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Urinary incontinence after robotic-assisted radical prostatectomy (RARP) has been associated with older age, a longer operative time, a higher BMI, a short membranous urethral length and preoperative erectile function. The authors sought to assess the association between the neuromuscular characteristics and postoperative urinary incontinence., Methods: RARP specimens from 29 men who underwent bilateral nerve sparing were reanalyzed. Urinary incontinence was evaluated using the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) at 6 weeks post surgery and last follow-up. Linear and logistic regression analyses were performed to assess neuromuscular characteristics and incontinence., Results: At the 1-year follow-up, 11 patients (38%) reported severe incontinence (>12 ICIQ-SF score). The median number of peripheral nerves observed at the base and apex in the specimens was 52 (IQR 13-139) and 59 (IQR: 28-129), respectively. Ganglia were present in 19 patients (65%) at the base and 12 patients (41%) at the apex. Additionally, the median proportional area of detrusor smooth muscle fibers at the base was 0.54 (IQR 0.31-1), while the median proportional area of striated muscle fibers at the apex was 0.13 (IQR 0.08-0.24). No statistically significant associations were found., Conclusions: Histologic neuromuscular characteristics were not associated with postoperative urinary incontinence. Enhanced intraoperative evaluation and larger-scale studies may prove useful for the prediction of postprostatectomy incontinence.

Introduction: Most men diagnosed with very-low and low-risk prostate cancer are candidates for active surveillance; however, there is still a misclassification risk. We examined whether PI-RADS category 4 or 5 combined with ISUP 1 on prostate biopsy predicts upgrading and/or adverse pathology at radical prostatectomy., Materials and Methods: A total of 127 patients had ISUP 1 cancer on biopsy after multiparametric MRI (mpMRI) and then underwent radical prostatectomy. We then evaluated them for ISUP upgrading and/or adverse pathology on radical prostatectomy., Results: Eight-nine patients (70%) were diagnosed with PI-RADS 4 or 5 lesions. ISUP upgrading was significantly higher among patients with PI-RADS 4-5 lesions (84%) compared to patients with equivocal or non-suspicious mpMRI findings (26%, p < 0.001). Both PI-RADS 4-5 lesions (OR 24.3, 95% CI 7.3, 80.5, p < 0.001) and stage T2 on DRE (OR 5.9, 95% CI 1.2, 29.4, p = 0.03) were independent predictors of upgrading on multivariate logistic regression analysis. Men with PI-RADS 4-5 lesions also had significantly more extra-prostatic extension (51% vs. 3%, p < 0.001) and positive surgical margins (16% vs. 3%. p = 0.03). The only independent predictor of adverse pathology was PI-RADS 4-5 (OR 21.7, 95% CI 4.8, 99, p < 0.001)., Conclusion: PI-RADS 4 or 5 lesions on mpMRI were strong independent predictors of upgrading and adverse pathology. Incorporating mpMRI findings when selecting patients for active surveillance must be further evaluated in future studies.

Background & Objectives: Tumor grade determines prognosis in urothelial carcinoma. The classification of low and high grade is based on nuclear morphological features that include nuclear size, hyperchromasia and pleomorphism. These features are subjectively assessed by the pathologists and are not numerically measured, which leads to high rates of interobserver variability. The purpose of this study is to assess the value of a computer-based image analysis tool for identifying predictors of tumor grade in bladder cancer., Methods: Four hundred images of urothelial tumors were graded by five pathologists and two expert genitourinary pathologists using a scale of 1 (lowest grade) to 5 (highest grade). A computer algorithm was used to automatically segment the nuclei and to provide morphometric parameters for each nucleus, which were used to establish the grading algorithm. Grading algorithm was compared to pathologists' agreement., Results: Comparison of the grading scores of the five pathologists with the expert genitourinary pathologists score showed agreement rates between 88.5% and 97.5%.The agreement rate between the two expert genitourinary pathologists was 99.5%. The quantified algorithm based conventional parameters that determine the grade (nuclear size, pleomorphism and hyperchromasia) showed > 85% agreement with the expert genitourinary pathologists. Surprisingly, the parameter that was most associated with tumor grade was the 10th percentile of the nuclear area, and high grade was associated with lower 10th percentile nuclei, caused by the presence of more inflammatory cells in the high-grade tumors., Conclusion: Quantitative nuclear features could be applied to determine urothelial carcinoma grade and explore new biologically explainable parameters with better correlation to grade than those currently used., (© 2024. The Author(s).)

