Virtual slide telepathology for an academic teaching hospital surgical pathology quality assurance program.

Virtual slide telepathology is an important potential tool for providing re-review of surgical pathology cases as part of a quality assurance program. The University of Arizona pathology faculty has implemented a quality assurance program between 2 university hospitals located 6 miles apart. The flagship hospital, University Medical Center (UMC), in Tucson, AZ, handles approximately 20 000 surgical pathology specimens per year. University Physicians Healthcare Hospital (UPHH) at Kino Campus has one tenth the volume of surgical pathology cases. Whereas UMC is staffed by 10 surgical pathologists, UPHH is staffed daily by a single part-time pathologist on a rotating basis. To provide same-day quality assurance re-reviews of cases, a DMetrix DX-40 ultrarapid virtual slide scanner (DMetrix, Inc, Tucson, AZ) was installed at the UPHH in 2005. Since then, glass slides of new cases of cancer and other difficult cases have been scanned the same day the slides are produced by the UPHH histology laboratory. The pathologist at UPHH generates a provisional written report based on light microscopic examination of the glass slides. At 2:00 pm each day, completed cases from UPHH are re-reviewed by staff pathologists, pathology residents, and medical students at the UMC using the DMetrix Iris virtual slide viewer. The virtual slides are viewed on a 50-in plasma monitor. Results are communicated with the UPHH laboratory by fax. We have analyzed the results of the first 329 consecutive quality assurance cases. There was complete concordance with the original UPHH diagnosis in 302 (91.8%) cases. There were 5 (1.5%) major discrepancies, which would have resulted in different therapy and/or management, and 10 (3.0%) minor discrepancies. In 6 cases (1.8%), the diagnosis was deferred for examination of the glass slides by the reviewing pathologists at UMC, and the diagnosis of another 6 (1.8%) cases were deferred pending additional testing, usually immunohistochemistry. Thus, the quality assurance program found a small number of significant diagnostic discrepancies. We also found that implementation of a virtual slide telepathology quality assurance service improved the job satisfaction of academic subspecialty pathologists assigned to cover on-site surgical pathology services at a small, affiliated university hospital on a rotating part-time basis. These findings should be applicable to some community hospital group practices as well.

Phase II study of erlotinib in patients with malignant pleural mesothelioma: a Southwest Oncology Group Study.

PURPOSE: Malignant pleural mesothelioma (MPM) expresses high levels of epidermal growth factor receptor (EGFR), and preclinical studies have identified antitumor activity of EGFR tyrosine kinase inhibitors (TKIs) in MPM. We conducted a phase II trial of the EGFR TKI erlotinib in previously untreated patients with MPM. PATIENTS AND METHODS: Patients with measurable and nonmeasurable disease were treated with erlotinib 150 mg/d on days 1 through 28 of each 28-day dosing cycle. Archived patient tumors were analyzed for immunohistochemical expression of EGFR, phospho-EGFR, human epidermal growth factor receptor 2 (HER2), phospho-extracellular signal-regulated kinase (ERK), and phosphatase and tensin homolog (PTEN) and phosphorylation of members of the phosphatidylinositol 3-kinase/Akt signaling pathway. RESULTS: Sixty-three patients were treated on the study. EGFR was highly expressed in 75% of patient tumors, as was phospho-ERK (82%), phospho-Akt (84%), phospho-mammalian target of rapamycin (74%), and phospho-forkhead (74%). HER2 was rarely expressed, and loss of PTEN was rare. For 33 patients with measurable disease, there were no objective responses; 14 patients (42%) had stable disease, 15 patients (45%) had disease progression, and four patients had inadequate assessments to determine response. Toxicities were mainly constitutional (51%), dermatologic (82%), and GI (52%); there was one death on trial, which was related to dyspnea. Median overall survival time was 10 months; 1-year survival rate was 43%; and median progression-free survival time was 2 months. CONCLUSION: Single-agent erlotinib was not effective in MPM, despite high expression of EGFR. Activation of the ERK and phosphatidylinositol 3-kinase/Akt downstream pathways are possible resistance mechanisms to EGFR TKI. The activated phosphatidylinositol 3-kinase/Akt pathway is a potential therapeutic target for MPM.

A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility.

