Association between Maternal Hemoglobin, other Maternal Attributes and Neonatal Birth Weight: A Case-control Study.

BACKGROUND: Although the burden of anemia in pregnant women and its consequences on the pregnancy outcome are well documented, there is limited evidence on the association between maternal hemoglobin concentration and low birth weight (LBW) in the study area. OBJECTIVES: This study aimed to determine the association between maternal hemoglobin concentration and neonatal birth weight (BW) in Qazvin, Iran, 2018-2019. MATERIALS AND METHODS: A case-control study was conducted among 450 neonates with BW < 2500 gm (LBW) and 451 neonates with BW > 2500 gm. In this study, neonates with BW less than 2500 gm were cases, while those who were greater than 2500 gm were considered as control. Multiple logistic regression model was used to calculate OR with 95% Confidence Interval (95 % CI) to determine the association between maternal anemia and other maternal attributes and neonate BW. RESULTS: Maternal hemoglobin (g/dl) was not significantly associated with neonate BW (OR: 1.03 (95 % CI: 0.58 - 1.81), p = 0.93). However, maternal initial weight (Kg) (OR: 0.96 (95 % CI: 0.94 - .098), p < 0.001), mother's age in year (OR: 1.04 (95 % CI: 1.00 - 1.09), p = 0.038), gestational age (OR: 0.49 (95 % CI: 0.43 - 0.57), p < 0.001) were significantly associated with neonatal BW. CONCLUSION: Maternal hemoglobin concentration was not significantly associated with LBW. However, other maternal attributes such as low initial maternal weight, low gestational age, low education status and old age were significantly associated with LBW. Intervention that targeted mothers with low initial weight, low educational status and older age is required to minimize LBW among neonates in the study area.

Diffuse and Localized SARS-CoV-2 Placentitis: Prevalence and Pathogenesis of an Uncommon Complication of COVID-19 Infection During Pregnancy.

Coronavirus disease 2019 (COVID-19) infection in pregnancy has been associated with preterm delivery and preeclampsia. A less frequent and underrecognized complication is extensive placental infection which is associated with high rates of perinatal morbidity and mortality. The frequency, early pathogenesis, and range of lesions associated with this infection are poorly understood. We conducted a population-based study of placental pathology from all mothers with COVID-19 (n=271) over an 18-month period delivering within our health system. The overall prevalence of diffuse severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) placentitis, as defined by typical histology and immunohistochemical (IHC) staining for SARS-CoV-2 spike protein, was 14.8/1000, but increased to 59/1000 in preterm births. We also identified 3 cases with isolated small foci of localized SARS-CoV-2 placentitis, characterized by focal perivillous fibrin and intervillositis, which illustrate the early pathogenesis and suggest that infection may be contained in some cases. Two other placental lesions were more common in mothers with COVID-19, high-grade maternal vascular malperfusion in preterm deliveries and high-grade chronic villitis at term (5/5 cases tested of the latter were negative by IHC for SARS-CoV-2). Additional investigation of diffuse and localized SARS-CoV-2 placentitis by IHC showed loss of BCL-2, C4d staining in surrounding villi, and an early neutrophil-predominant intervillous infiltrate that later became dominated by monocyte-macrophages. We propose a model of focal infection of syncytiotrophoblast by virally infected maternal leukocytes leading to loss of BCL-2 and apoptosis. Infection is then either contained by surrounding fibrinoid (localized) or initiates waves of aponecrosis and immune activation that spread throughout the villous parenchyma (diffuse).

Clinical and Pathologic Features Associated With Invasive Breast Carcinoma With 2018 American Society of Clinical Oncology/College of American Pathologists In Situ Hybridization Group 2 Results (Human Epidermal Growth Factor Receptor 2 [HER2]/Chromosome 17 Centromere [CEP17] Ratio ≥2.0 and Average HER2 Copy Number <4.0).

