Genomic landscape of endometrial polyps

The objective of this study was to explore the mechanism via which Raf kinase inhibitor protein (RKIP) suppresses the invasion of gastric cancer cells and promote apoptosis, with an attempt to provide evidences for the application of RKIP in treating gastric cancer. The recombinant plasmid pcDNA3.1-RKIP or RKIP-shRNA was transfected into the gastric cancer cell line SGC-7901 using liposome. Then, the messenger RNA (mRNA) and protein expressions of RKIP, HMGA2, and OPN were detected using qPCR and Western blotting. The effects of HMGA2 on the proliferation, apoptosis, and invasion of SGC-7901 cells were detected using flow cytometry and Transwell assay. To further explore the regulatory effect of PKIP on the biological activities of HMGA2, we over-expressed or knock down RKIP and HMGA2 simultaneously and detected its effects on the proliferation, apoptosis, and invasion of SGC-7901 cells. As shown by qPCR and Western blotting, after over-expression of RKIP in SGC-7901 cells, the mRNA and protein expressions of RKIP significantly increased (P < 0.01), whereas the mRNA and protein expressions of HMGA2 and OPN significantly decreased (P < 0.01). In contrast, the transfection of RKIP-shRNA in the SGC-7901 cells resulted in opposite results. After over-expression of HMGA2 in SGC-7901 cells, the protein expression of HMGA2 significantly increased (P < 0.01); however, it significantly decreased after the transfection of HMGA2-shRNA (P < 0.01). As shown by Transwell assay and flow cytometry, After the over-expression of HMGA2 in SGC-7901 cells, the (G2 + S) phase fraction significantly increased (P < 0.01); also, the percentage of the apoptotic cells significantly declined (P < 0.05) and the number of invasive cells significantly increased (P < 0.05). However, the interference of the expression of HMGA2 resulted in opposite results. The simultaneous over-expression of RKIP and HMGA2 in SGC-7901 cells or the simultaneous interference of RKIP and HMGA2 showed no significant difference with the control group in terms of (G2 + S) phase fraction, percentage of apoptotic cells, and number of invasive cells (P > 0.05). In conclusion, RKIP can inhibit the survival and invasion of gastric cancer cells and promote apoptosis, possibly by regulating the expression of HMGA2 or OPN.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Effects of Operative Hysteroscopy with Antiadhesion Solution in the Patients Who Have Abnormal Uterine Bleeding or Intrauterine Lesions

Lung cancer is still the leading cause of death from cancer worldwide primarily because of the fact that most lung cancers are diagnosed at advanced stages. Overexpression of the high mobility group protein HMGA2 has been observed in a variety of malignant tumors and often correlates with poor prognosis. Herein, HMGA2 expression levels were analyzed in matching cancerous and non-cancerous lung samples of 17 patients with adenocarcinoma (AC) and 17 patients with squamous cell carcinoma (SCC) with real-time quantitative RT-PCR (qRT-PCR). Transcript levels were compared to results obtained by immunohistochemistry (IHC). HMGA2 expression was detectable by qRT-PCR in all samples tested and varied from 5422 to 16 991 545 copies per 250 ng total RNA in the carcinoma samples and from 289 to 525 947 copies in the non-cancerous tissue samples. In 33/34 non-small cell lung cancer (NSCLC) samples tested, an overexpression of HMGA2 was revealed with statistically highly significant differences between non-neoplastic and tumor samples for both AC (P < 0.0001) as well as for SCC (P < 0.0001). Expression varies strongly and is increased up to 911-fold for AC and up to 2504-fold for SCC, respectively, with statistically significant higher increase in SCC (P < 0.05). The results presented herein indicate that HMGA2 overexpression is a common event in NSCLC and could serve as molecular marker for lung cancer.

