Search

Many institutions have cytopathology case archives for education. Unfortunately, these slides deteriorate over time and have limited accessibility. Whole slide imaging (WSI) can overcome these limitations. However, suboptimal image quality and scanning effort are barriers.We selected 123 slides from cytopathology study sets for WSI scanning at 400x magnification without z-stacking. The Ventana DP 200 scanner and Virtuoso software were used. Slides were scanned in 2 rounds: the first round of slides was prepared for scanning with light cleaning, and the second round was performed only on slides that had unacceptable WSI quality after thorough cleaning. Slides were assessed with a 4-tier grading system created by the authors. Time to scan each slide was recorded.Within the first round, 96 of 123 (78%) slides scanned were determined to be of acceptable quality. After the second round of scanning, in total, 118 of 123 (95.9%) slides were determined to be of acceptable quality. The average time needed to scan each slide was 213 seconds.The majority of slides scanned were of acceptable quality in the first round of scanning. After cleaning and rescanning, nearly every slide investigated was of acceptable quality. The primary objective is to provide other institutions that may be considering a similar project a benchmark so that they know what to expect in terms of slide scan success rate and the amount of time needed to digitize slides for educational archiving. This pilot study demonstrates the feasibility of using WSI for cytology education cases.

Many laboratories rescreen Papanicolaou test slides initially interpreted as negative, but positive for human papillomavirus (HPV) high-risk types, as a quality control measure. We have evaluated the utility of this practice in the era of HPV genotyping as a laboratory improvement project.Between August 2016 and October 2019, we identified 3618 rescreened Papanicolaou tests with follow-up biopsies. The biopsy results were put into 3 groups: 1) Negative; 2) LSIL: HPV changes or low-grade squamous intraepithelial lesion; and 3) HSIL: high-grade squamous intraepithelial lesion or carcinoma. HPV molecular testing results with subtyping for types 16 and 18 were available for 3117 of these cases.A total of 530 of 2812 Papanicolaou tests (18.8%) with positive HPV results were reinterpreted as cytologically abnormal after rescreening; 75 (14.2%) had a biopsy result of HSIL. The subset positive for HPV types 16/18 had 38 of 133 cytology positive cases diagnosed as HSIL on biopsy vs. 107 of 935 cytology negative cases diagnosed as HSIL on biopsy (28.6% vs. 11.4%, P0.0001). The subset positive for "other" (non-16/18) high-risk HPV types had 37 of 397 cytology positive follow-up HSIL vs. 84 of 1288 cytology negative follow-up HSIL (9.3% vs. 6.5%, P = 0.075).Rescreening has the highest yield in specimens positive for types 16/18. However, for this group colposcopy is recommended regardless of cytology findings, reducing the patient benefit. Routine rescreening of cytology negative/HPV positive Papanicolaou tests has reduced utility when HPV subtyping is performed and should be reconsidered.

Introduction The introduction of a new generation of core needle biopsies (CNBs) for endoscopic procedures has prompted reconsideration of the role of cytopathologists in the handling of small biopsies. The American Society of Cytopathology (ASC) has therefore conducted a survey with the intention of elucidating current practices regarding the handling of small CNBs. Materials and methods The membership of the ASC was invited by email to participate in an online survey over a 2-month period. The survey consisted of 20 multiple choice questions with 2-8 possible responses per question. Results Of 2651 members contacted by e-mail, 282 (10.6%) responded to the survey questions, including 196 pathologists (69.5%) and 86 cytotechnologists (30.5%). Of these, 265 respondents were from the US/Canada (94.0%), with 156 from academic institutions (58.9%) and 109 from non-academic practices (41.1%); 17 were from other countries (6.0%). In 18.8% of all practices, cytopathologists sign out >90% of small CNBs from endoscopic and radiologically guided procedures; in 36.5% of practices >90% are signed out by surgical pathologists; the remainder have such cases divided more evenly between cytopathologists and surgical pathologists. Responses show that 78.0% of all respondents are interested in signing out more small biopsies in the future, and 80.5% desire increased small biopsy-related resources from the ASC. Conclusions The survey responses indicate that practices currently vary widely across institutions. Most indicated an interest in greater incorporation of small biopsies into the practice of cytopathology.

Three main patterns of T cell receptor (TCR) expression are identified in peripheral T cell lymphoma (PTCL): TCR-αβ type, TCR-γδ type, and TCR silent type. Cases of PTCL that express both αβ and γδ receptors are rare. Here, we report a case of PTCL-NOS (not otherwise specified) with dual αβ and γδ expression in a 61-year-old female 6 years after orthotopic cardiac transplant. The patient presented with fatigue, fever, worsening anemia, and thrombocytopenia. A chest CT showed multiple irregular lung nodules and diffuse lymphadenopathy. Cytology evaluation of the lymph nodes revealed large atypical lymphocytes with immunopositivity for CD3, CD8, CD56, TIA1, and granzyme B and immunonegativity for CD5 and CD30. Epstein-Barr virus (EBV) was not detected by EBV-encoded RNA (EBER) in situ hybridization study. The corresponding flow cytometry demonstrated abnormal T cells with TCR-αβ and TCR-γδ expression and loss of CD5 expression. A TCR gene rearrangement study demonstrated the presence of a clonal lymphoid population with TCRβ and TCRγ rearrangements. The patient was deceased within 2 months despite aggressive treatment. The findings were consistent with an EBV-negative, monomorphic T cell post-transplant lymphoproliferative disorder (PTLD) with features of PTCL-NOS. A literature review of T cell lymphoma with co-expression of TCR-αβ and TCR-γδ was performed. A case of aggressive PTCL with dual expression of TCR-αβ and TCR-γδ in a post-transplant setting has not been previously reported.