CONTEXT.­: The American Society of Clinical Oncology/College of American Pathologists updated the human epidermal growth factor receptor 2 (HER2) breast carcinoma testing guideline in 2018 to address issues from uncommon HER2 fluorescence in situ hybridization (FISH) results. Based on the 2013 American Society of Clinical Oncology/College of American Pathologists guideline, cases wherein the HER2/chromosome 17 centromere (CEP17) ratio of 2.0 or more with an average HER2 copy number of less than 4.0 were considered in situ hybridization (ISH) positive. Under the 2018 guideline, such cases are classified as ISH Group 2 and are no longer considered eligible for anti-HER2 therapy when the corresponding HER2 immunohistochemistry result is 0, 1+, or 2+. OBJECTIVE.­: To assess the clinical, pathologic, and treatment aspects of patients with ISH Group 2 results. DESIGN.­: We retrospectively reviewed HER2 FISH results at our center between January 2012 and December 2014 and identified and characterized cases with ISH Group 2 results. RESULTS.­: Thirty-nine cases with ISH Group 2 results from 39 patients were reviewed. Twenty of 39 (51%) patients received anti-HER2 therapy. Patients treated with HER2-targeted therapy were less likely to have hormone receptor-positive tumors, compared with patients without anti-HER2 treatment, though not significantly (P = .30). The only significant difference between the 2 patient groups was receipt of cytotoxic chemotherapy treatment (P < .001). Overall, clinical outcome was similar between the 2 groups (P > .99). CONCLUSIONS.­: This retrospective study with median follow-up of at least 6 years shows patients with ISH Group 2 tumors had similar clinical outcomes, irrespective of HER2-targeted therapy. Further analysis in the prospective setting would provide valuable data that would potentially inform clinical decision making.

Prevalence of G6PD deficiency in Iranian neonates with jaundice: a systematic review and meta-analysis.

OBJECTIVES: This systematic review and meta-analysis study aimed to estimate the overall prevalence of Glucose-6-phosphate dehydrogenase (G6PD) deficiency in neonates with jaundice who were admitted to hospitals in Iran. MATERIALS AND METHODS: In this systematic review and meta-analysis, we searched PubMed/Medline, Scopus, ISI Web of Sciences, and Iranian Local databases up to December 2019.We calculated Prevalence and 95% Confidence Interval (95% CI) of G6PD deficiency as summary measures. We conducted subgroup analysis based on the sex and quality of studies, while meta-regression were applied for investigating the effect of years of studies and years of publication on the pooled prevalence. We applied sensitivity analysis to investigate the effect of excluding each study on the pooled prevalence estimation. RESULTS: The total sample size was 9799 people. The pooled prevalence of G6PD deficiency among neonates with jaundice in Iran was 7.0% (95% CI: 5.5-8.5%). The results of subgroup analysis showed that, pooled prevalence of G6PD deficiency among male neonate (12.1%, 95%CI: 7.6-16.7%) was more prevalent that female (3.00%, 95%CI: 1.1-4.9%). Based on the sensitivity analysis, lower and higher pooled prevalence of G6PD deficiency was observed 5.8% (95%CI: 4.7-6.9%) and 7.3% (95%CI: 5.7-8.8%) respectively by excluding each study. CONCLUSION: The overall prevalence of G6PD deficiency was 7% in Iranian neonates with Jaundice. Prevalence was high in male and early hours of life. We recommend screening test for G6PD deficiency in neonates to prevent its complications.

Four major patterns of placental injury: a stepwise guide for understanding and implementing the 2016 Amsterdam consensus.

The Amsterdam classification system defines four major patterns of placental injury, maternal vascular malperfusion, fetal vascular malperfusion, acute chorioamnionitis, and villitis of unknown etiology, and lists the histologic findings that characterize each. However, there continues to be uncertainty regarding specific definitions, histologic mimics, grading and staging, and what combination of findings is required to diagnose each pattern of injury in a reproducible fashion. The purpose of this review is to clarify some of these issues by suggesting a stepwise approach to more fully realize the potential of this new classification system. In our view, the critical steps for correctly identifying and communicating each pattern of injury are (1) familiarity with the underlying pathophysiology and known clinical associations, (2) incorporation of important gross findings, (3) learning to recognize underlying architectural alterations and defining features at low power, (4) using higher magnification to narrow the differential diagnosis and assess severity (grading) and duration (staging), and (5) adopting a template for generating standardized placental reports that succinctly provide useful information for patient care and research applications.

