Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone.

The widespread distribution of smartphones, with their integrated sensors and communication capabilities, makes them an ideal platform for point-of-care (POC) diagnosis, especially in resource-limited settings. Molecular diagnostics, however, have been difficult to implement in smartphones. We herein report a diffraction-based approach that enables molecular and cellular diagnostics. The D3 (digital diffraction diagnosis) system uses microbeads to generate unique diffraction patterns which can be acquired by smartphones and processed by a remote server. We applied the D3 platform to screen for precancerous or cancerous cells in cervical specimens and to detect human papillomavirus (HPV) DNA. The D3 assay generated readouts within 45 min and showed excellent agreement with gold-standard pathology or HPV testing, respectively. This approach could have favorable global health applications where medical access is limited or when pathology bottlenecks challenge prompt diagnostic readouts.

HER2 over-expressing high grade endometrial cancer expresses high levels of p95HER2 variant.

BACKGROUND: Subsets of high grade endometrial cancer (EnCa) over-express HER2 (ERBB2), yet clinical trials have failed to demonstrate any anti-tumor activity utilizing trastuzumab, an approved platform for HER2 positive breast cancer (BrCa). A truncated p95HER2 variant lacking the trastuzumab binding site may confer resistance. The objective of this investigation was to characterize the expression of the p95HER2 truncated variant in EnCa. MATERIALS AND METHODS: With institutional approval, 86 high grade EnCa tumors were identified with tumor specimens from surgeries performed between 2000 and 2011. Clinical data were collected and all specimens underwent tumor genotyping, HER2 immunohistochemistry (IHC, HercepTest®), HER2 fluorescent in situ hybridization (FISH), along with total HER2 (H2T) and p95HER2 assessment with VeraTag® testing. Regression models were used to compare a cohort of 86 breast tumors selected for equivalent HER2 protein expression. RESULTS: We identified 44 high grade endometrioid and 42 uterine serous carcinomas (USC). IHC identified high HER2 expression (2+ or 3+) in 59% of the tumors. HER2 gene amplification was observed in 16 tumors (12 USC, 4 endometrioid). Both HER2 gene amplification and protein expression correlated with H2T values. High p95HER2 expression above 2.8RF/mm2 was observed in 53% (n=54) with significant correlation with H2T levels. When matched to a cohort of 107 breast tumors based on HercepTest HER2 expression, high grade EnCa presented with higher p95 levels (p<0.001). CONCLUSIONS: These data demonstrate that compared to BrCa, high grade EnCa expresses higher levels of p95HER2 possibly providing rationale for the trastuzumab resistance observed in EnCa.

Inhibition of gamma-secretase activity impedes uterine serous carcinoma growth in a human xenograft model.

OBJECTIVE: Uterine serous carcinoma (USC) represents an aggressive subtype of endometrial cancer. We sought to understand Notch pathway activity in USC and determine if pathway inhibition has anti-tumor activity. METHODS: Patient USC tissue blocks were obtained and used to correlate clinical outcomes with Notch1 expression. Three established USC cell lines were treated with gamma-secretase inhibitor (GSI) in vitro. Mice harboring cell line derived or patient derived USC xenografts (PDXs) were treated with vehicle, GSI, paclitaxel and carboplatin (P/C), or combination GSI and P/C. Levels of cleaved Notch1 protein and Hes1 mRNA were determined in GSI treated samples. Statistical analysis was performed using the Wilcoxon rank sum and Kaplan-Meier methods. RESULTS: High nuclear Notch1 protein expression was observed in 58% of USC samples with no correlation with overall survival. GSI induced dose-dependent reductions in cell number and decreased levels of cleaved Notch1 protein and Hes1 mRNA in vitro. Treatment of mice with GSI led to decreased Hes1 mRNA expression in USC xenografts. In addition, GSI impeded tumor growth of cell line xenografts as well as UT1 USC PDXs. When GSI and P/C were combined, synergistic anti-tumor activity was observed in UT1 xenografts. CONCLUSIONS: Notch1 is expressed in a large subset of USC. GSI-mediated Notch pathway inhibition led to both reduced cell numbers in vitro and decreased tumor growth of USC some xenograft models. When combined with conventional chemotherapy, GSI augmented anti-tumor activity in one USC PDX line suggesting that targeting of the Notch signaling pathway is a potential therapeutic strategy for future investigation.

Transitional cell carcinoma of the ovary: a case-control study.