Extramedullary multiple myeloma (EMM) involving the liver as a focal space-occupying lesion is very rare, especially in the patients with cirrhosis. Here, we report a case of EMM in the liver and periportal lymph node, diagnosed by endoscopic ultrasound guided-fine-needle aspiration (EUS-FNA). A 57-year-old male patient, with history of cirrhosis, presented with abdominal pain and pancytopenia. The abdominal magnetic resonance imaging (MRI) demonstrated a 6.5 cm left hepatic mass with a 1.1 cm malignant-appearing periportal lymph node and diffuse osseous lesions. The cytology specimens from the hepatic mass and the periportal lymph node were obtained through EUS-FNA without rapid on-site evaluation (ROSE). The thin-layer preparations (ThinPrep) showed abundant plasmacytoid cells, which were confirmed to be Kappa-restricted neoplastic plasma cells by the cell block preparations. Later, his serum level of Kappa light chain was found significantly elevated by flow cytometry, which was identified as monoclonal IgA Kappa light chain by serum protein electrophoresis (SPEP) with immunofixation. The patient was diagnosed as IgA multiple myeloma with extramedullary involvement of the liver and periportal lymph node. This is the first case showing the ThinPrep cytomorphologic features of EMM in the liver and periportal lymph node. This case highlights the importance of distinguishing plasma cells from being hepatocytes and lymphocytes on the ThinPrep and also emphasizes the utility of the cell block in the diagnosis of plasma cell neoplasm.

Automated Papanicolaou test screening systems have now been available for over 25 years. Currently two automated screening systems are in widespread clinical use. These are the ThinPrep Imaging System and the FocalPoint GS Imaging System. In their current configurations, both facilitate faster screening by showing a limited number of fields of view (FOV) to cytotechnologists. The FOV are based on the use of proprietary algorithms applied to computerized images of the slide that determine the cells and cell groups with the highest likelihood of abnormality. If all of the FOV are deemed to be negative, the case can be signed out with no additional review; if one or more fields appear possibly abnormal, the entire slide must be manually screened. The United States Food and Drug Administration has ruled that for workload calculation purposes, looking at only the FOV review counts as one-half slide, potentially greatly increasing the number of slides that can be screened. However, follow-up studies of this technology have shown that screening accuracy declines when very large numbers of cases are reviewed per day. Recommendations designed to limit screening volumes to levels that do not jeopardize patient care have therefore been created. The development of fully automated screening that does not rely on human judgment remains an unrealized aspiration. This review covers the history of the development and clinical implementation of automated screening technology with descriptions of the various automated screening systems and their performance as reported in published literature.

Background Breast cancer mortality is most common in cancer in women, and there are no ex vivo models that can capture the primary growth of tumor with fidelity to the in vivo tumor growth. In this study, we grew human breast cancer cell lines in an acellular lung matrix of the ex vivo four-dimensional lung model to determine if they form primary tumor and the extent to which they mimic the histology and characteristics of the human tumors. Materials and methods Rat lungs were harvested, decellularized, and placed in a bioreactor. To study the primary tumor growth, we seeded the lung via the trachea with human breast cancer cells SUM159, MCF7, or MDMB231 and perfused the pulmonary artery with oxygenated media. Lobectomies were performed and processed for hematoxylin and eosin, Ki-67, caspase-3, estrogen receptor, and progesterone receptor antibodies. Results All three cell lines grew in the ex vivo four-dimensional model and formed perfusable tumor nodules with similar histology and morphology as the primary tumors. SUM159 and MDAMB231 showed higher proliferation and apoptotic indices than MCF7. In addition, MCF7 retained its estrogen receptor and progesterone receptor positivity, whereas SUM159 and MDAMB 231 did not have any staining. Conclusions Overall, our study showed that human breast cancer cells can be grown on the ex vivo four-dimensional lung model, which then form primary tumor nodules that mimic the morphology and histology of the original tumor.

Introduction Plasmacytoid and micropapillary variants of high-grade urothelial carcinoma (HGUC) exhibit unique histologic morphology and very aggressive clinical behavior. However, the morphology of these 2 variants in urinary cytology is not well studied and evaluated using The Paris System for reporting urinary cytology. Materials and methods A database search was performed in all patients with the diagnosis of plasmacytoid or micropapillary HGUC. A total of 5 patients with positive urinary cytology cases were identified. The cytomorphology of every urinary cytology case was correlated with the histologic features in the surgical specimens from the same patient. Results One urine and 4 bladder washings were evaluated. Cytologically, plasmacytoid HGUCs are characterized by single, large tumor cells with hyperchromasia, irregular nuclear membranes, and vacuolated cytoplasm. The nuclear-to-cytoplasmic (N:C) ratio was less than 0.5 in many of the malignant cells due to the abundant cytoplasm. The cytology features of micropapillary HGUC include the presence of micropapillae of tumor cells with no fibrovascular core. Individual high-grade urothelial cells were also identified in all 4 cases, but 1 (25%) of these had only rare cells meeting The Paris System criteria for HGUC due to abundant cytoplasm and lack of hyperchromasia in most malignant cells. Conclusions Plasmacytoid and micropapillary variants of HGUC have unique cytomorphologic features in urinary cytology specimens, which are reflective of the corresponding histological findings. These 2 clinically aggressive variants of HGUC may not be as readily interpreted as malignant using The Paris System for reporting urinary cytology, creating potential diagnostic pitfalls.