Simple SummaryUterine leiomyomas are benign smooth muscle tumors affecting millions of women globally. On a molecular level, leiomyomas can be classified into three main subtypes, each characterized by mutations affecting either MED12, HMGA2, or FH. Leiomyomas are still widely regarded as a single entity, although early observations suggest that different subtypes behave differently, in terms of both clinical outcomes and therapeutic requirements. The majority of classification studies on leiomyomas have been performed using fresh frozen tissue. Archival formalin-fixed paraffin-embedded (FFPE) tissue represents an invaluable source of biological material that can be studied retrospectively. Methods capable of generating high-quality data from FFPE material are in high demand. Here, we show that 3′RNA sequencing can accurately classify leiomyomas that have been stored as FFPE tissue in hospital archives for years. A targeted 3′RNA sequencing panel could provide researchers and clinicians with a cost-effective and scalable diagnostic tool for classifying smooth muscle tumors.Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3′RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3′RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3′RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.

Endometrioid endometrial carcinomas (EEC) are the most common malignant gynecologic tumors. Despite the increase in EEC molecular knowledge, the identification of new biomarkers involved in disease’s development and/or progression would represent an improvement in its course. High-mobility group A protein (HMGA) family members are frequently overexpressed in a wide range of malignancies, correlating with a poor prognosis. Thus, the aim of this study was to analyze HMGA1 and HMGA2 expression pattern and their potential role as EEC biomarkers. HMGA1 and HMGA2 expression was initially evaluated in a series of 46 EEC tumors (stages IA to IV), and the findings were then validated in The Cancer Genome Atlas (TCGA) EEC cohort, comprising 381 EEC tumors (stages IA to IV). Our results reveal that HMGA1 and HMGA2 mRNA and protein are overexpressed in ECC, but only HMGA1 expression is associated with increased histological grade and tumor size. Moreover, HMGA1 but not HMGA2 overexpression was identified as a negative prognostic factor to EEC patients. Finally, a positive correlation between expression of HMGA1 pseudogenes—HMGA1-P6 and HMGA1-P7—and HMGA1 itself was detected, suggesting HMGA1 pseudogenes may play a role in HMGA1 expression regulation in EEC. Thus, these results indicate that HMGA1 overexpression possesses a potential role as a prognostic biomarker for EEC.

Engineered uterine primary myometrial cells with high-mobility group AT-hook 2 overexpression display a leiomyoma-like transcriptional and epigenomic phenotype

High mobility group protein2 (HMGA2) and epithelial-to-mesenchymal transition are both related to progress of bladder cancer, however, the relationship between HMGA2, E-cadherin and vimentin in bladder cancer is not yet known. Thus, this study has examined expression of HMGA2, E-cadherin and vimentin in bladder cancer and investigated their relationship. The 5637 bladder cancer cell line and SV-HUC-1 normal uroepithelial cells were used to study expression of HMGA2, E-cadherin and vimentin using RT-PCR and western blotting. Paraffin wax-embedded bladder cancer tissues were used to study protein expression using immunohistochemistry and χ(2) analysis and Kendall's correlation were utilized statistical methods. Overexpression of HMGA2 was associated with down-regulation of E-cadherin and up-regulation of vimentin in the 5637 bladder cancer line. A total of 49 paraffin wax-embedded tissues of transitional cell bladder cancer were used. Positive expression levels of HMGA2 protein and vimentin were 41 and 43% in bladder tissues, respectively. No expression of E-cadherin was found in 43%. Expression of HMGA2, loss of E-cadherin and expression of vimentin are all significantly correlated with bladder cancer grade and stage. Loss of E-cadherin and expression of vimentin both correlated with recurrence of the bladder cancer. Expression of HMGA2 was closely associated with occurrence of epithelial-to-mesenchymal transition. Expression of HMGA2, loss of E-cadherin and expression of vimentin may indicate high degree malignancy of bladder cancer. Loss of E-cadherin expression and positive expression of vimentin may predict recurrence of bladder cancer.