RNA-Seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancers with and without Defective DNA-Mismatch Repair.

Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10-9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.

A global systematic review and meta-analysis on illicit drug consumption rate through wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) is a complementary, well-established comprehensive, cost-effective, and rapid technique for monitoring of illicit drugs used in a general population. This systematic review and meta-analysis is the first to estimate the rank and consumption rate of illicit drugs through WBE studies. In the current study, the related investigations regarding the illicit drug consumption rate based on WBE were searched among the international databases including Scopus, PubMed, Science direct, Google scholar, and local database, Magiran from 2012 up to May 2019. The illicit drug consumption rate with 95% confidence intervals was pooled between studies by using random effect model. The heterogeneity was determined using I2 statistics. Also, subgroup analyses were conducted to examine the possible effects of year and location of studies on observed heterogeneity. Meta-analysis of 37 articles indicates that the overall rank order of illicit drugs according to their pooled consumption rate can be summarized as tetrahydrocannabinol or cannabis (7417.9 mg/day/1000 people) > cocaine (655.7 mg/day/1000 people) > morphine (384.9 mg/day/1000 people) > methamphetamine (296.2 mg/day/1000 people) > codeine (222.7 mg/day/1000 people) > methadone (200.2 mg/day/1000 people) > 3,4-methylenedioxymethamphetamine (126.3 mg/day/1000 people) > amphetamine (118.2 mg/day/1000 people) > 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (33.7 mg/day/1000 people). The pooled level rate was 190.16 mg/day/1000 people for benzoylecgonine (main urinary cocaine metabolite), 137.9 mg/day/1000 people for 11-nor-9-carboxy-delta9-tetrahydrocannabinol (main metabolite of cannabis), and 33.7 mg/day/1000 people for 2-ethylidene-1,5-dimethyl-3, 3-diphenylpyrrolidine (main metabolite of methadone). The I2 values for all selected drugs were 100% (P value < 0.001). The results of year subgroup indicated that the changes of heterogeneity for all selected drugs were nearly negligible. The heterogeneity within studies based on continents subgroup just decreased in America for drugs like 11-nor-9-carboxy-delta9-tetrahydrocannabinol (I2 = 24.4%) and benzoylecgonine (I2 = 94.1%). The outcome of this meta-analysis can be used for finding the illicit drugs with global serious problem in view of consumption rate (i.e., cannabis and cocaine) and helping authorities to combat them.

Uterine Inflammatory Myofibroblastic Tumor Showing an Atypical ALK Signal Pattern by FISH and DES-ALK Fusion by RNA Sequencing: A Case Report.

Inflammatory myofibroblastic tumors (IMTs) are spindle cell neoplasms of intermediate (borderline) biologic potential with tendency for local recurrence but low risk of metastasis. They affect children more than adults. The most common sites of involvement are the lung, soft tissue, peritoneum, bladder, and less commonly the gynecologic tract. IMTs are characterized by spindle to epithelioid cells with myofibroblastic differentiation, some degree of smooth muscle differentiation, myxoid stroma and usually associated with brisk lymphoplasmacytic infiltrates. In about half of the cases, IMTs are associated with rearrangements of the anaplastic lymphoma kinase (ALK) gene located at chromosome 2p23. The ALK rearrangement can be detected by immunohistochemistry for ALK protein expression (mostly cytoplasmic with or without perinuclear accentuation) or by fluorescent in situ hybridization (FISH) using dual-color break-apart probes for which the typical pattern is seen as split 3' end (red) and 5' end (green) probe signals in addition to single normal, unsplit red-green signal pair (yellow). Herein we describe a case of uterine IMT initially misdiagnosed intraoperatively as leiomyoma which showed sparse lymphocytic infiltrates, positive ALK expression by immunohistochemistry, a predominantly atypical FISH signal pattern (1 yellow and 1 red signal only) and few typical signal patterns (1 yellow, 1 red, and 1 green signal) in a smaller population of tumor cells. The RNA sequencing showed a recently described DES-ALK fusion transcript in the tumor cells, suggesting an intrachromosomal inversion and deletion as the likely underlying mechanism for the atypical FISH pattern. Familiarity with the unusual morphology and atypical FISH pattern is crucial given that this tumor has an activating ALK rearrangement and may benefit from targeted tyrosine kinase inhibitors in the future.