OBJECTIVES: The aim of this study is to compare response to chemotherapy and survival between patients with transitional call carcinoma of the ovary (TCCO) and papillary serous ovarian cancer (PSOC). METHODS: We identified women with both pure and mixed TCCO who were treated between 2000 and 2010. Each case was matched to two women with PSOC by age, grade, stage, and year of diagnosis. Correlation between categorical variables was assessed with chi square test. The Kaplan-Meier survival analysis was used to generate overall survival data (OS). Factors predictive of outcome were compared using the log-rank test and Cox proportional hazards model. RESULTS: Eighty-one women diagnosed with TCCO were selected as cases and compared to 162 controls. Women with TCCO had a lower rate of platinum resistance compared to controls (9% vs. 25%; p=0.01). When multivariate logistic regression was used to control for other factors independently associated with platinum resistance, patients with TCCO had a significantly lower risk of platinum resistance compared to PSOC. Median progression-free survival was not significantly different (27 months vs. 22 months; p=0.15) for women with TCCO and PSOC, respectively. Median OS, however, was significantly different at 83 months vs. 52 months for the TCCO and PSOC groups, respectively (p=0.01). A Cox proportional hazards model identified optimal cytoreduction, transitional cell histology, age, stage, and platinum and paclitaxel chemotherapy as independent predictors of OS. CONCLUSIONS: Patients with TCCO are less likely to demonstrate resistance to platinum chemotherapy and have improved overall survival when compared to patients with PSOC.

The evolution of the Papanicolaou smear.

The Papanicolaou smear or cervicovaginal cytology has been the mainstay of screening for cervical cancer in women of over 60 years. In this article, the origins of the Pap smear as a screening method and the many changes associated with the use of the Pap smear are detailed.

Tissue-specific signatures of activating PIK3CA and RAS mutations in carcinosarcomas of gynecologic origin.

OBJECTIVES: Gynecologic carcinosarcoma is an aggressive malignancy that requires more effective treatment approaches. However, therapeutic implications regarding the specific gynecologic site of origin and the admixture of carcinomatous and sarcomatous elements that define this tumor remain uncertain. Therefore, broad genotyping was performed to identify tissue-specific somatic mutational profiles that may help direct targeted therapies in this complex neoplasia. METHODS: Genotyping was conducted on primary gynecologic carcinosarcomas arising from various disease sites (uterus, ovary, fallopian tube, vagina) and within isolated histological subcomponents. Nucleic acids extracted from diagnostic tissue were used in a genotyping platform that simultaneously queried >120 common mutations across 14 cancer genes. Mutational status was correlated with clinical variables using logistic regression and Kaplan-Meier survival estimates. RESULTS: Cancer gene mutations were identified in 46% of the 52 patient cohort and include TP53 (23%), PIK3CA (19%), KRAS (15%), CTNNB1 (4%) and NRAS (2%). Mutation in a single gene was observed in 31% of patient samples, while synchronous mutations involving 2 and 3 genes were noted in 13% and 2% of samples, respectively. Comparative evaluation of the carcinomatous and sarcomatous elements within a tumor demonstrated a similar mutation signature. Mutations in PIK3CA, KRAS and NRAS were exclusive to tumors of uterine origin and age-adjusted Cox proportional hazards modeling associated advanced age, stage and TP53 mutations with decreased survival in the uterine subset. CONCLUSION: While carcinosarcomas across gynecologic disease sites are histologically similar, therapeutically relevant mutations in the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways predominated in carcinosarcomas arising in the uterus.

Improving Malignancy Detection Rates in Body Fluids Submitted to the Hematology Laboratory for Nucleated Cell Count and Differential: A Quality Improvement Study.

CONTEXT.­: Body fluid specimens are regularly submitted to the hematology laboratory for cell count and differential. Unless there is high clinical suspicion for malignancy, most cases lack concurrent cytology review and may not benefit from more focused examination for malignancy. OBJECTIVE.­: To compare rates of malignancy detection before and after fluid-focused training for hematology technologists as part of a quality improvement initiative. DESIGN.­: During an 8-week pretraining period, body fluids submitted to the cytology laboratory were correlated with concurrent hematology specimens. After slide review and training sessions for the hematology technologists, the same data were collected for a 4-week period. Discrepant cases were reviewed by hematology laboratory supervisors and pathologists. RESULTS.­: We collected 465 pretraining and 249 posttraining body fluids with concurrent cytology and hematology evaluation. In the pretraining cohort, 48 cases (10.3%) were diagnosed as malignant by cytology; of those, 33 were detected by hematology. In the posttraining cohort, 30 cases (12.0%) were diagnosed as malignant by cytology of which 27 were detected by hematology. Of the 18 discrepant cases (all carcinomas), hematology slide review showed definite features of malignancy in 15 and no tumor cells in 3. The malignancy detection rate by the hematology laboratory significantly improved after training (68.8% versus 90.0%, P = .01). CONCLUSIONS.­: We demonstrate the comparatively lower malignancy detection rate for body fluid specimens processed in our hematology laboratory, particularly for carcinomas. Hematology technologist education/training improved the malignancy detection rate, an important quality improvement given the large proportion of body fluids undergoing hematology evaluation without concurrent cytology reviews.