The advent of US Food and Drug Administration (FDA)-approved molecular testing for human papillomavirus (HPV) has resulted in a dramatic shift from cytological testing alone to a combination of cytology and molecular testing for primary HPV screening. HPV testing has quickly become an essential component of daily practice in most laboratories and clinical practices. Although the principle of HPV testing is now familiar, it is important to understand the mechanisms behind these platforms in order to properly interpret the results and understand the limits of each method. HPV tests are more automated and reproducible than cytology, but are by no means perfect. None of these platforms will identify every HSIL/CIN2+ or cancer. This fact must be kept in mind when correlating the results of HPV testing with cytology or biopsy findings. The goal of this paper is to review the FDA- approved molecular testing platforms for HPV, including methodology, limitations, and specifications. The concordance between the platforms will also be discussed. Package inserts of the 5 FDA- approved molecular testing platforms for HPV, as well as a literature review of the platforms, were reviewed and assimilated into the article. Due to the multiple modalities available for detection of hrHPV, the concordance between these assays becomes important. Prior publications have compared HC2, Cervista, cobas, and Aptima, with most studies comparing to HC2 because it is considered the reference standard. With the newly approved BD platform, concordance studies were reviewed as well.

Introduction Pleural and peritoneal/ascites fluid samples with many lymphocytes are commonly received in the cytology laboratory. It is often difficult to distinguish reactive lymphocytes from hematopoietic malignancy based on morphology alone, however. Flow cytometry can be a useful adjunct in body fluids, although literature on this subject is limited. Materials and methods This study is a single-institution 5-year retrospective review of 377 fluid samples from 341 patients with corresponding flow cytometry analysis. The cytologic findings were correlated with the flow cytometry results and clinical data, as available. Results Of 4158 pleural fluids received over 5 years, 325 (7.8%) had corresponding flow cytometry analysis. Of these 325 samples, 57 (17.5%) were positive for hematopoietic malignancy by flow cytometry. Of the positive cases, only 24 (8.7%) represented a new diagnosis of hematopoietic malignancy (ie, did not have a known history). Of 3020 peritoneal/ascites fluids received over 5 years, 52 (2%) had corresponding flow cytometry. Of these, 8 were positive for hematopoietic malignancy, and only 2 represented a presumed new diagnosis. Conclusions Routine flow cytometry analysis for pleural and peritoneal/ascitic fluids is of limited utility, with only rare cases positive for hematopoietic malignancy without a known history. Of these cases, many had atypical cells that suggested a positive diagnosis. Conversely, in cases with a known history, about 75% were positive for hematopoietic malignancy. Our study suggests that the utility of flow cytometry for pleural and peritoneal/ascitic fluids is limited, and should be used sparingly in cases without atypical cytologic features, high clinical suspicion, or known history.

Introduction The roles of pathologists and cytotechnologists (CTs) continually evolve to optimize patient care, particularly with regard to rapid on-site evaluation (ROSE). Having ROSE performed helps ensure sufficient material is obtained for diagnosis and permits appropriate specimen triage for ancillary studies. At our institution, both on-site and telecytology evaluations are increasingly utilized, particularly in endobronchial ultrasound-guided procedures (EBUS). Consequently, time demands placed on the pathologist and CT staff has significantly increased, creating workload management challenges. Material and methods A consecutive number of ROSE procedures were documented for a 3-month time period at our institution. Case type and time spent for travel, adequacy assessment, processing, screening, and sign-out was recorded in order to assess time demands placed on staff by different procedures. Results Average travel/processing time by CTs was variable among ROSE procedures (72.9 minutes), as was adequacy assessment time by pathologists (16.9 minutes). EBUS posed the greatest time challenges with the longest CT travel/processing time as EBUS took almost 40% longer and adequacy assessment took the pathologist 3-4 times longer when compared with other procedures because of the targeting of multiple sites during EBUS with associated procedural delays. Using telecytology, average pathologist adequacy assessment time was reduced from 44.8 minutes to 24.6 minutes for EBUS. The provision of ROSE for EBUS is more challenging from a workload management perspective than for other procedures. Conclusions ROSE reimbursement is low, and no greater for EBUS than for other procedures. Use of telecytology can save time for pathologists and make the service more cost-effective if the number of procedures is sufficient to justify investment in the technology.

Objectives: The Lower Anogenital Squamous Terminology (LAST) group has recommended that cervical intraepithelial neoplasia (CIN) terminology be replaced by squamous intraepithelial lesion (SIL) terminology, with p16 immunohistochemistry used to separate lesions formerly diagnosed as CIN grade 2 into high-grade SILs (HSILs) and low-grade SILs. This study investigated the impact of these changes on the frequency of p16 testing and the diagnosis of high-grade lesions. Methods: Pathology reports for all cervical biopsy specimens in the 1 year before and after the introduction of LAST recommendations (July 2011 to June 2013) were examined. Results: Before and after the implementation of LAST, 365 (15.4%) of 2,376 cases were diagnosed as high grade (CIN 2/3) vs 486 (17.6%) of 2,761 cases diagnosed as HSILs ( P = .0343), and p16 was performed 79 (3.3%) times vs 383 (13.9%) times ( P

The traditional surgical pathology assessment requires tissue to be removed from the patient, then processed, sectioned, stained, and interpreted by a pathologist using a light microscope. Today, an array of alternate optical imaging technologies allow tissue to be viewed at high resolution, in real time, without the need for processing, fixation, freezing, or staining. Optical imaging can be done in living patients without tissue removal, termed in vivo microscopy, or also in freshly excised tissue, termed ex vivo microscopy. Both in vivo and ex vivo microscopy have tremendous potential for clinical impact in a wide variety of applications. However, in order for these technologies to enter mainstream clinical care, an expert will be required to assess and interpret the imaging data. The optical images generated from these imaging techniques are often similar to the light microscopic images that pathologists already have expertise in interpreting. Other clinical specialists do not have this same expertise in microscopy, therefore, pathologists are a logical choice to step into the developing role of microscopic imaging expert. Here, we review the emerging technologies of in vivo and ex vivo microscopy in terms of the technical aspects and potential clinical applications. We also discuss why pathologists are essential to the successful clinical adoption of such technologies and the educational resources available to help them step into this emerging role.