The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

Sonohysterography findings in patients with abnormal uterine bleeding at Kenyatta National Hospital

Prostate cancer is the most diagnosed cancer in men and the second leading cause of death in the United States. African American men have the highest incidence and mortality rates of prostate cancer compared to any other race. Epithelial-mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. Mesenchymal cells are migratory, invasive, and more resistant to apoptosis. Reactive Oxygen Species (ROS) has been shown to promote EMT. High mobility group A (HMGA2) is a non-histone protein that is highly expressed during the embryogenesis, whereas the gene expression is very low or absent during adulthood. Recent studies have been reported an overexpression of HMGA2 protein in malignant cancers. Loss of Let-7 miRNA (repressor of HMGA2) has been found to induce EMT via upregulation of HMGA2 in prostate cancer. There has been no link between ROS and HMGA2. We have reported that camalexin, a 3-thizol-2-yl-indole, may be a candidate treatment for aggressive prostate cancer cells by ROS-mediated apoptosis. The study demonstrated that treating the prostate cancer cell with camalexin increased ROS levels which contributed to decreased cell proliferation and increased apoptosis. We hypothesize that HMGA2 may regulate EMT in part by inducing ROS and that camalexin may antagonize HMGA2 signaling. We analysed HMGA2 and EMT marker expression in a panel of prostate cancer cell lines by western blot analysis. We also transiently and stably overexpressed wild-type HMGA2 and mutant HMGA2 (missing Let-7 binding site) in LNCaP cells. We measured ROS levels using DCFDA dye that detects hydrogen peroxide. We treated ARCaP-M (mesenchymal) cells with different concentrations of camalexin to analyse HMGA2 expression. Our results showed that HMGA2 is highly expressed in aggressive prostate cancer cell lines (C4-2, ARCaP, E006AA) as compared to RWPE1 and LNCaP cells. The transient overexpression of HMGA2 in LNCaP cells decreased E-cadherin more markedly than with mutant HMGA2 but did not show any changes in ROS. We will repeat this experiment with stable clones from HMGA2 overexpression. ARCaP-M cells treated with camalexin induced ROS and decreased HMGA2 expression. We are currently performing in vivo studies using ARCaP-M cells injected subcutaneously into nude mice followed by treatment with camalexin after the tumor grows to 50 mm3. In conclusion, HMGA2 promotes EMT, even more markedly if its Let-7 suppressor binding site is eliminated, and camalexin may target HMGA2 to decrease prostate cancer progression. GRANT SUPPORT: 1P20MD002285; 8G12MD007590 Citation Format: Ohuod A. hawsawi, Basil Smith, Jodi Dougan, Liza J. Burton, Valerie A. Odero-Marah. HMGA2 induces EMT in prostate cancer cells and may be antagonized by camalexin. [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 1595.

9521 Background: Relapsed ALL carries a very poor prognosis despite intensive therapy, indicating the need for new insights into disease mechanisms. We have previously used global gene expression profiling and copy number analysis in diagnosis/relapse pairs to better understand the biologic mechanisms leading to relapse. To create an integrated genomic profile we have now focused on high throughput RNA sequencing to detect mutations, insertions, deletions and fusion transcripts. Methods: To date we have sequenced 4 pediatric matched diagnosis/relapse pairs (i.e. 8 marrow samples) from B-precursor ALL patients. RNA sequencing was performed using the Illumina GAIIx Analyzer. Each sample was sequenced in 7 lanes using single-read 54 base pair sequencing to cover 52% of genes at 5X and 34% of genes at 20X. BWA (v0.5.5) and Samtools (v1.7) were used to align the reads to the human genome and predict single nucleotide variants (SNVs) and insertion/deletions (indels) across the genome. Known single nucleotide polymorphisms (SNPs) were filtered out by comparison to diagnostic samples and through use of dbSNP (r130) and 1,000 Genomes Project databases. We have focused our initial analysis on relapse specific lesions shared by samples presumably indicating selection for common chemoresistance pathways. Results: We found 232 variants that were relapse specific and shared among all 4 patients. Sixty-seven of the variants were within exons: 14 deletions, 6 indels, 34 insertions and 13 SNVs. There were 2,454 variants that were relapse specific and shared among 3 of 4 patients. Of these, 766 variants were within exons: 144 deletions, 69 indels, 344 insertions and 209 SNVs. There were 16,100 relapse specific variants found in 2 of 4 patients. Of these, 5,184 were within exons: 1,058 deletions, 300 indels, 2,146 insertions and 1,680 SNVs. Validation of a subset of these variants in a larger cohort of patients using Taqman assays and Sanger sequencing is underway. Conclusions: RNA sequencing in relapsed pediatric ALL reveals many novel relapse specific variants that are shared among patients. Furthur analysis of these variants will be necessary to determine their functional significance and potential therapeutic relevance. No significant financial relationships to disclose.