The classic morphology of clear cell renal cell carcinoma consists of nests of cells with clear cytoplasm. Nevertheless, other histologic patterns may be seen including cells with eosinophilic cytoplasm, bizarre multinucleated giant tumor cells and pseudopapillary structures. In this article, we present the first case of clear cell renal cell carcinoma with a prominent micropapillary pattern., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: To clinically and histologically characterize prostatic nodules resistant to morcellation ("beach balls," BBs)., Patients and Methods: We reviewed a consecutive cohort of 559 holmium laser enucleation of the prostate (HoLEP) procedures performed between January 2020 and November 2023. The BBs group comprised 55 men (10%) and the control group comprised 504 men (90%). The clinical, intraoperative, outcome, and histologic data were statistically processed for the prediction of the presence of BBs and their influence on the perioperative course and outcome., Results: The BBs group in comparison to the controls was older (75 vs 73 years, respectively, p = 0.009) and had higher rates of chronic retention (51 vs 29%, p = 0.001), larger prostates on preoperative abdominal ultrasound (AUS) (140 vs 80 cc, p = 0.006 E-16 ), longer operating time (120 vs 80 min, p = 0.001), higher weights of removed tissue (101 vs 60 gr, p = 0.008 E-10 ), higher complication rates (5 vs 1%, p = 0.03), and longer hospitalization (p = 0.014). A multivariate analysis revealed that larger prostates on preoperative AUS and older age independently predicted the presence of BBs which would prolong operating time. ROC analyses revealed that a threshold of 103 cc on AUS predicted BBs with 94% sensitivity and 84% specificity. BBs were mostly characterized histologically by stromal component (p = 0.005)., Conclusions: BBs are expected in older patients and cases of chronic retention. Prostatic volume is the most reliable predictor of their presence. They contribute to prolonged operating time and increased risk of complications. The predominantly stromal composition of the BBs apparently confers their resistance to morcellation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction:   Prostate cancer screening with PSA is associated with low specificity; furthermore, little is known about the optimal timing of biopsy.  We aimed to evaluate whether a risk classification system combining PSA density (PSAD) and mpMRI can predict clinically significant cancer and determine biopsy timing., Materials and Methods:  We reviewed the medical records of 256 men with a PI-RADS ≥ 3 lesion on mpMRI who underwent transperineal targeted and systematic biopsies of the prostate between 2017-2019.  Patients were stratified into three risk groups based on PSAD and mpMRI findings. The study endpoint was clinically significant prostate cancer (CSPC).  The association between the risk groups and CSPC was evaluated., Results:  Based on the proposed risk stratification system 42/256 men (16%) were high-risk (mpMRI finding of extra-prostatic extension and/or seminal vesicle invasion and/or a PI-RADS 5 lesion with a PSAD > 0.15 ng/mL²), 164/256 (64%) intermediate-risk (PI-RADS 4-5 lesions and/or PSAD > 0.15ng/mL² with no high-risk features) and 50/256 (20%) low-risk (PI-RADS 3 lesions and PSAD ≤ 0.15 ng/mL²).  High-risk patients had significantly higher rates of CSPC (76%) when compared to intermediate-risk (26%) and low-risk (4%).  On multivariable logistic regression analysis adjusted for age, previous biopsy, and clinical T-stage we found an association between intermediate-risk (OR = 4.84, p = 0.038) and high-risk (OR = 40.13, p < 0.001) features and CSPC.  High-risk patients had a shorter median biopsy delay time (110 days) compared to intermediate- and low-risk patients (141 and 147 days, respectively).  We did not find an association between biopsy delay and CSPC., Conclusions:   Our findings suggest that a three-tier risk classification system based on mpMRI and PSAD can identify patients at high-risk for CSPC who may benefit from earlier biopsy.