Background We determined the effect of human lung fibroblasts (LFs) in metastatic lesion formation in a four-dimensional (4D) lung cancer model. Methods Human cancer-associated fibroblasts (CAFs) were isolated from the primary tumor, and normal LFs were isolated from adjacent lung using fluorescence-activated cell sorting. The 4D metastatic lung cancer model was seeded with the human lung cancer cell lines (H460, A549, and H1299) alone or was seeded with CAFs or LFs. In addition, the 4D model seeded with human lung cancer cell lines was also treated with conditioned media of LFs or CAFs grown on the 4D model. We determined the number of metastatic tumor cells per high-power field on the model. Results There were significantly fewer metastatic lesions per high-power field in the 4D model seeded with the H460 cell line and LFs compared with H460 alone on day 15 ( p = 0.008) or compared with H460 and CAFs ( p = 0.002). This relationship was also seen with A549 and H1299 tumor cells. Moreover, the H460 cell line treated with conditioned media from the 4D model seeded with LFs had significantly fewer metastatic lesions than the 4D model seeded with CAFs. This was also seen with two other pairs of human fibroblasts obtained from patients. Conclusions The secreted factor from LFs grown on the 4D model inhibits the formation of metastatic lesions. The 4D model may be used to determine the role of different components of the tumor's microenvironment in metastatic lesion formation, and this secreted factor may provide a novel therapy for treatment of cancer metastasis.

Background We have developed a four-dimensional (4D) lung cancer model that forms perfusable tumor nodules. We determined if the model could be modified to mimic metastasis. Methods We modified the 4D lung cancer model by seeding H1299, A549, or H460 cells through the trachea only to the left lobes of the acellular lung matrix. The model was modified so that the tumor cells can reach the right lobes of the acellular lung matrix only through the pulmonary artery as circulating tumor cells (CTC). We determined the gene expressions of the primary tumor, CTCs, and metastatic lesions using the Human OneArray chip. Results All cell lines formed a primary tumor in the left lobe of the ex vivo 4D lung cancer model. The CTCs were identified in the media and increased over time. All cell lines formed metastatic lesions with H460 forming significantly more metastatic lesions than H1299 and A549 cells. The CTC gene signature predicted poor survival in lung cancer patients. Unique genes were significantly expressed in CTC compared with the primary tumor and metastatic lesion. Conclusions The 4D lung cancer model can isolate tumor cells in 3 phases of tumor progression. This 4D lung cancer model may mimic the biology of lung cancer metastasis and may be used to determine its mechanism and potential therapy in the future.

Background Currently, there is no in vitro or ex vivo model that can isolate circulating tumor cells (CTCs). Recently, we developed a four-dimensional (4D) lung cancer model that allows for the isolation of CTCs. We postulated that these cells have different properties than parental (2D) cells.

Introduction The Bethesda System (TBS) guidelines for reporting the presence of endometrial cells on Papanicolaou tests increased the reporting age from 40 (TBS 2001) to 45 (TBS 2014) years. Exfoliated endometrial cells (EMC) are usually a normal finding. Nevertheless, benign-appearing EMC occasionally correspond to endometrial hyperplasia or malignancy, especially in older, postmenopausal women. This study assesses the impact of this age cutoff change. Materials and Methods This retrospective review compares endometrial biopsies following TBS 2001 and TBS 2014. Papanicolaou tests with EMC reported in women older than age 40 or 45 years were correlated with follow-up endometrial biopsies performed between May 25, 2014, to May 26, 2015, and May 27, 2015, to May 26, 2016, respectively. Results The number of reported EMC declined from 770 to 492 (a 36.1% decrease). The follow-up endometrial biopsy rate for Papanicolaou tests reporting EMC using TBS 2001 was 13.6% (105 of 770) versus TBS 2014 at 13.8% (68 of 492; P = 0.92). For TBS 2001, 15% of women aged 45 and older had follow-up biopsies (65 of 434; P = 0.62). Most follow-up biopsies showed benign endometrium. In the TBS 2001 group, 1 biopsy showed malignancy and another showed complex hyperplasia with atypia. Both patients were older than 45 years. The TBS 2014 group contained 1 biopsy of malignancy and 1 with simple hyperplasia with focal atypia. Conclusions The implementation of TBS 2014 reduced the frequency of reporting benign-appearing endometrial cells. The follow-up biopsy rate has remained essentially the same, but the total number of biopsies performed decreased, with a similar low yield of significant abnormalities.

The Papanicolaou (Pap) test category of “low grade squamous intraepithelial lesion, cannot exclude high grade squamous intraepithelial lesion” (LSIL-H) is not recognized by The Bethesda System but is commonly used. It is essentially an amalgamation of the official LSIL and ASC-H categories. Since these two categories have similar follow-up algorithms, the clinical utility of the combined LSIL-H category is unclear. We have therefore studied follow-up patterns for these three entities in our laboratory to determine the real-world impact of each in our patient population. We searched our pathology database over an 18-month period to find Pap tests (predominantly ThinPrep) interpreted as LSIL-H (137), LSIL (2,189), and ASC-H (101). Like other studies, we found that the discovery rate of high grade dysplasia in biopsies after LSIL-H (31.9%) was similar to ASC-H (35.3%) and was higher than LSIL (7.6%; P

Multiple intestinal lipomas (lipomatous polyposis) are quite rare, and they can be quite challenging to diagnose because this condition may be clinically confused with familial adenomatous polyposis with a suggestive family history. Herein, we present a case of lipomatous polyposis that was presented with abdominal pain and, in colonoscopy, had more than 100 polyps. The patient was admitted for surgery with diagnosis of familial polyposis. Resected colon specimen had multiple polyps ranging from 0.1 to 1.5 cm. Microscopically, the polyps were composed of mature adipose tissue with normal overlying mucosa. There were also increased fat cells in the submucosa of the colon adjacent to the polyps. Lipomatous polyposis rarely occurs and can be confused with familial polyposis. Polypectomy is a simple and cost-effective procedure to help in diagnosis and prevent a major surgery.