Three-dimensional saline infusion sonography compared to two-dimensional saline infusion sonography for the diagnosis of focal intracavitary lesions.

Objective To evaluate the value of hysteroscopy in elder women with abnormal uterine bleeding (AUB) and asymptomatic postmenopausal women with a thickened endometrium. Methods Fifty-three cases in the AUB group and seventy-eight cases in the endometrial hyperplasia group underwent hysteroscopy examination and hysteroscopy-guided biopsy, then the hysteroscopic and histopathological results were compared between the two groups. Results Of the 131 cases, the normal endometrium accounted for 29.8% (n=39), endometrial polyp for 49.6% (n=65), submucous myomas for 4.6% (n=6), hyperplasia endometrii for 6.1%(n=8) and endometrial carcinoma for 9.9% (n=13). Both the AUB group and the endometrial hyperplasia group had 8 cases of endometrial carcinoma (15.1%, 6.4%, respectively). For the diagnosis of normal endometrium with hysteroscopy, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 88%, 97%, 94% and 95%, respectively, in the AUB group, versus 82%, 95%, 86% and 93%, respectively, in the endometrial hyperplasia group. For the endometrial polyps, hysteroscopy showed a sensitivity, specificity, PPV and NPV of 100%, 79%, 74%, 100%, respectively, in the AUB group and 98%, 88%, 92%, 97%, respectively, in the endometrial hyperplasia group. For the endometrial cancer, hysteroscopy had a sensitivity, specificity, PPV and NPV of 75%, 100%, 100% and 96%, respectively, in the AUB group; while in the endometrial hyperplasia group, the sensitivity was 80%, the specificity and PPV were 100%, and the NPV was 99%. Conclusions In elder females, hysteroscopy allows for an accurate diagnosis in endometrial disease, and hysteroscopically directed sampling is mandatory, even if the uterine cavity appears normal at hysteroscopy, to rule out endometrial neoplasms. Key words: Hysteroscopy; Endometrial; Uterine hemorrhage

Conclusion: The overexpression of HMGA1 or Ezrin may contribute to the carcinogenesis, development, and metastasis of laryngeal squamous cell carcinoma (LSCC). Objective: To investigate the expression of HMGA1 and Ezrin in LSCC and analyze their clinical significance. Methods: The expression of HMGA1 and Ezrin was analyzed by immunohistochemistry (IHC) in 50 cases of LSCC. Thirty cases of laryngeal polyp and 30 cases of atypical hyperplasia of larynx were studied as controls. The expression of HMGA1 and Ezrin was analyzed by real-time PCR and by Western blot in 30 cases of LSCC; samples from adjacent normal epithelial tissues in 30 cases were studied as controls. Results: (1) IHC revealed that the positive rate of HMGA1 protein was 68.0% (34/50), 53.3% (16/30), and 13. 3% (4/30) in LSCC, atypical hyperplasia of larynx, and laryngeal polyp (p < 0.05), and the positive rate of Ezrin protein was 64.0% (32/50), 50.0% (15/30), and 23.3% (7/30) (p < 0.01), respectively. (2) Real-time PCR demonstrated that the mean relative mRNA expression levels of HMGA1 in LSCC and in normal tissues were 2.41 ± 0.40 and 1.05 ± 0.18, respectively (p < 0.01). The mRNA levels of Ezrin in LSCC and in normal tissues were 1.79 ± 0.27 and 1.04 ± 0.22, respectively (p < 0.05). (3) Western blotting revealed that the mean relative protein expression levels of HMGA1 in LSCC and in normal tissues were 1.73 ± 0.60 and 0.35 ± 0.17, respectively (p < 0.01). The protein levels of Ezrin in LSCC and in normal tissues were 1.82 ± 0.77 and 0.42 ± 0.20, respectively (p < 0.01).