Clinicopathologic Evaluation and Molecular Profiling of Recurrent Stage IA Endometrial Endometrioid Carcinoma: A Case-control Study.

Most low-grade, early-stage endometrial endometrioid carcinomas (EEC) have an excellent prognosis; however, recurrences occur in a small subset with several studies reporting an increase in CTNNB1 exon 3 mutations in this population. Herein we evaluated 10 recurrent low-grade (FIGO 1 or 2), early-stage (FIGO IA) EECs matched to 10 nonrecurrent EECs to further characterize their clinicopathologic features and molecular phenotype. Cases were matched to controls based on size, grade, and depth of invasion. All tumors were evaluated for specific clinicopathologic parameters followed by next-generation sequencing using a 1213 gene panel. Recurrent EECs demonstrated no significant clinicopathologic differences when compared with nonrecurrent EECs, in terms of age, body mass index, pattern of invasion, presence of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia, associated metaplastic changes, peritumoral lymphocytes, mitoses, and tumor-infiltrating lymphocytes. Both cohorts also showed a similar number of pathogenic mutations, including CTNNB1 exon 3 mutations, as well as tumor mutational burden and microsatellite profiles. Although in this particular study, the lack of correlation between CTNNB1 exon 3 mutation and recurrence might be secondary to a small sample size, it also suggests the presence of other contributing factors. Thus, it helps set the foundation for larger series incorporating whole genome, transcriptome, proteome, and epigenome analyses to answer this clinically important question.

Quantitative next-generation sequencing-based analysis indicates progressive accumulation of microsatellite instability between atypical hyperplasia/endometrial intraepithelial neoplasia and paired endometrioid endometrial carcinoma.

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

Sonographically Determined Fecal Width: An Objective Indicator of Management of Bladder and Bowel Dysfunction in Children.

OBJECTIVE: To test whether sonographically determined fecal width (SDFW) correlates with symptom improvement in a population of children with bladder and bowel dysfunction (BBD) managed with standard urotherapy (SU), even for those patients lacking initial bowel complaints. METHODS: We retrospectively analyzed 200 pediatric BBD patients managed with SU for at least 3 months. Self-reported symptom improvement (complete, partial, no response) following International Children's Continence Society guidelines was tabulated. Patients with complex urologic diagnoses other than vesicoureteral reflux (VUR) were excluded. Pharmacotherapy choice, physical therapy (PT), urinary tract infection (UTI) occurrence, and VUR status were tabulated. SDFW was recorded. Non-parametric analysis of variants (ANOVA) and parametric/non-parametric t testing were used for analysis. RESULTS: Patients had a mean age of 9.5 years (4-12). Forty-eight patients had no gastrointestinal complaints at presentation. Urotherapy yielded complete, partial, and no responses in 14% (n = 27), 33% (n = 67), and 53% (n = 106) of patients, respectively. The average SDFW for those patients with complete response (2.6 cm) was smaller than the SDFW of those with a partial response (3.1 cm) or no response (3.3 cm) (P = .0001). Non-compliance led to greater SDFW compared to compliant patients (3.7 cm and 3.1 cm, respectively, P = .0001). Fecal width was unaffected by VUR, UTI, PT, or pharmacotherapy. CONCLUSION: SDFW correlates well with symptom improvement in pediatric patients managed for BBD, confirming our hypothesis. SDFW is reasonable as single objective parameter to identify successful management in patients with BBD, extending to those without bowel complaints at presentation.

Endometrial Carcinomas With Subclonal Loss of Mismatch Repair Proteins: A Clinicopathologic and Genomic Study.