Aimed at bridging imaging technology development with cancer diagnosis, this paper first presents the prevailing challenges of lung cancer detection and diagnosis, with an emphasis on imaging techniques. It then elaborates on the working principle of coherent anti-Stokes Raman scattering microscopy, along with a description of pathologic applications to show the effectiveness and potential of this novel technology for lung cancer diagnosis. As a nonlinear optical technique probing intrinsic molecular vibrations, coherent anti-Stokes Raman scattering microscopy offers an unparalleled, label-free strategy for clinical cancer diagnosis and allows differential diagnosis of fresh specimens based on cell morphology information and patterns, without any histology staining. This powerful feature promises a higher biopsy yield for early cancer detection by incorporating a real-time imaging feed with a biopsy needle. In addition, molecularly targeted therapies would also benefit from early access to surgical specimen with high accuracy but minimum tissue consumption, therefore potentially saving specimens for follow-up diagnostic tests. Finally, we also introduce the potential of a coherent anti-Stokes Raman scattering-based endoscopy system to support intraoperative applications at the cellular level.

Context.—Previous work has reported that most high-grade cervical neoplasia is seen in patients with preceding Papanicolaou test results of atypical squamous cells of undetermined significance. This information was based on conventional test results and the Bethesda 1991 reporting system and was determined before the current treatment guidelines.Objective.—Our objective was to perform a retrospective review of all histologically confirmed, high-grade cervical neoplasia to determine the diagnosis of the preceding liquid-based Papanicolaou test.Design.—A total of 189 histologically confirmed, high-grade cervical intraepithelial neoplasia (CIN) cases grade 2 and greater were identified for a 1-year period.Results.—Of the 189 cases, 10 (5.3%) had a previous diagnosis of atypical squamous cells of undetermined significance; 55 (29.1%) had low-grade squamous intraepithelial lesions; 31 (16.4%) had low-grade squamous intraepithelial lesions, unable to rule out a high-grade squamous intraepithelial lesion; 21 (11.1%) had atypical squamous cells, unable to rule out a high-grade squamous intraepithelial lesion; 68 (36%) had high-grade squamous intraepithelial lesions; 1 (0.5%) had atypical glandular cells; 1 (0.5%) had adenocarcinoma in situ; and 2 (1%) had invasive carcinoma. Combined “low grade” Papanicolaou test results (atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesion) preceded 51 of 103 cases of CIN 2 (49.5%) and 14 of 103 cases (13.6%) of CIN 3/cancer, whereas “high grade” Papanicolaou test results (atypical squamous cells, unable to rule out a high-grade squamous intraepithelial lesion; low-grade squamous intraepithelial lesions, unable to rule out a high-grade squamous intraepithelial lesion; high-grade squamous intraepithelial lesions; atypical glandular cells; adenocarcinoma in situ; and invasive carcinoma) preceded 52 of 103 CIN 2 cases (50.5%) and 72 of 103 CIN 3/cancer cases (69.9%).Conclusions.—Our data show that we can now more-reliably predict high-grade dysplasia on routine Papanicolaou tests. Only a small fraction of histologically confirmed CIN 2/3 cases are found following a Papanicolaou test diagnosis of atypical squamous cells of undetermined significance.

Context.—Atypical has served as a descriptive term in cytology since the birth of the specialty by Dr Papanicolaou. This indeterminate diagnosis often results in repeat biopsies or additional tissue sampling and a needless delay in patient care if used inappropriately. Because of the definitional ambiguity of this term and the associated physician frustration, we have made a concerted effort at Methodist Hospital since 1995 to minimize the use of atypical as a diagnostic category. Objective.—To evaluate whether the dissolution of the atypical category has increased our cytologic-histologic discordance rate to more than the published reference range. Design.—From March 3, 2006, through December 31, 2008, all nongynecologic cases with atypical/indeterminate listed as the general diagnostic category were identified and retrieved from our laboratory data files. We then assessed the cytologic-histologic correlation rate during the corresponding time frame. Results.—A total of 48 atypical cases (0.2%) from 19 347 nongynecologic specimens were identified. Of the atypical cases, 52% (25 of 48) had intradepartmental consultation, 58% (28 of 48) had additional preparations examined, and 29% (14 of 48) documented limitations because of poor preservation. Our cytologic-histologic discrepancy rate for the period was 5.5% (214 of 3912 cases), with 89.3% (191 of 214 cases) resulting from sampling issues. On review of the small percentage of cytologic interpretative discrepancies, only one case was unhampered by less than 10% tumor cellularity or poor preservation. Conclusions.—Not using atypical as a diagnostic category, unless defined by Bethesda guidelines, has not affected our cytologic-histologic correlation rate.