Background & Objective: Assessment of muscularis propria invasion is a crucial step in the management of urothelial carcinoma since it necessitates aggressive treatment. The diagnosis of muscle invasion is a challenging process for pathologists. Artificial intelligence is developing rapidly and being implemented in various fields of pathology. The purpose of this study was to develop an algorithm for the detection of muscularis propria invasion in urothelial carcinoma. Methods: The Training cohort consisted of 925 images from 50 specimens of urothelial carcinoma. Ninety-seven images from 10 new specimens were used as a validation cohort. Clinical validation used 127 whole specimens with a total of 617 slides. The algorithm determined areas where tumor and muscularis propria events were in nearest proximity, and presented these areas to the pathologist. Results: Analytical evaluation showed a sensitivity of 72% for muscularis propria and 65% for tumor, and a specificity of 46% and 77% for muscularis propria and tumor detection, respectively. The incorporation of the spatial proximity factor between muscularis propria and tumor in the clinical validation significantly improved the detection of muscularis propria invasion, as the algorithm managed to identify all except for one case with muscle invasive bladder cancer in the clinical validation cohort. The case missed by the algorithm was nested urothelial carcinoma, a rare subtype with unusual morphologic features. The pathologist managed to identify muscle invasion based on the images provided by the algorithm in a short time, with an average of approximately 5 s. Conclusion: The algorithm we developed may greatly aid in accurate identification of muscularis propria invasion by imitating the thought process of the pathologist., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Prostatic neuroendocrine malignancies represent a spectrum of diseases. Treatment-induced neuroendocrine differentiation (tiNED) in hormonally treated adenocarcinoma has been the subject of a large amount of recent research. However, the identification of neuroendocrine features in treatment-naïve prostatic tumor raises a differential diagnosis between prostatic adenocarcinoma with de novo neuroendocrine differentiation (dNED) versus one of the primary prostatic neuroendocrine tumors (P-NETs) and carcinomas (P-NECs). While [ 18 F]FDG is being used as the main PET radiotracer in oncologic imaging and reflects cellular glucose metabolism, other molecules labeled with positron-emitting isotopes, mainly somatostatin-analogues labeled with 68 Ga and prostate-specific membrane antigen (PSMA)-ligands labeled with either 18 F or 68 Ga, are now routinely used in departments of nuclear medicine and molecular imaging, and may be advantageous in imaging prostatic neuroendocrine malignancies. Still, the selection of the preferred PET radiotracer in such cases might be challenging. In the current review, we summarize and discuss published data on these different entities from clinical, biological, and molecular imaging standpoints. Specifically, we review the roles that [ 18 F]FDG, radiolabeled somatostatin-analogues, and radiolabeled PSMA-ligands play in these entities in order to provide the reader with practical recommendations regarding the preferred PET radiotracers for imaging each entity. In cases of tiNED, we conclude that PSMA expression may be low and that [ 18 F]FDG or radiolabeled somatostatin-analogues should be preferred for imaging. In cases of prostatic adenocarcinoma with dNED, we present data that support the superiority of radiolabeled PSMA-ligands. In cases of primary neuroendocrine malignancies, the use of [ 18 F]FDG for imaging high-grade P-NECs and radiolabeled somatostatin-analogues for imaging well-differentiated P-NETs is recommended. KEY POINTS: • The preferred PET radiotracer for imaging prostatic neuroendocrine malignancies depends on the specific clinical scenario and pathologic data. • When neuroendocrine features result from hormonal therapy for prostate cancer, PET-CT should be performed with [ 18 F]FDG or radiolabeled somatostatin-analogue rather than with radiolabeled PSMA-ligand. • When neuroendocrine features are evident in newly diagnosed prostate cancer, differentiating adenocarcinoma from primary neuroendocrine malignancy is challenging but crucial for selection of PET radiotracer and for clinical management., (© 2023. The Author(s), under exclusive licence to European Society of Radiology.)

