and miR-214 in mixed epithelial–mesenchymal tumors. All the significantly changed miRNAs were down-regulated in the malignant tissues as compared to their normal counterparts. This may suggest their tumor suppressor role in these malignancies. No statistically significant changes in miRNA expression levels were found between leiomyosarcoma tumors and controls. The identified miRNAs warrant further studies as valuable candidate markers for the differential diagnosis of uterine sarcomas from benign uterine lesions and between uterine sarcoma subtypes.

Identification of suitable reference genes for gene expression measurement in uterine sarcoma and carcinosarcoma tumors

OBJECTIVEnTo date no suitable reference genes have been identified in carcinosarcomas and non-epithelial malignant tumors of corpus uteri for normalizing real-time RT-PCR (qRT-PCR) assays. The purpose of this study was to select appropriate references for gene expression studies in these tumors.nnnMATERIALS AND METHODSnWe used RNA extracts from 75 tissue samples, representing 50 tumors and 25 fragments of normal uterine tissues obtained from 50 patients treated for mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. qRT-PCR for five potential reference (housekeeping) genes, namely B2M, HMBS, HPRT1, TBP and UBC, was performed. The expression stability of these genes was assayed using geNorm software application.nnnRESULTSnThe analysis of gene expression data with geNorm identified HPRT1 as the most stable reference gene, followed by UBC and HMBS, for all the investigated tissues. When stratified by disease, the results still pointed at HPRT1 as the gene that retained the greatest robustness in mixed tumors as well as in smooth muscle and stromal sarcomas.nnnCONCLUSIONSnOur work is the first report on reference gene selection for qRT-PCR applications in mixed tumors, smooth muscle sarcoma and stromal sarcoma of the uterus. A ranking of candidate genes stability values for the three types of tumors is provided and might serve as a valuable guide for future gene expression studies of these rare entities.

Uterine tumors resembling ovarian sex cord tumors, a clinicopathologic study of six cases

Uterine tumors resembling ovarian sex cord tumors (UTROSCTs) cause difficulties, both with respect to diagnosis as well as to the nomenclature. They belong to the group of low-grade malignant neoplasms, and their clinical course likely depends on the percentage of the sex cord-like component. Morphologically, they can be divided into type I and type II with less or more than 50% of sex cord-like areas, respectively. Six patients with an age range of 24 to 63 years underwent the treatment for primary UTROSCT at the Cancer Center and Institute of Oncology in Warsaw, Poland, between 2000 and 2011. In addition to the surgery, 4 patients were treated with gestagens. Biopsies or excisions from the tumors were examined microscopically and immunohistochemically. Two cases were classified as type I, and 4 cases, as type II tumors. The tumor size ranged from 3 to 24 cm. The sex cord component varied from 25% to 70%. By immunohistochemical examination, the sex cord-like component was calretinin positive, whereas the stromal component was positive for CD10 and negative for h-caldesmon in all the cases studied. In addition, progesterone receptor positivity was found in all the cases, and 4 tumors were positive for smooth muscle actin, cytokeratin AE1/3, and inhibin. No recurrences were noted in any of the 6 patients over 3 to 14.5 years of follow-up period. A correct subclassification of sarcomas of UTROSCT type is of crucial importance because most patients with this rare neoplasm respond well to gestagen therapy and have a good prognosis, compared with other uterine stromal sarcomas.

Macrophage infiltration and genetic landscape of undifferentiated uterine sarcomas

Endometrial stromal tumors include translocation-associated low- and high-grade endometrial stromal sarcomas (ESS) and highly malignant undifferentiated uterine sarcomas (UUS). UUS is considered a poorly defined group of aggressive tumors and is often seen as a diagnosis of exclusion after ESS and leiomyosarcoma (LMS) have been ruled out. We performed a comprehensive analysis of gene expression, copy number variation, point mutations, and immune cell infiltrates in the largest series to date of all major types of uterine sarcomas to shed light on the biology of UUS and to identify potential novel therapeutic targets. We show that UUS tumors have a distinct molecular profile from LMS and ESS. Gene expression and immunohistochemical analyses revealed the presence of high numbers of tumor-associated macrophages (TAMs) in UUS, which makes UUS patients suitable candidates for therapies targeting TAMs. Our results show a high genomic instability of UUS and downregulation of several TP53-mediated tumor suppressor genes, such as NDN, CDH11, and NDRG4. Moreover, we demonstrate that UUS carry somatic mutations in several oncogenes and tumor suppressor genes implicated in RAS/PI3K/AKT/mTOR, ERBB3, and Hedgehog signaling.