High-risk human papillomavirus (HR-HPV) testing for colposcopy triage of low grade squamous intraepithelial lesion (LSIL) is not recommended because of high positive rates in young women. It remains unclear whether HR-HPV testing may be useful for triage of older women. We compiled HR-HPV data for women aged >or=30 years with LSIL for the period March 1, 2006 to February 28, 2008. Follow-up cervical biopsy information was collected for the period March 1, 2006 to August 15, 2008. We used the Hybrid Capture II test performed on residual material from liquid-based Pap tests. Of 735 women, 254 had HR-HPV testing, and of these 144 had positive HR-HPV results. Among women with positive HR-HPV results 79 underwent biopsy (54.9%) and 11 had cervical intraepithelial neoplasia (CIN) 2 or 3 (13.9% of women with biopsy follow-up). A total of 481 women did not undergo HR-HPV testing, of whom 192 underwent biopsy (39.9%) and 11 had CIN 2 or 3 (5.7% of biopsied women [P = 0.04]). Among women who tested negative for HR-HPV and had follow-up biopsies, only one had a high grade lesion found (CIN 2). The overall HR-HPV positive rate in tested women >or=30 years old with LSIL was 56.7% if women who had reflex HR-HPV testing for ASC-US are included. The HR-HPV positive rate in residual material from Pap tests interpreted as LSIL was 63.8%. Among women >or=30 years of age with LSIL, CIN 2-3 is significantly more likely in HR-HPV positive women. Relatively few older women with LSIL test positive for HR-HPV. Colposcopy triage using HR-HPV may be justified in this population.

ContextThe Papanicolaou (Pap) test has indisputably decreased cervical cancer mortality, as rates have declined by up to 80% in the United States since its implementation. However, the Pap test is considered less sensitive for detecting glandular lesions than for detecting those of squamous origin. Some studies have even suggested an increasing incidence of cervical adenocarcinoma, which may be a consequence of a relatively reduced ability to detect glandular lesions with cervical cancer screening techniques.ObjectiveTo evaluate the detection rate of glandular lesions with screening techniques currently used for cervical cancer screening and to provide insight as to which techniques are most efficacious in our study population.DesignWe retrospectively reviewed any available cytology, human papillomavirus (HPV), and histologic malignancy data in patients diagnosed with adenocarcinoma in situ and adenocarcinoma from 2 geographically and socioeconomically disparate hospital systems. Identified patients having had a negative/unsatisfactory Pap test within 5 years of adenocarcinoma in situ or adenocarcinoma tissue diagnosis were considered Pap test screening failures. Patients with negative HPV tests on cytology samples were considered HPV screening failures.ResultsOne hundred thirty cases were identified (age range, 22–93 years); 39 (30%) had no Pap history in our files. Eight of 91 remaining cases (8.8%) were screening failures. The detected sensitivity for identifying adenocarcinoma in situ/adenocarcinoma in this study was 91.2% by cytology alone and 92.3% when incorporating HPV testing. The most common cytologic diagnosis was atypical glandular cells (25 cases), and those diagnosed with adenocarcinoma were 7.4 years older than those diagnosed with adenocarcinoma in situ (50.3 versus 42.9 years). Nine of 24 HPV-tested cases (37.5%) were called atypical squamous cell of undetermined significance on cytology.ConclusionsOur results highlight the importance of combined Pap and HPV cotesting. Although the number of cases identified is relatively small, our data suggest screening for squamous lesions facilitates the recognition of glandular lesions in the cervix. Additionally, increased use of combined Pap and HPV cotesting may decrease detection failure rates with regard to glandular lesions.

ContextWhole slide imaging (WSI) produces a virtual image that can be transmitted electronically. This technology has clinical applications in situations in which glass slides are not readily available.ObjectiveTo examine the results of a validation study performed using the draft version of the WSI clinical validation guideline recently released by the College of American Pathologists.DesignTen iScan Coreo Au scanners (Ventana Medical Systems, Tucson, Arizona) were validated, 6 with one set of 100 cases and 4 with a different set of 100 cases, for 1000 case examinations. The cases were selected consecutively from the following case types: internal consultations and malignancies and cases with frozen sections, special stains, and/or immunohistochemistry. Only key slides were scanned from each case. The slides were scanned at ×20 magnification. Pathologists reviewed the cases as both glass slides and WSI, with at least a 3-week washout period between viewings.ResultsIntraobserver agreement between glass slides and WSI was present for 786 (79%) of the 1000 cases. Major discrepancies occurred in 18 cases (1.8%). κ statistics compiled for the subset of cases (n = 504; 50%) with concern for neoplasia showed excellent agreement (κ = 0.8782). Individual scanners performed similarly to one another. Analysis of the results revealed an area of concern: small focal findings.ConclusionsThe results were felt to validate the use of WSI for the intended applications in our multiinstitutional laboratory system, although scans at ×20 magnification may be insufficient for cases hinging on small focal findings, such as microorganisms and inflammatory processes.

ObjectiveTo determine the effect of cisplatin on circulatory tumor cells (CTC) and tumor nodules in a four-dimensional (4D) lung cancer model.MethodsCTCs from the 4D model seeded with H1299, A549, or H460 and respective cells that were grown under two-dimensional conditions in a Petri dish were treated with 50 μM cisplatin for 24 and 48 hours and cell viability was determined. The lung nodules in the 4D model were then treated with different continuous or intermittent doses of cisplatin and the nodule size, the number of CTCs, and the level of matrix metalloproteinase (MMP) were determined.ResultsCisplatin led to a significant decrease in the viability of tumor cells grown under 2D conditions (P

A Coherent Anti-Stokes Raman Scattering (CARS) microendoscope probe for early stage label-free prostate cancer diagnosis at single cell resolution is presented. The handheld CARS microendoscope probe includes a customized micro-electromechanical systems (MEMS) scanning mirror as well as miniature optical and mechanical components. In our design, the excitation laser (pump and stokes beams) from the fiber is collimated, reflected by the reflecting mirror, and transmitted via a 2D MEMS scanning mirror and a micro-objective system onto the sample; emission in the epi-direction is returned through the micro-objective lens, MEMS and reflecting mirror, and collimation system, and finally the emission signal is collected by a photomultiplier tube (PMT). The exit pupil diameter of the collimator system is designed to match the diameter of the MEMS mirror and the entrance pupil diameter of the micro-objective system. The back aperture diameter of the micro-objective system is designed according to the largest MEMS scanning angle and the distance between the MEMS mirror and the back aperture. To increase the numerical aperture (NA) of the micro-objective system in order to enhance the signal collection efficiency, the back aperture diameter of the micro-objective system is enlarged with an upfront achromatic wide angle Keplerian telescope beam expander. The integration of a miniaturized micro-optics probe with optical fiber CARS microscopy opens up the possibility of in vivo molecular imaging for cancer diagnosis and surgical intervention.