Subclonal loss of mismatch repair (MMR) proteins has been described in a small subset of endometrial carcinomas (ECs), but the genomic basis for this phenomenon has received limited attention. Herein, we retrospectively evaluated all ECs with MMR immunohistochemistry (n=285) for subclonal loss, and in those (n=6), performed a detailed clinicopathologic and genomic comparison of the MMR-deficient and MMR-proficient components. Three tumors were FIGO stage IA, and one each stage IB, II, and IIIC2. Patterns of subclonal loss were as follows: (1) 3 FIGO grade 1 endometrioid carcinomas with subclonal MLH1/PMS2, MLH1 promoter hypermethylation, and no MMR gene mutations; (2) POLE -mutated FIGO grade 3 endometrioid carcinoma with subclonal PMS2, and PMS2 and MSH6 mutations limited to the MMR-deficient component; (3) dedifferentiated carcinoma with subclonal MSH2/MSH6, as well as complete loss of MLH1/PMS2, MLH1 promoter hypermethylation, and PMS2 and MSH6 mutations in both components; (4) dedifferentiated carcinoma with subclonal MSH6, and somatic and germline MSH6 mutations in both components, but with a higher allele frequency in MMR-deficient foci. Recurrences occurred in 2 patients, one consisted of the MMR-proficient component from a FIGO 1 endometrioid carcinoma, while the other was from the MSH6 -mutated dedifferentiated endometrioid carcinoma. At the last follow-up (median: 44 mo), 4 patients were alive and disease-free and 2 were alive with disease. In summary, subclonal MMR loss reflects subclonal and often complex genomic and epigenetic alterations, which may have therapeutic implications and therefore must be reported when present. In addition, subclonal loss can occur in both POLE -mutated and Lynch syndrome-associated ECs.

High Rates of Ultraviolet-Signature Mutations in Squamous Cell Carcinomas of the Parotid Gland and Prognostic Implications.

In the absence of clear pathologic differences, clinical history may differentiate potential primary parotid squamous cell carcinomas (SCC) from metastases. The presence of an ultraviolet (UV) signature can distinguish between tumors of cutaneous and non-cutaneous origin. This study aimed to investigate rates of UV signature mutations in squamous cell carcinomas of the parotid gland as well as differences in clinical features between tumors of cutaneous and non-cutaneous origin. Clinical and pathologic data were collected from 71 patients with SCC involving the parotid gland, of which 48 had cutaneous, 10 had mucosal, and 13 had no history of SCC. In 34 available cases, genomic DNA was isolated from formalin-fixed paraffin-embedded tissue specimens and sequenced using a targeted hybrid capture 1213 gene panel. Tumor mutational burden and COSMIC (Catalogue of Somatic Mutations in Cancer) mutational signatures were calculated. Most (74%) were UV-positive. Patients with UV-positive tumors were significantly older, white, and had higher rates of sun exposure. Patients with UV-negative tumors had a significantly higher mortality rate and shorter time to death: 6 (67%) died of disease with a median time to death of 9 months compared to 5 (20%) UV-positive patients who died of disease with a median time to death of 32 months. Pathologic features did not significantly vary by clinical history or UV status. The presence of a UV-signature combined with clinical history can be used to determine the primary source of SCC involving the parotid gland. UV-positivity may reflect a less aggressive disease course in an older population.

CDKN2A loss-of-function predicts immunotherapy resistance in non-small cell lung cancer.

Immune checkpoint blockade (ICB) improves outcomes in non-small cell lung cancer (NSCLC) though most patients progress. There are limited data regarding molecular predictors of progression. In particular, there is controversy regarding the role of CDKN2A loss-of-function (LOF) in ICB resistance. We analyzed 139 consecutive patients with advanced NSCLC who underwent NGS prior to ICB initiation to explore the association of CDKN2A LOF with clinical outcomes. 73% were PD-L1 positive (≥ 1%). 48% exhibited high TMB (≥ 10 mutations/megabase). CDKN2A LOF was present in 26% of patients and was associated with inferior PFS (multivariate hazard ratio [MVA-HR] 1.66, 95% CI 1.02-2.63, p = 0.041) and OS (MVA-HR 2.08, 95% CI 1.21-3.49, p = 0.0087) when compared to wild-type (WT) patients. These findings held in patients with high TMB (median OS, LOF vs. WT 10.5 vs. 22.3 months; p = 0.069) and PD-L1 ≥ 50% (median OS, LOF vs. WT 11.1 vs. 24.2 months; p = 0.020), as well as in an independent dataset. CDKN2A LOF vs. WT tumors were twice as likely to experience disease progression following ICB (46% vs. 21%; p = 0.021). CDKN2A LOF negatively impacts clinical outcomes in advanced NSCLC treated with ICB, even in high PD-L1 and high TMB tumors. This novel finding should be prospectively validated and presents a potential therapeutic target.