The cytologic criteria of malignancy.

Cytology and cell biology are two separate fields that share a focus on cancer. Cancer is still diagnosed based on morphology, and surprisingly little is known about the molecular basis of the defining structural features. Cytology uses the smallest possible biopsy for diagnosis by reducing morphologic "criteria of malignancy" to the smallest scale. To begin to develop common ground, members of the American Society of Cytopathology Cell Biology Liaison Working Group classify some of the "criteria of malignancy" and review their relation to current cell biology concepts. The criteria of malignancy are extremely varied, apparently reflecting many different pathophysiologies in specific microenvironments. Criteria in Group 1 comprise tissue-level alterations that appear to relate to resistance to anoikis, alterations in cell adhesion molecules, and loss of apical-basal polarity. Criteria in Group 2 reflect genetic instability, including chromosomal and possibly epigenetic instability. Criteria in Groups 3 are subcellular structural changes involving cytoplasmic components, nuclear lamina, chromatin and nucleoli that cannot be accounted for by genetic instability. Some distinct criteria in Group 3 are known to be induced by cancer genes, but their precise structural basis remains obscure. The criteria of malignancy are not closely related to the histogenetic classification of cancers, and they appear to provide an alternative, biologically relevant framework for establishing common ground between cytologists and cell biologists. To understand the criteria of malignancy at a molecular level would improve diagnosis, and likely point to novel cell physiologies that are not encompassed by current cell biology concepts.

The utility of electron microscopy in cytopathology.

BACKGROUND: The use of ultrastructural analysis in the diagnostic work-up of histologic specimens has been well studied but less is known about the utility of electron microscopy (EM) in cytopathology. DESIGN: 149,006 non-gynecologic cytology cases at the Massachusetts General Hospital between the years 1993 and 2006 were searched to identify those in which material had been submitted for EM. Cytologic and EM diagnoses were correlated with available histologic diagnoses. The results were put into one of three categories: confirmatory, diagnostic, or insufficient material for diagnosis (IMFD). RESULTS: Material for EM was obtained from 178 cytology cases that included 131 fine-needle aspirates (FNA) and 47 exfoliative specimens. EM provided additional diagnostic information beyond that offered by cytologic examination alone in 32% of cases, and in 48% of cases EM confirmed the cytologic findings. Insufficient material and discrepant results were noted for EM evaluation in 19% of cases and in 1% cases respectively. EM was most useful when applied to FNAs for subclassifying tumors as epithelial or mesenchymal (45.6%), for the diagnosis of non-neoplastic processes (15.7%) such as alveolar proteinosis and amyloidosis, and for the identification of microorganisms (12.2%). In our study, although EM was infrequently applied to exfoliative specimens to distinguish mesothelioma from adenocarcinoma, it proved to be very useful in this setting. CONCLUSION: When adequate material is obtained, EM can contribute significantly to the evaluation of both FNA and exfoliative cytology cases, including the diagnosis and subclassification of epithelial and mesenchymal tumors, non-neoplastic processes, and the identification of microorganisms.

Abdominal radical trachelectomy: Success and pitfalls in a general gynecologic oncology practice.

OBJECTIVE: To report our successes and complications with a series of abdominal radical trachelectomies performed to preserve fertility in young women at the Massachusetts General Hospital (MGH). METHODS: Institutional review board (IRB) permission was obtained for retrospective record review. Data were collected regarding patient age and parity, tumor stage and histology, surgical time and complications, post-operative complications, follow-up, and pregnancy. RESULTS: Ten patients underwent radical abdominal trachelectomy, 9 by the same surgeon (LD). Surgery was essentially identical to that of radical hysterectomy with the exception of re-anastomosis of the uterine fundus to the vagina and placement of cerclage. Pre-operative evaluation and post operative follow-up was for the most part identical for all patients. Two patients achieved pregnancy, with 1 twin delivery and 1 patient had 2 pregnancies. Two patients experienced cervical stenosis with regular menses and the same 2 patients passed their abdominal cerclage vaginally. To date there have been no cancer recurrences. Pap smear follow-up has been complicated by difficulty in reading smears from the lower uterine segment (LUS). CONCLUSION: Radical abdominal trachelectomy can be successfully performed by any gynecologic oncologist who is trained in radical pelvic surgery. Pre-operative counseling is crucial in obtaining informed consent. Patients must be aware of potential post-operative complications, including pre-term delivery. Cytology department needs to be aware of potential pitfalls in reading Pap smears from the LUS.