The development of real-time, label-free imaging techniques has recently attracted research interest for in situ differentiation of cancerous lesions from normal tissues. Molecule-specific intrinsic contrast can arise from label-free imaging techniques such as Coherent Anti-Stokes Raman Scattering (CARS), Two-Photon Excited AutoFluorescence (TPEAF), and Second Harmonic Generation (SHG), which, in combination, would hold the promise of a powerful label-free tool for cancer diagnosis. Among cancer-related deaths, lung carcinoma is the leading cause for both sexes. Although early treatment can increase the survival rate dramatically, lesion detection and precise diagnosis at an early stage is unusual due to its asymptomatic nature and limitations of current diagnostic techniques that make screening difficult. We investigated the potential of using multimodality nonlinear optical microscopy that incorporates CARS, TPEAF, and SHG techniques for differentiation of lung cancer from normal tissue. Cancerous and non-cancerous lung tissue samples from patients were imaged using CARS, TPEAF, and SHG techniques for comparison. These images showed good pathology correlation with hematoxylin and eosin (H and E) stained sections from the same tissue samples. Ongoing work includes imaging at various penetration depths to show three-dimensional morphologies of tumor cell nuclei using CARS, elastin using TPEAF, and collagen using SHG and developing classification algorithms for quantitative feature extraction to enable lung cancer diagnosis. Our results indicate that via real-time morphology analyses, a multimodality nonlinear optical imaging platform potentially offers a powerful minimally-invasive way to differentiate cancer lesions from surrounding non-tumor tissues in vivo for clinical applications.

Background: Our institution's pathology unknown conferences provide educational cases for our residents. However, the cases have not been previously available digitally, have not been collated for postconference review, and were not accessible to a wider audience. Our objective was to create an inexpensive whole slide image (WSI) education suite to address these limitations and improve the education of pathology trainees. Materials and Methods: We surveyed residents regarding their preference between four unique WSI systems. We then scanned weekly unknown conference cases and study set cases and uploaded them to our custom built WSI viewer located at RecutClub.com. We measured site utilization and conference participation. Results: Residents preferred our OpenLayers WSI implementation to Ventana Virtuoso, Google Maps API, and OpenSlide. Over 16 months, we uploaded 1366 cases from 77 conferences and ten study sets, occupying 793.5 GB of cloud storage. Based on resident evaluations, the interface was easy to use and demonstrated minimal latency. Residents are able to review cases from home and from their mobile devices. Worldwide, 955 unique IP addresses from 52 countries have viewed cases in our site. Conclusions: We implemented a low-cost, publicly available repository of WSI slides for resident education. Our trainees are very satisfied with the freedom to preview either the glass slides or WSI and review the WSI postconference. Both local users and worldwide users actively and repeatedly view cases in our study set.

Objective: Mouse lung cancer cell lines 393P and 344SQ are derived from KrasLA1/+ p53R172HΔG mice and have different metastatic potential in immunocompetent mice. 393P cells have local primary tumor growth but do not form metastatic lesions when injected into the subcutaneous space of 129sv mice. However, 344SQ cells form a primary tumor and metastatic lesions when injected into the subcutaneous space. Our aim is to determine if lymphocytes play a role in this differential metastatic lung cancer formation between the two cell lines. Methods: Both 393P and 344SQ cells were grown in the ex vivo 4D lung cancer model. We determined the amount of primary tumor growth, the number of circulating tumor cells (CTCs), and metastatic lesion formation. The CTCs from the 4D model seeded with either cells were injected into the tail veins of immunocompetent 129sv mice (wt) or immunodeficient mice (nu/nu). We calculated the Kaplan Meier survival curve. Next, we added lymphocytes derived from a 129sv mouse spleen to an ex vivo 4D lung cancer model seeded with 393P or 344SQ and isolated the CTCs. We plated the CTCs on a petri dish and compared the survival of these cells. Results: Both 393P and 344SQ cells grew in the 4D model. There were no significant differences in the primary tumor growth and circulating tumor cell formation between the two cell lines. There were no significant differences in the number of metastatic lesion formation between the two cell lines (p=0.5). When CTCs from the 4D model seeded with 344SQ were injected into the tail vein of nu/nu mice or wt mice, there were no differences in the survival rates between the two types of mice (p=0.3). However, nu/nu mice that were injected with CTC from the 4D model seeded with 393P were less likely to survive compared to the wt mice (p=0.002). No metastatic lesions formed in the wt mice injected with 393P CTC for 8 months. The CTC from the 4D model seeded with 344SQ and treated with lymphocytes had no significant difference in cell growth when cultured on a petri dish compared to the control group (p=0.9). However, the CTC from the 4D model seeded with 393P treated with lymphocytes had significantly less live cells compared to the control group (p=0.01). Conclusions: Lymphocytes inhibit metastatic lesion formation in 393P cell lines but not 344SQ cell lines. The ex vivo 4D lung cancer model allows for the identification of the cell type that is responsible for inhibition of metastasis. The model can be used to study the role of different components of a tumor's microenvironment in tumor metastasis. Citation Format: Dhruva K. Mishra, Humberto Juarez, Michael J. Thrall, Min P. Kim. Lymphocytes lead to circulating tumor cell death from 4D model seeded with non-metastatic lung cancer cell line. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A03.