Pathologic examination of prostate biopsies is time consuming due to the large number of slides per case. In this retrospective study, we validate a deep learning-based classifier for prostate cancer (PCA) detection and Gleason grading (AI tool) in biopsy samples. Five external cohorts of patients with multifocal prostate biopsy were analyzed from high-volume pathology institutes. A total of 5922 H&E sections representing 7473 biopsy cores from 423 patient cases (digitized using three scanners) were assessed concerning tumor detection. Two tumor-bearing datasets (core n = 227 and 159) were graded by an international group of pathologists including expert urologic pathologists (n = 11) to validate the Gleason grading classifier. The sensitivity, specificity, and NPV for the detection of tumor-bearing biopsies was in a range of 0.971-1.000, 0.875-0.976, and 0.988-1.000, respectively, across the different test cohorts. In several biopsy slides tumor tissue was correctly detected by the AI tool that was initially missed by pathologists. Most false positive misclassifications represented lesions suspicious for carcinoma or cancer mimickers. The quadratically weighted kappa levels for Gleason grading agreement for single pathologists was 0.62-0.80 (0.77 for AI tool) and 0.64-0.76 (0.72 for AI tool) for the two grading datasets, respectively. In cases where consensus for grading was reached among pathologists, kappa levels for AI tool were 0.903 and 0.855. The PCA detection classifier showed high accuracy for PCA detection in biopsy cases during external validation, independent of the institute and scanner used. High levels of agreement for Gleason grading were indistinguishable between experienced genitourinary pathologists and the AI tool., (© 2023. Nature Publishing Group UK.)

Objective: Multiparametric magnetic resonance imaging (mpMRI) is central to diagnosing prostate cancer; however, not all imaged lesions represent clinically significant tumors. We aimed to evaluate the association between the relative tumor volume on mpMRI and clinically significant prostate cancer on biopsy., Materials and Methods: We retrospectively reviewed the medical records of 340 patients who underwent combined transperineal targeted and systematic prostate biopsies between 2017 and 2021. Tumor volume was estimated based on the mpMRI diameter of suspected lesions. Relative tumor volume (tumor density) was calculated by dividing the tumor and prostate volumes. The study outcome was clinically significant cancer on biopsy. Logistic regression analyses were used to evaluate the association between tumor density and the outcome. The cutoff for tumor density was determined with ROC curves., Results: Median estimated prostate and peripheral zone tumor volumes were 55cm 3 and 0.61cm 3 , respectively. Median PSA density was 0.13 and peripheral zone tumor density was 0.01. Overall, 231 patients (68%) had any cancer and 130 (38%) had clinically significant cancer. On multivariable logistic regression age, PSA, previous biopsy, maximal PI-RADS score, prostate volume, and peripheral zone tumor density were significant predictors of outcome. Using a threshold of 0.006, the sensitivity, specificity, positive and negative predictive values of peripheral zone tumor density were 0.9, 0.51, 0.57, and 0.88, respectively., Conclusion: Peripheral zone tumor density is associated with clinically significant prostate cancer in patients with PI-RADS 4 and 5 mpMRI lesions. Future studies are required to validate our findings and evaluate the role of tumor density in avoiding unnecessary biopsies., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Context.—: Medical education in pathology relies on the accumulation of experience gained through inspection of numerous samples from each entity. Acquiring sufficient teaching material for rare diseases, such as Hirschsprung disease (HSCR), may be difficult, especially in smaller institutes. The current study makes use of a previously developed decision support system using a decision support algorithm meant to aid pathologists in the diagnosis of HSCR., Objective.—: To assess the effect of a short training session on algorithm-assisted HSCR diagnosis., Design.—: Five pathologists reviewed a data set of 568 image sets (1704 images in total) selected from 50 cases by the decision support algorithm and were tasked with scoring the images for the presence or absence of ganglion cells. The task was repeated a total of 3 times. Each pathologist had to complete a short educational presentation between the second and third iterations., Results.—: The training resulted in a significantly increased rate of correct diagnoses (true positive/negative) and a decreased need for referrals for expert consultation. No statistically significant changes in the rate of false positives/negatives were detected., Conclusions.—: A very short (<10 minutes) training session can greatly improve the pathologist's performance in the algorithm-assisted diagnosis of HSCR. The same approach may be feasible in training for the diagnosis of other rare diseases., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article.