Somatic mutation profiling of vulvar cancer: Exploring therapeutic targets

BACKGROUNDnVulvar squamous cell carcinoma (VSCC) constitutes over 90% of vulvar cancer. Its pathogenesis can follow two different pathways; high risk human papillomavirus (hrHPV)-dependent and HPV-independent. Due to the rarity of VSCC, molecular mechanisms underlying VSCC development remain largely unknown. The study aimed to identify pathogenic mutations implicated in the two pathways of VSCC development.nnnMETHODSnUsing next generation sequencing, 81 VSCC tumors, 52 hrHPV(+) and 29 hrHPV(-), were screened for hotspot mutations in 50 genes covered by the Ion AmpliSeq Cancer Hotspot Panel v2 Kit (Thermo Fisher Scientific).nnnRESULTSnMutations of TP53 (46% and 41%, of hrHPV(+) and hrHPV(-) cases respectively) and CDKN2A (p16) (25% and 21%, of hrHPV(+) and hrHPV(-) cases respectively) were the most common genetic alterations identified in VSCC tumors. Further mutations were identified in PIK3CA, FBXW7, HRAS, FGFR3, STK11, AKT1, SMAD4, FLT3, JAK3, GNAQ, and PTEN, albeit at low frequencies. Some of the identified mutations may activate the PI3K/AKT/mTOR pathway. The activation of mTOR was confirmed in the vast majority of VSCC samples by immunohistochemical staining.nnnCONCLUSIONSnDetecting pathogenic mutations in 13/50 genes examined at comparable frequencies in hrHPV(+) and hrHPV(-) tumors suggest that genetic mechanisms of the two routes of VSCC pathogenesis may be similar, despite being initiated from different premalignant lesions. Importantly, our data provide a rationale for new anti-VSCC therapies targeting the PI3K/AKT/mTOR pathway.

Normalizers for microRNA quantification in plasma of patients with vulvar intraepithelial neoplasia lesions and vulvar carcinoma

The role of circulating microRNAs as a promising tool for diagnosing cancer and monitoring anticancer therapies has been widely studied in the past decades. To date, no suitable reference microRNAs for normalizing quantitative real-time polymerase chain reaction assays has been identified in vulvar intraepithelial neoplasia lesions and vulvar squamous cell carcinoma. The purpose of this study was to select appropriate references for gene expression studies in plasma of patients with these lesions. Expression levels of six microRNAs—hsa-miR-425-5p, hsa-miR-191-5p, hsa-miR-93-5p, hsa-miR-423-5p, hsa-miR-103a-3p, and hsa-miR-16-5p—were analyzed by quantitative reverse transcription polymerase chain reaction in plasma samples obtained from 17 patients with vulvar intraepithelial neoplasia lesion and 27 patients with vulvar squamous cell carcinoma. The expression stability of these candidate normalizers was assayed using geNorm algorithm. hsa-miR-93-5p was revealed as the most stably expressed reference in plasma samples of both vulvar intraepithelial neoplasia lesion and vulvar squamous cell carcinoma patients. The results pointed at hsa-miR-93-5p and hsa-miR-425-5p as microRNAs that retained the greatest robustness in plasma of vulvar intraepithelial neoplasia lesion and vulvar squamous cell carcinoma patients, respectively. Our work is the first report on reference microRNA selection for quantitative real-time polymerase chain reaction applications in vulvar intraepithelial neoplasia lesion and vulvar squamous cell carcinoma. The candidate microRNA stability values for the two types of lesions are provided and might serve for normalization of the future novel microRNA biomarkers in these rare entities.

Abstract 3182: Tumor associated macrophages in undifferentiated uterine sarcoma: association with angiogenesis and therapeutic implications