We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture.

CASE REPORT 60-year-old woman presented to our clinic for evaluation of a right renal mass, incidentally Adiscovered on a computed tomography (CT) scan obtained during a workup for shortness of breath. Her medical history was significant for hypertension, hyperlipidemia, hypothyroidism, and asthma. She had undergone several abdominal surgeries, including laparoscopic gastric banding, splenectomy, hysterectomy, and bilateral salpingo-oophorectomy. She denied a history of tobacco, heavy alcohol, or illicit drug use. She had no family history of kidney disease or genitourinary malignancy. On a review of systems, she reported intermittent nausea, vomiting, and dyspepsia but denied fevers, night sweats, weight loss, gross hematuria, dysuria, or a change in bowel habits. Her vitals signs were within normal limits, and her body mass index was 32.4 kg/m. Her abdomen was soft, not distended, and without palpable masses, although several surgical scars were present. Routine laboratory tests, including complete blood count, electrolytes, creatinine, coagulation studies, and urinalysis, were performed. The complete blood count with differential demonstrated mild thrombocytosis (473 K/ mL) and leukocytosis (13 K/mL) but was otherwise normal. The results from the remainder of the laboratory studies were also within normal limits. A CT scan with intravenous contrast of the abdomen and pelvis obtained 1 month before her clinic evaluation revealed a 3.4 3.0-cm, enhancing mass located in the posterior mid-pole cortex of the right kidney (Fig. 1). No additional masses were seen in the remainder of the right kidney. The left kidney and both ureters appeared normal. No retroperitoneal lymphadenopathy or other intra-abdominal abnormalities were identified.

Background Extracellular matrix allows lung cancer to form its shape and grow. Recent studies on organ reengineering for orthotopic transplantation have provided a new avenue for isolating purified native matrix to use for growing cells. Whether human lung cancer cells grown in a decellularized rat lung matrix would create perfusable human lung cancer nodules was tested. Methods Rat lungs were harvested and native cells were removed using sodium dodecyl sulfate and Triton X-100 in a decellularization chamber to create a decellularized rat lung matrix. Human A549, H460, or H1299 lung cancer cells were placed into the decellularized rat lung matrix and grown in a customized bioreactor with perfusion of oxygenated media for 7 to 14 days. Results Decellularized rat lung matrix showed preservation of matrix architecture devoid of all rat cells. All three human lung cancer cell lines grown in the bioreactor developed tumor nodules with intact vasculature. Moreover, the lung cancer cells developed a pattern of growth similar to the original human lung cancer. Conclusions Overall, this study shows that human lung cancer cells form perfusable tumor nodules in a customized bioreactor on a decellularized rat lung matrix created by a customized decellularization chamber. The lung cancer cells grown in the matrix had features similar to the original human lung cancer. This ex vivo model can be used potentially to gain a deeper understanding of the biologic processes involved in human lung cancer.

Telecytology is the interpretation of cytology material at a distance using digital images. For more than a decade, pioneering efforts to introduce telecytology into clinical practice have been reported. A Medline search for "telecytology" and "cytology" reveals a voluminous literature, though much of what has been published to date is based on technologies that are rapidly becoming obsolete. The technological limitations of previous techniques, including the transmission of static digital images and dynamic streaming images, have limited telecytology to minor niches. The primary problem with these technologies is that the remote viewer can only see a small fraction of the material on the original slides, introducing the possibility of diagnostic error based not only on image quality but also on image selection. Remote robotic microscopy offers one possible solution to this problem, but to date has found limited acceptance, principally attributable to slow operating times. Whole slide imaging seems to be a much more promising solution, though cytology-specific literature regarding its use is still scant. The advent of whole slide imaging opens up new possibilities for telecytology by enabling high-quality images of entire cytology specimens to be available to anyone, anywhere via the Internet. Although challenges remain, especially with regard to capturing the full microscopy experience including multiple planes of focus and sharp high-powered images, rapidly advancing technology promises to overcome these limitations. Increasing application of whole slide imaging technology in surgical pathology will undoubtedly also increase its application to cytology due to the increasing affordability and practicality of the equipment as it serves a larger number of useful roles within a pathology department. The current and expanding applications of telecytology for clinical practice, education, quality assurance, and testing will be reviewed.

We report the development and application of a knowledge-based coherent anti-Stokes Raman scattering (CARS) microscopy system for label-free imaging, pattern recognition, and classification of cells and tissue structures for differentiating lung cancer from non-neoplastic lung tissues and identifying lung cancer subtypes. A total of 1014 CARS images were acquired from 92 fresh frozen lung tissue samples. The established pathological workup and diagnostic cellular were used as prior knowledge for establishment of a knowledge-based CARS system using a machine learning approach. This system functions to separate normal, non-neoplastic, and subtypes of lung cancer tissues based on extracted quantitative features describing fibrils and cell morphology. The knowledge-based CARS system showed the ability to distinguish lung cancer from normal and non-neoplastic lung tissue with 91% sensitivity and 92% specificity. Small cell carcinomas were distinguished from nonsmall cell carcinomas with 100% sensitivity and specificity. As an adjunct to submitting tissue samples to routine pathology, our novel system recognizes the patterns of fibril and cell morphology, enabling medical practitioners to perform differential diagnosis of lung lesions in mere minutes. The demonstration of the strategy is also a necessary step toward in vivo point-of-care diagnosis of precancerous and cancerous lung lesions with a fiber-based CARS microendoscope.