Introduction: Concurrent systematic biopsies during image-guided targeted biopsies of the prostate were found to improve the detection rate of clinically significant prostate cancer (CSPC). However, these biopsies do not routinely include anterior or apical sampling. We aimed to evaluate the significance of anterior and apical samplings during combined biopsies., Methods: After obtaining institutional review board approval we identified 303 consecutive patients who underwent transperineal combined biopsies of the prostate between 2017-2020. Systematic biopsies were obtained from the peripheral zone, anterior zone, and apex. Study outcomes included CSPC and any cancer on anterior or apical biopsies. Logistic regression analyses were used to evaluate the association between pre-biopsy characteristics and study outcomes., Results: Median prostatic-specific-antigen value was 6.8 ng/dL. Most patients had stage T1c disease (77%). Overall, combined biopsies detected CSPC in 87 patients (29%). Any cancer and CSPC in the anterior zone were found in 54 (18%) and 19 (6%) patients, respectively. Any cancer and CSPC in the apex were found in 54 (18%) and 16 (5%) patients, respectively. Anterior/apical samplings upgraded the pathological result in 19 patients (6%). Logistic regression analyses demonstrated that PI-RADS 5 lesions predicted the presence of CSPC in both the anterior zone (OR = 8, 95%CI = 3-22, P <0.001) and apex (OR = 4, 95%CI = 1-10, P = 0.01)., Conclusions: Avoiding anterior and apical samplings during prostate biopsy does not result in substantial under-diagnosis of significant cancer. However, these areas are easily accessible using the transperineal approach and should be sampled in selected patients, particularly those with PI-RADS 5 lesions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: High-risk prostate cancer is associated with adverse pathology and unfavorable outcomes after radical prostatectomy. 68 Ga-PSMA PET/CT is more accurate than conventional imaging for preoperative staging. We aimed to evaluate whether lymph node involvement on 68 Ga-PSMA PET/CT prior to radical prostatectomy in patients with high-risk prostate cancer is associated with worse short-term oncologic outcomes., Methods: We retrospectively reviewed 149 patients with high-risk localized or locoregional prostate cancer who underwent 68 Ga-PSMA PET/CT prior to radical prostatectomy between 2015 and 2020. None of the patients received neoadjuvant or adjuvant treatment. The study endpoints were PSA persistence and biochemical recurrence. Logistic regression models were used to identify preoperative predictors of PSA persistence. Kaplan-Meier analyses were used to estimate biochemical recurrence-free survival., Results: Of 149 identified patients, 19 (13%) were found to have lymph node involvement on preoperative 68 Ga-PSMA PET/CT. The sensitivity, specificity, and accuracy of 68 Ga-PSMA PET/CT for identifying pathologic lymph node involvement were 68%, 95%, and 92%, respectively. PSA persistence rate was lower among patients with PET-negative lymph nodes than those with PET-positive nodes (15 vs. 84%, p < 0.001). Positive nodes on imaging (OR = 41.03, p < 0.001) and clinical T2c-T3 stage (OR = 6.96, p = 0.002) were associated with PSA persistence on multivariable analysis. Among patients with PET-negative nodes the 1- and 2-year biochemical recurrence-free survival rates were 87% and 76%, respectively., Conclusions: Preoperative staging with 68 Ga-PSMA PET/CT may identify a subgroup of high-risk prostate cancer patients with favorable short-term outcomes after radical prostatectomy without adjuvant treatment. Future studies will evaluate whether these results are sustained during long-term follow-up., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The JAK/STAT3 signaling pathway may be aberrantly activated and have various and conflicting roles in breast cancer. The current study explored prognostic implications of activated STAT3 in human epidermal growth factor receptor 2 (HER2)-positive primary breast cancers in the context of a large prospective study (ALTTO). Activated STAT3 was determined by immunohistochemical analysis of STAT3 phosphorylation (Y705) performed on the primary tumors. This analysis evaluated whether patients with activated STAT3 had disease-free survival (DFS) and overall survival (OS) different from patients without activated STAT3. A total of 5694 patients out of the 8381 patients enrolled in ALTTO were included in this analysis (67.9%), and 2634 of them (46%) had evidence of STAT3 activation (minimum tumor Allred score ≥2). The median follow-up was 6.93 years (6.85-6.97 years), at the end of which 1035 (18.18%) and 520 (9.13%) patients experienced DFS and OS events, respectively. Patients with STAT3 activation experienced improved DFS compared to those without it (multivariable hazard ratio [HR], 0.84; 95% confidence interval [CI] 0.74-0.95; P = .006). There were no group differences in OS (multivariable HR, 0.92; 95% CI 0.78-1.10; P = .37). This effect was limited to ER-positive tumors. In conclusion, these findings support the role of STAT3 activation as a marker of favorable outcome in ER-positive/HER2-positive breast cancer patients., (© 2020 Union for International Cancer Control.)