The 2 methodologies in current clinical use to assess HER2 status in breast cancer are: fluorescence in situ hybridization (FISH) (gene amplification) and immunohistochemistry (protein over-expression). A consistent finding has been that 3% to 15% of breast cancers over-express HER2 protein without evidence for gene amplification. Accurate determination of the HER2 status has implications for selecting patients most likely to respond to trastuzumab. We report here our preliminary experience with a new anti-HER2 rabbit monoclonal antibody, 4B5. The evaluation of HER2 status in 2 different cohorts of breast cancer cases (Single Institution (SI) and Multinational (MN)) with a total of 322 breast cancer cases was performed on an automated staining system (Ventana Medical Systems, Inc, Tucson, AZ) and scored by 3 pathologists (0-3+), for comparison with CB11 staining results (PATHWAY) and FISH (PathVysion). Interlaboratory reproducibility of automated staining results and interpretation was determined on a subset of the SI cohort at 3 separate laboratories. Rabbit monoclonal 4B5 demonstrated sharper membrane staining with less cytoplasmic and stromal background staining than CB11. In the SI cohort, the staining results for 4B5 were highly comparable with those obtained for CB11 with an overall concordance of 93.3%. In the multinational cohort, the overall concordance with CB11 was 84.7%. This lower level of concordance was associated with a much higher overall agreement of 4B5 with FISH (89.5%), compared with agreement of CB11 with FISH (81.2%). The difference in the performance of CB11 in the MN cohort versus the SI cohort may be due to differences in tissue fixation and processing in a centralized, high volume laboratory in an academic medical center versus multiple sites in the international community with potentially nonstandardized techniques. The staining results with 4B5 indicate that it has a more robust performance than CB11 because the correlation of 4B5 with FISH was nearly equivalent (88.2% MN; 89.3% SI) in both cohorts. Interlaboratory reproducibility was also excellent (kappa 1.0). RMoAb 4B5 provides excellent sensitivity, specificity, and interlaboratory reproducibility for the detection of HER2 status in breast cancer.

Eye-movement study and human performance using telepathology virtual slides: implications for medical education and differences with experience.

A core skill in diagnostic pathology is light microscopy. Remarkably little is known about human factors that affect the proficiency of pathologists as light microscopists. The cognitive skills of pathologists have received relatively little attention in comparison with the large literature on human performance studies in radiology. One reason for this lack of formal visual search studies in pathology has been the physical restrictions imposed by the close positioning of a microscope operator's head to the microscope's eyepieces. This blocks access to the operator's eyes and precludes assessment of the microscopist's eye movements. Virtual slide microscopy now removes this barrier and opens the door for studies on human factors and visual search strategies in light microscopy. The aim of this study was to assess eye movements of medical students, pathology residents, and practicing pathologists examining virtual slides on a digital display monitor. Whole histopathology glass slide digital images, so-called virtual slides, of 20 consecutive breast core biopsy cases were used in a retrospective study. These high-quality virtual slides were produced with an array-microscope equipped DMetrix DX-40 ultrarapid virtual slide processor (DMetrix, Tucson, Ariz). Using an eye-tracking device, we demonstrated for the first time that when a virtual slide reader initially looks at a virtual slide his or her eyes are very quickly attracted to regions of interest (ROIs) within the slide and that these ROIs are likely to contain diagnostic information. In a matter of seconds, critical decisions are made on the selection of ROIs for further examination at higher magnification. We recorded: (1) the time virtual slide readers spent fixating on self-selected locations on the video monitor; (2) the characteristics of the ways the eyes jumped between fixation locations; and (3) x and y coordinates for each virtual slide marking the sites the virtual slide readers manually selected for zooming to higher ROI magnifications. We correlated the locations of the visually selected fixation locations and the manually selected ROIs. Viewing profiles were identified for each group. Fully trained pathologists spent significantly less time (mean, 4.471 seconds) scanning virtual slides when compared to pathology residents (mean, 7.148 seconds) or medical students (mean, 11.861 seconds), but had relatively prolonged saccadic eye movements (P < .0001). Saccadic eye movements are defined as eye movements between fixation locations. On the other hand, the pathologists spent significantly more time than trainees dwelling on the 3 locations they subsequently chose for zooming. Unlike either the medical students or the residents, the pathologists frequently choose areas for viewing at higher magnification outside of areas of foveal (central) vision. Eye movement studies of scanning pathways (scan paths) may be useful for developing eye movement profiles for individuals and for understanding the difference in performances between novices and experts. They may also be useful for developing new visual search strategies for rendering diagnoses on telepathology virtual slides.

Evaluation of polyethylene glycol based hydrogel for tissue sealing after laparoscopic partial nephrectomy in a porcine model.