Clinicopathologic, Immunohistochemical, and Molecular Characteristics of Ovarian Serous Carcinoma With Mixed Morphologic Features of High-grade and Low-grade Serous Carcinoma.

Despite the current classification of high-grade serous carcinoma (HGSCA) and low-grade serous carcinoma (LGSCA) as mutually exclusive diseases based on morphology and molecular pathogenesis, cases with mixed morphologic features of HGSCA and LGSCA have been reported. Herein we assess the clinicopathologic, immunohistochemical (IHC), and molecular genetic characteristics of a group of these cases, which we termed indeterminate grade serous carcinoma (IGSCA) in comparison with groups of HGSCA and LGSCA. Using the World Health Organization (WHO) classification criteria, we selected 27 LGSCA and 19 IGSCA for detailed morphologic study. Thirteen classic HGSCA, 19 classic LGSCA, and 19 IGSCA were selected for p53 and BRAF V600E IHC and molecular genetic testing by next-generation sequencing. IGSCA showed the architectural patterns of invasion of LGSCA, but with higher grade nuclear features focally and a mitotic index intermediate between LGSCA and HGSCA. Few cases in the IGSCA group showed mutant TP53 by IHC or sequencing (4/18, 22.2%), 1 case had mutant BRAF non-V600E by sequencing, and 1 had an NRAS mutation. When present, the mutations were identical in the low-grade and high-grade areas. The IGSCA group had a long-term survival similar to the classic HGSCA group. IGSCA with mixed morphologic features of HGSCA and LGSCA is a rare and potentially clinically aggressive variant of serous carcinoma. Their distinct morphologic, but heterogenous molecular features, including low frequency of TP53 and BRAF mutations suggest that these rare tumors may have a different pathogenesis pathway compared with classic HGSCA and classic LGSCA.

Mutational Profile in Vulvar, Vaginal, and Urethral Melanomas: Review of 37 Cases With Focus on Primary Tumor Site.

Melanomas of female genital tract are rare tumors with poor prognosis. While BRAF-V600E is the most common pathogenic mutation seen in cutaneous sun-exposed melanomas, mucosal and anogenital melanomas usually lack BRAF mutations and instead they harbor KIT alterations. The American Joint Committee on Cancer staging guideline (AJCC eighth edition) recommends using cutaneous melanoma guidelines for vulvar melanoma staging and does not provide any recommendations for vaginal melanoma staging. The aim of this study is to investigate the mutational status of invasive melanomas arising from different anatomic sites in lower female genital tract (vulvar hair-bearing skin, glabrous skin, vagina and urethra) in a group of 37 patients. Tumors were analyzed using a DNA targeted next-generation sequencing panel covering the 21 most common genes and mutation hotspots in melanomas. The most common genetic alterations in invasive melanomas of lower female genital tract are KIT (32%), TP53 (22%), and NF1 (19%). Overall 66% (21/32) of cases showed a pathogenic alteration in at least one of the MAPK pathway genes. No statistical significance seen between different primary tumor sites and the frequency of the oncogenic mutations, nor were any significant differences found by mutation status. Only one case of urethral melanoma showed a BRAF non-V600E mutation (D594G). Our results suggest a similar molecular pathogenesis and overall survival in melanomas arising from lower female genital tract, irrespective of their exact location in the urogenital area. Future classifications of melanoma should consider grouping vulvar melanomas with mucosal rather than cutaneous melanomas.

Fabrication of novel 2D Ag-TiO2/γ-Al2O3/Chitosan nano-composite photocatalyst toward enhanced photocatalytic reduction of nitrate.