The most common site of breast cancer metastasis is the bone, occurring in approximately 70% of patients with advanced disease. Bone metastasis is associated with severe morbidities and high mortality. Therefore, deeper understanding of the mechanisms that enable bone-metastatic relapse are urgently needed. We report the establishment and characterization of a bone-seeking variant of breast cancer cells that spontaneously forms aggressive bone metastases following surgical resection of primary tumor. We characterized the modifications in the immune milieu during early and late stages of metastatic relapse and found that the formation of bone metastases is associated with systemic changes, as well as modifications of the bone microenvironment towards an immune suppressive milieu. Furthermore, we characterized the intrinsic changes in breast cancer cells that facilitate bone-tropism and found that they acquire mesenchymal and osteomimetic features. This model provides a clinically relevant platform to study the functional interactions between breast cancer cells and the bone microenvironment, in an effort to identify novel targets for intervention.

Tumor-to-tumor metastasis is being described in different types of tumors and in increasing amount of cases. Being aware of this phenomenon is important, as it affects disease stage and treatment approach. In this report, we descried an incidental histopathologic finding of metastatic adenocarcinoma to an ovarian cystadenofibroma and review cases published previously in the literature.

18 F-PSMA-1007 is a novel prostate-specific membrane antigen (PSMA)-based radiopharmaceutical for imaging prostate cancer (PCa). The aim of this study was to compare the diagnostic accuracy of 18 F-PSMA-1007 with 68 Ga-PSMA-11 PET/CT in the same patients presenting with newly diagnosed intermediate- or high-risk PCa. Methods: Sixteen patients with intermediate- or high-risk PCa underwent 18 F-PSMA-1007 and 68 Ga-PSMA-11 PET/CT within 15 d. PET findings were compared between the 2 radiotracers and with reference-standard pathologic specimens obtained from radical prostatectomy. The Cohen κ-coefficient was used to assess the concordance between 18 F-PSMA-1007 and 68 Ga-PSMA-11 for detection of intraprostatic lesions. The McNemar test was used to assess agreement between intraprostatic PET/CT findings and histopathologic findings. Sensitivity, specificity, positive predictive value, and negative predictive value were reported for each radiotracer. SUV max was measured for all lesions, and tumor-to-background activity was calculated. Areas under receiver-operating-characteristic curves were calculated for discriminating diseased from nondiseased prostate segments, and optimal SUV cutoffs were calculated using the Youden index for each radiotracer. Results: PSMA-avid lesions in the prostate were identified in all 16 patients with an almost perfect concordance between the 2 tracers (κ ranged from 0.871 to 1). Aside from the dominant intraprostatic lesion, similarly detected by both radiotracers, a second less intense positive focus was detected in 4 patients only with 18 F-PSMA-1007. Three of these secondary foci were confirmed as Gleason grade 3 lesions, whereas the fourth was shown on pathologic examination to represent chronic prostatitis. Conclusion: This pilot study showed that both 18 F-PSMA-1007 and 68 Ga-PSMA-11 identify all dominant prostatic lesions in patients with intermediate- or high-risk PCa at staging. 18 F-PSMA-1007, however, may detect additional low-grade lesions of limited clinical relevance., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Objective: Circulating tumor DNA is a promising noninvasive tool for cancer monitoring. One of the challenges in applying this tool is the detection of low-frequency mutations. The detection limit of these mutations varies between different molecular methods. The aim of this study is to characterize the factors affecting the limit of detection for epidermal growth factor receptor p.T790M mutation in circulating tumor DNA of patients with lung adenocarcinoma., Methods: DNA was extracted from plasma samples of 102 patients. For sequencing the DNA, we used 2 different next-generation sequencing-based platforms: Ion Torrent Personal Genome Machine (56 cases) and Roche/454 (46 cases). Serially diluted synthetic DNA samples carrying the p.T790M mutation were sequenced using the Ion Torrent Personal Genome Machine for validation. Limit of detection was determined through the analysis of non-hot-spot nonreference reads, which were regarded as sequencing artifacts., Results: The frequency of the non-hot-spot nonreference reads was higher in Ion Torrent Personal Genome Machine compared to Roche/454 (0.07% ± 0.08% and 0.03% ± 0.06%, respectively, P < .001). We found that different base type substitutions occur with different frequency. Since the base substitution leading to p.T790M mutation is C>T transition, its frequency was used to determine the limit of detection for the assay. Based on the C>T non-hot-spot nonreference allele frequency, we found that the limit of detection is 0.18% in Ion Torrent Personal Genome Machine and 0.1% in Roche/454. Based on these values, 48% and 56% of the cases were positive for T790M mutation in Ion Torrent Personal Genome Machine and Roche/454 groups, respectively. Agreement between duplicates was 76% in Ion Torrent Personal Genome Machine and 72% in Roche/454. Using serially diluted synthetic DNA samples carrying the p.T790M mutation, we could identify mutations with allele frequency of 0.18% or more using the Ion Torrent Personal Genome Machine, supporting our approach to determine the detection limit., Conclusion: Both the sequencing platform and the specific nucleotide change affect the limit of detection and should therefore be determined in the validation process of new assays.