PURPOSE: Polyethylene glycol (PEG) based hydrogel is available as a tissue sealant and hemostatic aid. We determined the long-term safety and efficacy of its use as a tissue sealant for laparoscopic partial nephrectomy in a porcine model. MATERIALS AND METHODS: A total of 16 swine were cycled to 1 control group and 3 treatment groups, which underwent laparoscopic partial nephrectomy with hemostasis achieved only with application of a biodegradable PEG based hydrogel. The 3 treatment groups were sacrificed at 2, 6 and 12 weeks, respectively. Humoral immune response to the hydrogel used in the porcine abdomen was examined using enzyme-linked immunosorbent assay to detect antibodies in the serum at 0, 2, 6 and 12 weeks. Cell mediated immune response was examined using a lymphocyte proliferation assay to measure the response of leukocytes to various mitogens and antigens, including the polymerized hydrogel, at the same intervals. RESULTS: Hemostasis was satisfactory after hydrogel application. No adverse effects in the immediate and delayed periods were noted. At 2, 6 and 12 weeks there were no significant differences in hemoglobin or creatinine levels, or in the humoral immune response by enzyme-linked immunosorbent assay. There was no significant difference between test and control pig reactivity to hydrogel as an antigen in the lymphocyte proliferation assay at any time point. Histologically by 6 weeks the animals had almost absorbed the hydrogel with acute inflammation and foreign body reaction resolving by 6 to 12 weeks. No deleterious effect to renal tubules was seen. CONCLUSIONS: Biodegradable PEG based hydrogel is effective for long-term use as an agent for hemostasis. There was no detectable humoral immune response and no cell mediated immune response to sealant after 2 weeks. This represents promising sealant technology and it should be further investigated for human use.

Kimura disease with advanced renal damage with anti-tubular basement membrane antibody.

Kimura disease (KD) is an autoimmune eosinophilic granulomatous disorder with generalized lymphadenopathy. A handful of pediatric patients with renal disease have been described, none of whom have been African-American (AA). We present an AA boy with KD and nephrotic syndrome (NS). Two months after stopping steroids, fever, asthma, eczema, and proteinuria recurred. His NS did not relapse but his platelet count decreased to 51,000/microl (x10(6)/l). On restarting prednisone, his platelet count normalized. A kidney biopsy revealed 23 of 37 glomeruli obsolescent and advanced damage with over 50% of cortical tissue replaced by interstitial fibrosis and chronic inflammation. Glomerular immunofluorescence was largely negative; very intense linear anti-tubular basement membrane (TBM) deposits of IgA, IgG, C3, and C4 were noted. At present, 36 months from onset, serum creatinine is 1.2 mg/dl (106 micromol/l). We present a 4-year-old AA boy with KD, NS, relapsing thrombocytopenia, and renal damage with anti-TBM antibody.

An array microscope for ultrarapid virtual slide processing and telepathology. Design, fabrication, and validation study.

This paper describes the design and fabrication of a novel array microscope for the first ultrarapid virtual slide processor (DMetrix DX-40 digital slide scanner). The array microscope optics consists of a stack of three 80-element 10 x 8-lenslet arrays, constituting a "lenslet array ensemble." The lenslet array ensemble is positioned over a glass slide. Uniquely shaped lenses in each of the lenslet arrays, arranged perpendicular to the glass slide constitute a single "miniaturized microscope." A high-pixel-density image sensor is attached to the top of the lenslet array ensemble. In operation, the lenslet array ensemble is transported by a motorized mechanism relative to the long axis of a glass slide. Each of the 80 miniaturized microscopes has a lateral field of view of 250 microns. The microscopes of each row of the array are offset from the microscopes in other rows. Scanning a glass slide with the array microscope produces seamless two-dimensional image data of the entire slide, that is, a virtual slide. The optical system has a numerical aperture of N.A.= 0.65, scans slides at a rate of 3 mm per second, and accrues up to 3,000 images per second from each of the 80 miniaturized microscopes. In the ultrarapid virtual slide processing cycle, the time for image acquisition takes 58 seconds for a 2.25 cm2 tissue section. An automatic slide loader enables the scanner to process up to 40 slides per hour without operator intervention. Slide scanning and image processing are done concurrently so that post-scan processing is eliminated. A virtual slide can be viewed over the Internet immediately after the scanning is complete. A validation study compared the diagnostic accuracy of pathologist case readers using array microscopy (with images viewed as virtual slides) and conventional light microscopy. Four senior pathologists diagnosed 30 breast surgical pathology cases each using both imaging modes, but on separate occasions. Of 120 case reads by array microscopy, there were 3 incorrect diagnoses, all of which were made on difficult cases with equivocal diagnoses by light microscopy. There was a strong correlation between array microscopy vs. "truth" diagnoses based on surgical pathology reports. The kappa statistic for the array microscopy vs. truth was 0.96, which is highly significant (z=10.33, p <0.001). There was no statistically significant difference between rates of agreement with truth between array microscopy and light microscopy (z=0.134, p >0.05). Array microscopy and light microscopy did not differ significantly with respect to the number/percent of correct decisions rendered (t=0.552, p=0.6376) or equivocal decisions rendered (t=2.449, p=0.0917). Pathologists rated 95.8% of array microscopy virtual slide images as good or excellent. None were rated as poor. The mean viewing time for a DMetrix virtual slide was 1.16 minutes. The DMetrix virtual slide processor has been found to reduce the virtual slide processing cycle more than 10 fold, as compared with other virtual slide systems reported to date. The virtual slide images are of high quality and suitable for diagnostic pathology, second opinions, expert opinions, clinical trials, education, and research.