The Difference Between Unfixed and Postfixation Placental Weight.

OBJECTIVES: Reference values for placental weights correlated with gestational age are used in surgical pathology. Most reference values were established for fresh placentas. Some laboratories routinely fix all placentas, bringing into question the accuracy of the reference weight values. We wanted to determine the impact of fixation on placental weight. METHODS: One hundred placentas from uncomplicated pregnancies were weighed in the fresh state, after removal of the cord and membranes. After fixation in formalin for 1 day and 5 days, the placentas were reweighed. The change in weight for each placenta was analyzed by a two-tailed paired t test. RESULTS: Statistically, a small but significant gain in weight occurred after 24 hours (3.7%, P << .001), and there was no significant change identified in the additional 4 days (P = .51). Nine placentas lost weight with fixation; the weight of four was unchanged. CONCLUSIONS: We consider formalin fixation to add a statistically significant but clinically negligible amount of weight to the placenta.

Cytologic features of endocervical glandular lesions: comparison of SurePath, ThinPrep, and conventional smear specimen preparations.

The cytologic features of endocervical neoplasia have been well-described for conventional and ThinPrep, but not for SurePath, methods. This study is designed to ascertain if cytological features are similar in SurePath specimens. Conventional, ThinPrep and SurePath specimens with either endocervical adenocarcinoma in situ or invasive endocervical adenocarcinoma were evaluated for architectural and cytological features previously described for endocervical neoplasia. A generalized linear model was used to assess the differences of ordinal multinomial data. Of 18 evaluated, the only features showing statistical differences were architectural: large groups of cells and single cells were more frequent in SurePath preparations and conventional smears. Feathering was more frequently noted in conventional smears. Individual cytological features were identical across all groups. Mitoses and apoptotic debris were seen with equal frequency in all preparations. The architectural and cytologic features of endocervical glandular neoplasia in liquid-based specimens show only subtle architectural differences when compared with conventional smears. Keeping these differences in mind, virtually the same criteria can be used to identify endocervical glandular lesions in all three specimen types.

Modified Plasma-Thrombin Method of Cell Block Preparation for Fine-Needle Aspiration Biopsies in Resource-Limited Settings.

OBJECTIVES: The plasma-thrombin method is commonly used to make cell blocks from fine-needle aspiration (FNA) samples but requires centrifugation. We describe a modification to this method that does not require centrifugation for use in resource-limited settings. METHODS: Pooled fresh plasma is aliquoted into 2-mL Eppendorf tubes and the FNA sample directly rinsed into the plasma. Two drops of reconstituted thrombin are added and gently mixed. A cell clot is transferred to a tissue bag, fixed in formalin, and processed. This method was applied to FNA samples from 44 patients presenting to the Mbarara University of Science and Technology FNA clinic. RESULTS: The cell blocks were less cellular than the smears but contained adequate material to confirm morphologic impression or perform immunocytochemistry in 36 of 44 cases (82% adequacy rate). CONCLUSIONS: The modified plasma-thrombin method is a reliable cell block preparation method that can be easily applied in resource-limited settings.

Integrated genomic analysis of clear cell ovarian cancers identified PRKCI as a potential therapeutic target.

Clear cell ovarian cancer (CCOC) is an epithelial ovarian cancer histotype with unique pathologic, biologic and clinical features. Despite its worse prognosis than serous ovarian cancer (SOC), the genomic landscape of CCOC is less well defined. Integrated genomic analysis of CCOC allows the identification of potential therapeutic targets to improve the treatment of this tumor. Using comparative genomic hybridization and gene expression profiling, we have screened 12 CCOC cell lines and 40 tumors to identify 45 amplified and overexpressed genes. Pathways analysis of these genes identified 19 genes with cancer-related functions. Of these, PRKCI is one of the most frequently amplified and overexpressed genes and its expression induced cancer cell proliferation and migration/invasion in vitro as well as tumor growth in vivo. Targeting PRKCI by small molecule inhibitor, sodium aurothiomalate (ATM), significantly reduced the in vivo tumor growth and may be a new therapeutic strategy to improve the treatment of CCOC.

Smears are important for adequate cytologic diagnosis of kidney lesions.