The presence of excess nitrate in groundwater limits it use as a drinking water supply and its removal is critical to balance the nitrogen cycle in aquatic systems. In this study, ultra-thin 2-dimensional Ag-TiO2/γ-Al2O3/Chitosan (Ag-TiO2/Al2O3/CS) nano-composite was synthesized for the fast reduction of nitrate under UVA irradiation from aqueous solutions. As-synthesized nano-composite was well characterized by X-ray diffraction, transmission electron microscopy, N2 adsorption-desorption isotherms, Fourier-transform infrared spectroscopy and UV-vis diffuse reflectance spectroscopy. Experimental variables including pH, nitrate concentration, photocatalyst dose and contact time were considered to demonstrate their effect on the rate of nitrate reduction. Formic acid was used as a radical scavenger at optimal concentration of 2:1 (formic acid:nitrate). The results showed that upon UVA irradiation, the synthesized nano-composite exhibited fast nitrate reduction in broad pH range (about 74% removal at pH 11 in 5 min reaction time) in diverse water chemical conditions. The Ag-doped and hybrid heterostructures can effectively utilize UV-visible-light to remove nitrate and degrade formic acid. For the 3 cycles the photocatalyst efficiency remained same and after the third cycle, its efficacy decreased gradually. This work suggests 2D Ag-TiO2/Al2O3/CS nano-composite for the fast removal of nitrate in drinking water treatment.

Large Chromosomal Rearrangements Yield Biomarkers to Distinguish Low-Risk From Intermediate- and High-Risk Prostate Cancer.

OBJECTIVE: To test the hypothesis that chromosomal rearrangements (CRs) can distinguish low risk of progression (LRP) from intermediate and high risk of progression (IHRP) to prostate cancer (PCa) and if these CRs have the potential to identify men with LRP on needle biopsy that harbor IHRP PCa in the prostate gland. PATIENTS AND METHODS: Mate pair sequencing of amplified DNA from pure populations of Gleason patterns in 154 frozen specimens from 126 patients obtained between August 14, 2001, and July 15, 2011, was used to detect CRs including abnormal junctions and copy number variations. Potential CR biomarkers with higher incidence in IHRP than in LRP to cancer and having significance in PCa biology were identified. Independent validation was performed by fluorescence in situ hybridization in 152 specimens from 124 patients obtained between February 12, 2002, and July 12, 2008. RESULTS: The number of abnormal junctions did not distinguish LRP from IHRP. Loci corresponding to genes implicated in PCa were more frequently altered in IHRP. Integrated analysis of copy number variations and microarray data yielded 6 potential markers that were more frequently detected in Gleason pattern 3 of a Gleason score 7 of PCa than in Gleason pattern 3 of a Gleason score 6 PCa. Five of those were cross-validated in an independent sample set with statistically significant areas under the receiver operating characteristic curves (AUCs) (P≤.01). Probes detecting deletions in PTEN and CHD1 had AUCs of 0.87 (95% CI, 0.77-0.97) and 0.73 (95% CI, 0.60-0.86), respectively, and probes detecting gains in ASAP1, MYC, and HDAC9 had AUCs of 0.71 (95% CI, 0.59-0.84), 0.82 (95% CI, 0.71-0.93), and 0.77 (95% CI, 0.66-0.89), respectively (for expansion of gene symbols, use search tool at www.genenames.org). CONCLUSION: Copy number variations in regions encompassing important PCa genes were predictive of cancer significance and have the potential to identify men with LRP PCa by needle biopsy who have IHRP PCa in their prostate gland.

Extension of the mutational and clinical spectrum of SOX2 related disorders: Description of six new cases and a novel association with suprasellar teratoma.

SOX2 is a transcription factor that is essential for maintenance of pluripotency and has several conserved roles in early embryonic development. Heterozygous loss-of-function variants in SOX2 are identified in approximately 40% of all cases of bilateral anophthalmia/micropthalmia (A/M). Increasingly SOX2 mutation-positive patients without major eye findings, but with a range of other developmental disorders including autism, mild to moderate intellectual disability with or without structural brain changes, esophageal atresia, urogenital anomalies, and endocrinopathy are being reported, suggesting that the clinical phenotype associated with SOX2 loss is much broader than previously appreciated. In this report we describe six new cases, four of which carry novel pathogenic SOX2 variants. Four cases presented with bilateral anophthalmia in addition to extraocular involvement. Another individual presented with only unilateral anophthalmia. One individual did not have any eye findings but presented with a suprasellar teratoma in infancy and was found to have the recurrent c.70del20 mutation in SOX2 (c.70_89del, p.Asn24Argfs*65). This is this first time this tumor type has been reported in the context of a de novo SOX2 mutation. Notably, individuals with hypothalamic hamartomas and slow-growing hypothalamo-pituitary tumors have been reported previously, but it is still unclear how SOX2 loss contributes to their formation.