Multicentric papillary renal carcinoma in renal allograft.

A renal transplant recipient with 13 years of excellent allograft function was found incidentally to have a malignant mass in his transplanted kidney. After resection, pathological analysis showed 29 separate lesions of renal cell carcinoma. All tumors were confined within the renal capsule. The majority of tumors (21 of 29 tumors) were chromophil basophilic carcinoma with papillary architecture, 5 tumors were clear cell, 2 tumors were mixed cell type, and 1 tumor was chromophil eosinophilic papillary carcinoma. These histological findings are similar to those reported in hereditary papillary renal carcinoma. To our knowledge, this is the first case of multicentric papillary renal carcinoma occurring in the renal allograft. We speculate that the allograft in this case is predisposed to malignant changes because of preexisting genetic mutations, as well as prolonged immunosuppression.

Acute myelo-monocytic infiltrate of the lower esophagus in a 4-year-old renal transplant recipient.

BACKGROUND: Acute myeloid leukemia after solid organ transplantation is a rare phenomenon. Few achieve remission; most succumb to relapse and infection. METHODS: A 4-year-old male renal transplant recipient on triple immunosuppression had culture-negative high spiking fever, persistent leukopenia, anemia and severe gastritis. Upper endoscopy showed 2 ulcerating masses in the lower esophagus. RESULTS: Esophageal biopsy showed a highly atypical myelo-monocytic infiltrate. A blastic population of cells featuring convoluted nuclear envelopes with an open chromatin pattern and abundant cytoplasm were filling the submucosa and infiltrating into the muscularis propria. Extensive investigation including bone marrow aspiration showed no spread. Drastic reduction of immunosuppression except 4-mg/d (0.22 mg/kg/d) prednisone for 2 to 3 weeks led to resolution of the leukemic process proven on repeat biopsy. The patient still is in remission 2 years later. CONCLUSION: This case provides evidence that early diagnosis and aggressive reduction of immunosuppression may remit a posttransplant locally invasive acute myelo-monocytic infiltrative process.

Different phenotypes in human prostate cancer: alpha6 or alpha3 integrin in cell-extracellular adhesion sites.

The distribution of alpha6/alpha3 integrin in adhesion complexes at the basal membrane in human normal and cancer prostate glands was analyzed in 135 biopsies from 61 patients. The levels of the polarized alpha6/alpha3 integrin expression at the basal membrane of prostate tumor glands were determined by quantitative immunohistochemistry. The alpha6/alpha3 integrin expression was compared with Gleason sum score, pathological stage, and preoperative serum prostate-specific antigen (PSA). The associations were assessed by statistical methods. Eighty percent of the tumors expressed the alpha6 or alpha3 integrin and 20% was integrin-negative. Gleason sum score, but not serum PSA, was associated with the integrin expression. Low Gleason sum score correlated with increased integrin expression, high Gleason sum score with low and negative integrin expression. Three prostate tumor phenotypes were distinguished based on differential integrin expression. Type I coexpressed both alpha6 and alpha3 subunits, type II exclusively expressed alpha6 integrin, and type III expressed alpha3 integrin only. Fifteen cases were further examined for the codistribution of vinculin, paxillin, and CD 151 on frozen serial sections using confocal laser scanning microscopy. The alpha6/alpha3 integrins, CD151, paxillin, and vinculin were present within normal glands. In prostate carcinoma, alpha6 integrin was colocalized with CD 151, but not with vinculin or paxillin. In tumor phenotype I, the alpha6 subunit did not colocalize with the alpha subunit indicating the existence of two different adhesion complexes. Human prostate tumors display on their cell surface the alpha6beta1 and/or alpha3beta1 integrins. Three tumor phenotypes associated with two different adhesion complexes were identified, suggesting a reorganization of cell adhesion structures in prostate cancer.