INTRODUCTION: Traditionally at our institution, smears with or without liquid-based cytology (LBC) and core biopsies (CBs) have been obtained by radiologists performing image-guided fine-needle aspiration biopsies (FNABs) of deep organs. Since 2015, however, there has been a shift to providing cytology with samples for LBC only when obtaining CBs. The impression among our institution's cytologists is that LBC alone is less often adequate for diagnosis compared with smears and LBC together. We examined a series of kidney FNABs pre- and post-"LBC only" to evaluate this impression. MATERIALS AND METHODS: With institutional review board approval, we compared all kidney FNABs from 2012 to those from 2015. We recorded the type(s) of cytology preparation(s), the number of cytology slides, the cytology diagnosis, the concurrent CB diagnosis, and whether there was a subsequent excision and the excision diagnosis. We examined cytology and CB slides as needed. RESULTS: In 2012, 105 patients underwent 111 kidney biopsies, 109 with smears made. In 2015, 58 patients underwent 62 kidney biopsies, 7 with smears made. In 2012, there were 13 (12%) nondiagnostic (ND) cytology cases and 19 (17%) cases where the cytology and CB diagnoses were discrepant. By comparison, in 2015, there were 20 (32%) ND cytology cases and 21 (33%) discrepant cases. CONCLUSIONS: There were more cytology slides per case and fewer ND diagnoses in 2012 compared with 2015 (12% versus 32%, respectively, P = 0.001). Concordance was also better in 2012 (83% versus 67%, P = 0.015). We believe that our metrics would improve if we returned to the procedures followed in 2012.

Aorta-Lesion-Attenuation-Difference (ALAD) on contrast-enhanced CT: a potential imaging biomarker for differentiating malignant from benign oncocytic neoplasms.

OBJECTIVE: To evaluate whether the Aorta-Lesion-Attenuation-Difference on contrast-enhanced CT can aid in the differentiation of malignant and benign oncocytic renal neoplasms. MATERIALS AND METHODS: Two independent cohorts-an initial (biopsy) dataset and a validation (surgical) dataset-with oncocytomas and chromophobe renal cell carcinomas (chRCC) were included in this IRB-approved retrospective study. A region of interest was placed on the renal mass and abdominal aorta on the same CT image slice to calculate an Aorta-Lesion-Attenuation-Difference (ALAD). ROC curves were plotted for different enhancement phases, and diagnostic performance of ALAD for differentiating chRCC from oncocytomas was calculated. RESULTS: Seventy-nine renal masses (56 oncocytomas, 23 chRCC) were analyzed in the initial (biopsy) dataset. Thirty-six renal masses (16 oncocytomas, 20 chRCC) were reviewed in the validation (surgical) cohort. ALAD showed a statistically significant difference between oncocytomas and chromophobes during the nephrographic phase (p < 0.001), early excretory phase (p < 0.001), and excretory phase (p = 0.029). The area under the ROC curve for the nephrographic phase was 1.00 (95% CI: 1.00-1.00) for the biopsy dataset and showed the narrowest confidence interval. At a threshold value of 25.5 HU, sensitivity was 100 (82.2%-100%) and specificity was 81.5 (61.9%-93.7%). When tested on the validation dataset on measurements made by an independent reader, the AUROC was 0.93 (95% CI: 0.84-1.00) with a sensitivity of 100 (80.0%-100%) and a specificity of 87.5 (60.4%-97.8%). CONCLUSIONS: Nephrographic phase ALAD has potential to differentiate benign and malignant oncocytic renal neoplasms on contrast-enhanced CT if histologic evaluation on biopsy is indeterminate.

Touch Preps of Products of Conception for Rule-Out Ectopic Pregnancy: A Viable Alternative to Frozen Section.

OBJECTIVES: Tissue confirmation of the intrauterine location of a pregnancy can be difficult grossly, necessitating a frozen section diagnosis. First-trimester tissue is often scant and can be exhausted by frozen section. We examined 39 endometrial curettings performed for rule-out ectopic pregnancy by touch prep to examine the utility of cytopathology in documenting trophoblast or chorionic villi (products of conception [POC]). METHODS: First-trimester curettage specimens sent to the Massachusetts General Hospital Pathology Departmentreceived a gross examination followed by a touch prep of the area most suspicious for villi. Touch preps were reviewed blinded to the final diagnosis and then compared. RESULTS: Thirty-three of the 39 touch preps and histology specimens were concordant, including all 11 negative histology specimens and 22 of the 28 positive histology specimens. CONCLUSIONS: POC can be diagnosed by touch prep and may offer confirmation of grossly identified villi. Positive predictive value is 100%. Negative touch preps should receive further evaluation.