Sarcomas of the uterus are derived from uterine smooth muscle (leiomyosarcomas) or from endometrial stromal cells. The latter group includes three subtypes: the low-grade (LG) and high-grade (HG) endometrial stromal sarcomas (ESS), each defined by characteristic chromosomal translocations, and undifferentiated uterine sarcoma (UUS), the most aggressive form. The majority of UUS patients present with high-stage disease and even patients with stage I tumors often die within 2 years from diagnosis. Adjuvant radio- and chemotherapy do not improve the clinical outcome. No prior comprehensive molecular analysis of all three types of endometrial stromal tumors has been reported. We performed genomic analysis (whole exome sequencing and aCGH), gene expression profiling (RNA-seq and microarrays), immunohistochemistry (IHC) and FISH on 31 endometrial stromal tumors, including 17 UUS and 14 LG and HG ESS. All ESS cases carried characteristic JAZF1, PHF1, MBTD1 or YWHAE rearrangements. Selected findings were validated by IHC, qRT-PCR and Sanger sequencing. Genomic analysis revealed that UUS are characterized by complex chromosomal aberrations. The most frequent somatic mutations (in TP53, KRAS, FBXW7, PIK3CA and ERBB3) and copy number changes (e.g. gains of 19q, 8q11.1-q24.3 and 3q26.2-q29) in UUS resemble those seen in uterine carcinosarcomas and carcinomas. UUS over-expressed numerous genes encoding M2 macrophage-specific markers, including CD163, CCL18, mannose receptor, stabilin-1, and macrophage galactose-type C-type lectin 2. Immunohistochemistry for CD163 and CD68 confirmed high tumor-associated macrophages (TAM) counts in UUS tissues compared to the ESS. In UUS, we also observed significantly higher mRNA expression for genes implicated in angiogenesis (CD34, MMP9), immunosuppression and tumor invasion (e.g. CCR2, IL10RA, IRAK3, CCL13, CXCL9, CXCL10). TAMs are associated with progression, resistance to cytotoxic therapy and metastatic spread in most human tumor types. In animal models, TAMs were shown to induce increased angiogenesis. CSF1 is a major regulator of macrophage function and is implicated in these tumor-promoting effects. Clinical trials have shown an effect of inhibitors of the CSF1 pathway in tenosynovial giant cell tumors and our data indicate that these inhibitors may be considered for the treatment of UUS. Our study also showed significantly elevated expression in UUS of two additional therapeutic targets involving macrophage pathways: CCR2 (C-C chemokine receptor type 2) that can be targeted by monoclonal antibodies, and BTK (Bruton9s tyrosine kinase) that can be targeted by ibrutinib. In conclusion, our findings demonstrate abundant presence of TAMs and over-expression of TAM-associated markers in UUS compared to the other tumors derived from endometrial stroma. Moreover, our results indicate novel potential therapeutic targets for UUS patients management. Citation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Sushama Varma, Robert T. Sweeney, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Matt van de Rijn, Maria Debiec-Rychter. Tumor associated macrophages in undifferentiated uterine sarcoma: association with angiogenesis and therapeutic implications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3182.

Abstract 4698: Gene expression profiling distinguishes between endometrial stromal tumors subtypes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnIntroductionnnEndometrial stromal tumors (EST) are the second most prevalent uterine sarcomas. EST are divided into three histosubtypes: benign endometrial stromal nodules (ESN), low-grade endometrial stromal sarcomas (ESS) and the most malignant undifferentiated stromal sarcomas (UES). Moreover, due to clinicopathological features intermediate between classical low-grade ESS and UES, some tumors have been described in the literature as “high-grade ESS”. The histological distinction between these three subtypes has important prognostic implications. The molecular biology of these tumors remains poorly understood because of their low incidence.nnPatients and MethodsnnIn the present study, we performed gene expression profiling of 22 EST using microarray, RNA-Seq and qRT-PCR techniques. Our cohort included 10 classical low-grade ESS (8 with ESS-associated gene fusions), 8 UES and 4 tumors which were classified as high-grade ESS based on the presence of YWHAE-FAM22A/B fusion and histological features.nnResultsnnUsing gene expression microrarrays, we have identified 81 entities differentially expressed in analyzed samples (fold change > 2, p < 0.005). Hierarchical clustering analysis revealed that high-grade ESS formed a separate sub-cluster with a distinct molecular profile as compared with the low-grade ESS and UES cases. Low-grade ESS cases formed 2 distinct sub-clusters, each containing 5 cases, one of which presented gene expression profile partially corresponding to high-grade ESS cases. GO analysis of genes strongly up-regulated in UES specimens showed over-representation of immune response (GO:0006955) and immune system process (GO:0002376) pathways (MARCO, MYO1F, FCN1, GPR183, HLA-DPB1, FCGR2B, NCF1, CCL18, CD28, PTAFR, CCL13, STK4, CD14, NCF4 and CTLA4 genes) (p < 0.05). UES cases were also characterized by significant down-regulation of WT1 tumor suppressor gene as compared to the low- and high-grade ESS. Moreover, both high-grade ESS and UES cases were characterized by significantly lower expression level of PEG3 gene, which has been reported as a tumor suppressor gene. All low-grade ESS cases presented significant over-expression of estrogen receptor ESR1 gene. Microarray results were validated by RNA-Seq and qRT-PCR experiments.nnConclusionsnnThis study provides gene expression profiles that distinguish between EST subtypes and sheds light on the biology of these tumors. Our results point to immune system involvement in the pathogenesis of UES and support the notion that the current EST classification should discriminate high-grade ESS as a distinct subtype.nnCitation Format: Joanna Przybyl, Magdalena Kowalewska, Anna Quattrone, Barbara Dewaele, Vanessa Vanspauwen, Julio Finalet-Ferreiro, Michal Swierniak, Elwira Bakula-Zalewska, Janusz A. Siedlecki, Mariusz Bidzinski, Jan Cools, Maria Debiec-Rychter. Gene expression profiling distinguishes between endometrial stromal tumors subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4698. doi:10.1158/1538-7445.AM2014-4698