With the increasing usage of sensitive PCR technology for pharmacogenetics, cross contamination becomes a significant concern. Researchers employed techniques which basically include replacing laboratory equipment after each sample preparation; however, there are no recommended guidelines. In the present work we wanted to evaluate the risk of cross contamination during tissue processing using the routine precaution measures. Twenty-one surgical samples of lung adenocarcinoma were used, of which 7 contained EGFR exon 19 mutation, 7 contained EGFR exon 21 mutation (p.L858R) and 7 were EGFR wild-type. The samples were ordered by alternating the mutation group to maximize the potential for cross contamination and underwent tissue sectioning and de-paraffinization. The entire process was performed using the same tools. Following DNA extraction all samples underwent PCR amplification and were scrutinized for small fractions of EGFR mutation using deep sequencing with the Ion torrent PGM technology. Twenty samples yielded results. The fraction of mutated copies was 41 ± 23% (range 11-66) for the cases with known exon 19 mutation and 48±24% (range 0-65) for the cases with known exon 21 mutations. No in-frame exon 19 deletion mutations were identified in the wild-type (WT) and exon 21 groups. The fraction of EGFR exon 21 (codon 858) mutations was 0.018±0.014% (range 0-0.05%) in the WT and exon 19 groups, which was not statistically different than the background sequencing artifact noise for the same base-pair alteration (p = 0.21). Our results suggest that standard precautions are sufficient for molecular pathology diagnosis of surgical samples and are not associated with increased risk of cross contamination.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma (NHL), and constitutes 30-40% of all cases in adults. DLBCL is heterogeneous in terms of its clinical course, and related molecular and genetic features. In the current era of appropriate chemo-immunotherapy, approximately 50%-60% of patients are cured after treatment. DLBCL is characterized by numerous chromosomal changes which are encountered in a high proportion of cases., Objective: To examine whether the identification of chromosomal changes at time of diagnosis has prognostic significance in DLBCL., Methods and Study Population: The study cohort included those patients with DLBCL, diagnosed and treated during 1996-2012 at the Hematology unit in Bnai-Zion Medical Center, Haifa, who had a cytogenetic study performed based on G-banding analysis from the original biopsy at the time of diagnosis., Results: One hundred and twenty one patients with DLBCL were included and of these 59 also had chromosomal analysis performed from the tumor tissue at diagnosis. The average age of the cohort was 60.9 (21-87) years, and 59.3% were men. Average follow-up was 50.6 months (1-240 months). In 16 of the 59 biopsies (27.1%) lymphoma cells did not grow in vitro and analysis was not performed in these cases. Of the remaining 43 cases with chromosomal analysis: 36 (83.7%) had chromosomal aberrations, which most frequently involved chromosomes 14, 18, 1 and X. There was no difference in outcome between patients with chromosomal changes, including the presence of complex karyotype or hyperdiploidy (defined as > 50 chromosomes), and those with normal karyotype., Conclusions: Although complex changes in basic chromosomal structure and altered numbers of chromosomes were identified in patients with DLBCL, none of these features were of prognostic significance in terms of overall survival. In the light of these findings, we confirm that in common practice for DLBCL outside of clinical trials there appears to be no advantage to performing routine chromosomal studies on biopsy specimens obtained from patients with newly diagnosed DLBCL.