Rate and associations of epididymal cysts on pediatric scrotal ultrasound.

PURPOSE: We established the baseline occurrence of epididymal cysts, and the correlation between epididymal cysts and testicular size. MATERIALS AND METHODS: We retrospectively reviewed all pediatric scrotal ultrasounds done at our institution in 8 years. We analyzed the proportion of cysts by patient age and compared testicular size in boys with vs without epididymal cysts. RESULTS: Of all patients 14.4% had epididymal cysts. The cyst incidence increased with age, ie 35.3% of boys older than 15 years had cysts. Boys with epididymal cysts had larger testes than boys without cysts regardless of side or age (p <0.001). CONCLUSIONS: Epididymal cysts are more common in older boys. Boys with epididymal cysts had larger testes than boys without cysts.

Is age at toilet training associated with the presence of vesicoureteral reflux or the occurrence of urinary tract infection?

PURPOSE: We sought to determine whether age at toilet training is influenced by a history of vesicoureteral reflux or urinary tract infection. MATERIALS AND METHODS: We reviewed records on 1,184 patients treated at a pediatric urology practice. All patients had information available regarding age at toilet training, renal sonography and voiding cystourethrography, and presence or absence of urinary tract infection. We evaluated possible associations between vesicoureteral reflux and urinary tract infection, and age at toilet training. RESULTS: Of 1,184 patients 280 had unilateral reflux, 339 had bilateral reflux and 565 had normal anatomy. Also, 926 patients had urinary tract infections. Girls tended to be toilet trained 3 months earlier than boys (p <0.001) in all subgroups (normal anatomy, unilateral reflux, bilateral reflux). Children with and without urinary tract infections were toilet trained at similar ages. However, timing of the first urinary tract infection seemed to be associated with age at toilet training. For girls a urinary tract infection occurring earlier tended to delay toilet training, while earlier toilet training seemed to be associated with a later urinary tract infection (p <0.001). The patterns were similar for boys but were not statistically significant. CONCLUSIONS: Age at toilet training seems to be independent of the presence of vesicoureteral reflux. Urinary tract infection itself is not necessarily associated with age at toilet training. However, timing of the first urinary tract infection seems to be related to age at toilet training.

Measurable residual disease monitoring for patients with acute myeloid leukemia following hematopoietic cell transplantation using error corrected hybrid capture next generation sequencing.

Improved systems for detection of measurable residual disease (MRD) in acute myeloid leukemia (AML) are urgently needed, however attempts to utilize broad-scale next-generation sequencing (NGS) panels to perform multi-gene surveillance in AML post-induction have been stymied by persistent premalignant mutation-bearing clones. We hypothesized that this technology may be more suitable for evaluation of fully engrafted patients following hematopoietic cell transplantation (HCT). To address this question, we developed a hybrid-capture NGS panel utilizing unique molecular identifiers (UMIs) to detect variants at 0.1% VAF or below across 22 genes frequently mutated in myeloid disorders and applied it to a retrospective sample set of blood and bone marrow DNA samples previously evaluated as negative for disease via standard-of-care short tandem repeat (STR)-based engraftment testing and hematopathology analysis in our laboratory. Of 30 patients who demonstrated trackable mutations in the 22 genes at eventual relapse by standard NGS analysis, we were able to definitively detect relapse-associated mutations in 18/30 (60%) at previously disease-negative timepoints collected 20-100 days prior to relapse date. MRD was detected in both bone marrow (15/28, 53.6%) and peripheral blood samples (9/18, 50%), while showing excellent technical specificity in our sample set. We also confirmed the disappearance of all MRD signal with increasing time prior to relapse (>100 days), indicating true clinical specificity, even using genes commonly associated with clonal hematopoiesis of indeterminate potential (CHIP). This study highlights the efficacy of a highly sensitive, NGS panel-based approach to early detection of relapse in AML and supports the clinical validity of extending MRD analysis across many genes in the post-transplant setting.

Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry.

Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored.

Epididymal Cysts: Are They Associated With Infertility?

Scrotal sonography is commonly used for evaluation of the infertile male. While epididymal cysts are frequently observed during sonographic assessment, their presence has uncertain import. This study is a retrospective case-control sonographic and chart review comparison of infertile men and fertile volunteers to clarify the possible association of epididymal cysts and infertility. The study included 91 consecutively recruited patients from January 2012 to December 2014. The infertile group consisted patients with male factor infertility who underwent scrotal sonography ( n = 67). The fertile group consisted of men requesting vasectomy who were recruited for study involvement and consented to undergo scrotal sonography ( n = 24). The main outcome measure was infertility. The existence of epididymal cysts on scrotal sonography was the main risk factor. Predictably, the only sonographic findings associated with infertility were small testes (right: t(df = 89) = -2.52; left: t(df = 89) = -2.28, both p = .01) and the presence of a varicocele, χ2(df = 1) = 5.766 with p = .02. The infertile men were also younger and more likely to use alcohol. Of the 91 men studied, 71% demonstrated epididymal cysts (73% of infertile and 67% of fertile men). Epididymal cysts were not be associated with infertility, χ2(df = 1) = 0.362 with p = .55. This occurrence of epididymal cysts is the highest ever reported (71% of all men). While the occurrence of epididymal cysts in this cohort is unexplained, our observation that these cysts are not associated with infertility will be useful for those clinicians counseling patients observed to have these structures.

Which Objective Parameters Are Associated with a Positive Urine Culture in the Setting of Ureteral Calculi: The Ureteral Calculi Urinary Culture Calculator.

OBJECTIVE: To determine associations between laboratory values and subsequent culture positivity in the acute ureteral calculi patient. Specifically, we aim to develop a predictive model to assist with optimization of patient outcomes and improvement of antimicrobial stewardship. METHODS: Utilizing the electronic medical record system, we conducted a retrospective review of 3888 patients with ureteral calculi. Relevant demographic information, vital signs, and laboratory parameters obtained in the emergency department were tabulated. We applied a combination of analysis of variance and Pearson Fisher's Exact test for the analysis. RESULTS: A total of 3888 patients were included in the analysis of whom 3171 (81.6%) had a negative urine culture and 717 (18.4%) had a positive urine culture. Basic vital signs and laboratory parameters, such as heart rate, temperature, white blood cell (WBC) count, platelet count, and neutrophil differential only varied slightly in the positive and negative culture groups. Urinary nitrite was found to have specificity of 97.2% with a negative predictive value of 83.7%. Urinary leukocyte esterase was found to have a sensitivity of 86.8% and positive predictive value of 46.9%. On microscopy analysis, WBCs per high power field (WBCs/hpf) varied directly with likelihood of a positive urine culture; >150 WBCs/hpf had an 86.1% likelihood of positive urine culture. CONCLUSION: With the data provided from this large cohort analysis, we were able to create the ureteral calculi urinary culture calculator. With this calculator, urologists are better equipped to stratify a patient's risk of having a positive urine culture in the setting of a ureteral calculus.

The limitations of simple gene set enrichment analysis assuming gene independence.

Since its first publication in 2003, the Gene Set Enrichment Analysis method, based on the Kolmogorov-Smirnov statistic, has been heavily used, modified, and also questioned. Recently a simplified approach using a one-sample t-test score to assess enrichment and ignoring gene-gene correlations was proposed by Irizarry et al. 2009 as a serious contender. The argument criticizes Gene Set Enrichment Analysis's nonparametric nature and its use of an empirical null distribution as unnecessary and hard to compute. We refute these claims by careful consideration of the assumptions of the simplified method and its results, including a comparison with Gene Set Enrichment Analysis's on a large benchmark set of 50 datasets. Our results provide strong empirical evidence that gene-gene correlations cannot be ignored due to the significant variance inflation they produced on the enrichment scores and should be taken into account when estimating gene set enrichment significance. In addition, we discuss the challenges that the complex correlation structure and multi-modality of gene sets pose more generally for gene set enrichment methods.