A data science approach for the classification of low-grade and high-grade ovarian serous carcinomas.

BACKGROUND: Copy Number Alternations (CNAs) is defined as somatic gain or loss of DNA regions. The profiles of CNAs may provide a fingerprint specific to a tumor type or tumor grade. Low-coverage sequencing for reporting CNAs has recently gained interest since successfully translated into clinical applications. Ovarian serous carcinomas can be classified into two largely mutually exclusive grades, low grade and high grade, based on their histologic features. The grade classification based on the genomics may provide valuable clue on how to best manage these patients in clinic. Based on the study of ovarian serous carcinomas, we explore the methodology of combining CNAs reporting from low-coverage sequencing with machine learning techniques to stratify tumor biospecimens of different grades. RESULTS: We have developed a data-driven methodology for tumor classification using the profiles of CNAs reported by low-coverage sequencing. The proposed method called Bag-of-Segments is used to summarize fixed-length CNA features predictive of tumor grades. These features are further processed by machine learning techniques to obtain classification models. High accuracy is obtained for classifying ovarian serous carcinoma into high and low grades based on leave-one-out cross-validation experiments. The models that are weakly influenced by the sequence coverage and the purity of the sample can also be built, which would be of higher relevance for clinical applications. The patterns captured by Bag-of-Segments features correlate with current clinical knowledge: low grade ovarian tumors being related to aneuploidy events associated to mitotic errors while high grade ovarian tumors are induced by DNA repair gene malfunction. CONCLUSIONS: The proposed data-driven method obtains high accuracy with various parametrizations for the ovarian serous carcinoma study, indicating that it has good generalization potential towards other CNA classification problems. This method could be applied to the more difficult task of classifying ovarian serous carcinomas with ambiguous histology or in those with low grade tumor co-existing with high grade tumor. The closer genomic relationship of these tumor samples to low or high grade may provide important clinical value.

Lineage relationship of Gleason patterns in Gleason score 7 prostate cancer.

Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2-ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2-ERG fusion-positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression.

Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.

High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.

Prognostic significance of measured depth of invasion of urothelial carcinoma of the bladder compared to the 2010 American Joint Committee on Cancer pT2 and pT3 classifications.

PURPOSE: We compared the prognostic ability of the current American Joint Committee on Cancer (AJCC) staging system to direct measurement of the depth of tumor invasion into the muscularis propria and perivesical fat. MATERIALS AND METHODS: We identified 148 patients with pT2N0 and 206 with pT3N0 who underwent radical cystectomy between 1990 and 2003. Clinicopathological features were reviewed. A measurement in mm was recorded of the depth of tumor invasion into the muscularis propria for pT2 cases and into perivesical fat for pT3 cases. Cancer specific survival between the pT2a and pT2b, and the pT3a and pT3b patient groups was estimated using the Kaplan-Meier method and compared with the log rank test. Optimal cutoff points for invasion depth in mm were estimated using an iterative estimation process to find the minimum p value with the maximum HR. RESULTS: Of 148 patients with pT2 bladder cancer, including 76 with pT2a and 72 with pT2b, and 206 with pT3 bladder cancer, including 144 with pT3a and 62 with pT3b, there was no significant difference in cancer specific survival between the substages (p = 0.94 and 0.37, respectively). However, patients with measured invasion less than 4.5 mm into perivesical fat had significantly improved cancer specific survival compared to that in patients with invasion 4.5 mm or greater (5-year cancer specific survival 53% vs 40%, p = 0.02). CONCLUSIONS: We found no significant difference in cancer specific survival when pT2 and pT3 tumors were stratified by AJCC substage. However, for pT3 tumors direct measurement of the depth of tumor invasion into perivesical fat identified a significant stratification of cancer specific survival at 4.5 mm.