Abstract poster sessions were conducted at the College of American Pathologists Annual Meeting on September 10–14, 2003 in San Diego, California.

Congenital Diaphragmatic Hernia and Chromosomal Anomalies

Dariusz Borys, MD ([email protected])1; Jerome B. Taxy, MD.21Department of Pathology, Metro Group Hospitals at University of Illinois at Chicago; 2Department of Pathology, Advocate Lutheran General Hospital, Park Ridge, Ill.

Context: Congenital diphragmatic hernia (CDH) may be associated with chromosomal anomalies in 3% to 40% of cases. Most of the malformations are associated with changes in chromosome number, especially trisomy 18, but more recent studies report higher incidence of structural chromosomal abnormalities, such as tetrasomy 12p or 4p and 8p deletion. None of the presently recognized anomalies are considered causative.

Design: We reviewed our institutional autopsy experience with CDH for the years 1992–2002. Thirteen cases were found. The fetuses ranged between 21 and 35 weeks of gestation. Only 3 were born alive and 5 were diagnosed prenatally.

Results: Eleven cases (84%) had left diaphragmatic hernia. Cytogenetic study revealed 5 cases (38%) with normal karyotypes, 3 cases (23%) with complex karyotypes, and 5 cases (38%) in which no karyotype was performed. The complex karyotypes were: 46,XX,del(8)p(23.1), 47,XX,+i(12)(p10)(6)/46XX (Pallister-Killian syndrome), and 47,XY,+der(22)t(11:22)(q23.3:q11.2). The unbalanced translocation of chromosomes 11 and 22 in congenital diaphragmatic hernia has not been previously described. Three (23%) had heart abnormalities, 1 associated with an 8p deletion. The other 2 had no karyotype study.

Conclusion(s): Neither in this study nor in the literature is there a consistent or prevailing association between a specific chromosomal anomaly and CDH. Previously described 8p deletions (21–22) are in different locations than the 8p(23.1) deletion previously described in 2 cases and that we encountered in 1 case. Only a few previous Pallister-Kilian syndromes associated with CDH have been described. If structural chromosomal anomalies are etiologically related to this syndrome, the embryologic closure of the diaphragmatic leaflets may be mediated by more than one gene and/or may be related to chromosomal abnormalities not currently detectable.

Fine-Needle Aspiration Cytology of Synovial Sarcoma With an Emphasis on Histopathologic Correlation

Geetika Khanna, MD ([email protected])1; Sonal Sharma, MD2; Rajni Prasad, MD1; Gita Ashok Raj, MD.11Department of Pathology, Safdarjung Hospital, New Delhi, India; 2Department of Pathology, Vallabhbhai Patel Chest Institute, Delhi, India.

Context: Fine-needle aspiration (FNA) is an effective modality in the diagnosis of soft tissue lesions. There are very few studies describing the cytologic features of synovial sarcoma. We report 20 FNA specimens from 15 patients with histologically proven synovial sarcoma.

Design: May-Grunwald Giemsa, Papanicolaou, and hematoxylin-eosin (H&E)–stained FNA smears and corresponding H&E sections from 15 cases of synovial sarcoma were retrieved from archival material and examined by light microscopy.

Results: Thirteen cases were soft tissue masses without any obvious bony involvement. Two cases showed pressure atrophy and lytic destruction of bone. The majority of the cytology specimens were similar, exhibiting moderate to marked cellularity with 3-dimensional microtissue fragments of spindle-shaped cells. Within fragments polarity was discernible, facilitating the recognition of a characteristic fascicular and whorled pattern. The nuclei were round to ovoid to elongated, hyperchromatic and with inconspicuous nucleoli. Epithelial elements were marked by round to ovoid cells with few glandlike formations within otherwise solid tissue fragments. Areas with predominance of epithelial elements showed a large number of single cells and loose clusters. One case showed a large number of calcific spherules. No giant cells, necrosis, or atypical mitotic figures were seen. The biphasic nature of the tumor could be appreciated on cytology in 10 cases. Extensive histopathologic sampling revealed few epithelial areas in the 3 cases diagnosed as monophasic spindle cell synovial sarcoma on cytology. Two cases found to have only epithelial elements on cytology were confirmed as monophasic epithelial synovial sarcoma on histopathology.

Conclusion(s): In biphasic synovial sarcoma, there is a predominance of neoplastic spindle cells, making distinction from other soft tissue sarcomas difficult. However, a false diagnosis may be avoided if adequate needle sampling is ensured and clinical correlation considered.

Ossifying Renal Tumor of Infancy

Atilano G. Lacson, MD ([email protected])1; Yves L. Homsy, MD2; Richard Benator, MD.31Department of Pathology, 2Division of Urology (Surgery), and 3Division of Radiology (Medicine), University of South Florida at All Children's Hospital, St Petersburg.

Context: Ossifying renal tumor of infancy, a rare benign tumor of the kidney, leads to early onset hematuria with few exceptions. The tumor develops in utero but may be occult and evade detection at birth, only to show up as hilar calcification with or without a mass and with or without hydronephrosis a month to a few years later. An ultrasound-visible hilar or palpable abdominal mass invites a more sinister differential diagnosis, especially later in infancy and early childhood, including neuroblastoma and Wilms tumor. Intraoperative sampling is critical in reaching an accurate frozen section diagnosis.

Design: We reviewed 11 examples of ossifying renal tumor of infancy and childhood (ORTI) from the literature and added 2 cases of our own using immunohistochemistry, Ki-67 proliferation index, and electron microscopy.

Results: The consistent histologic appearance of all tumors reviewed leaves no room for another diagnosis when sufficient representative tissue samples are examined. As previously reported, “osteoblast-like” cells lining the osteoid matrix are EMA+ and AE1,3+. The cellular blastema-like component beneath the urothelium is similarly EMA+ and AE1,3+, with a Ki-67 proliferation index as high as 25%. The osteoblast-like cells showed ultrastructural features of a “hybrid” appearance between “fibroblasts” and “epithelial cells,” while the blastemal-like cells showed few specialized cytoplasmic or membrane features.

Conclusions: Intraoperative sampling for frozen sections can create confusion if the cellular portion alone is submitted. Observations to date imply the benign nature of this lesion even on long-term follow-up. However, examples of recurrences after partial removal argue for nephrectomy at the outset, especially with reports of significant reduction in renal function following surgery. Potential for recurrence in our cases would have been high if renal-sparing tumor resection was performed, because these lesions developed from the pelvic urothelium, extend in a subepithelial manner to adjacent calyces beyond the stalk, and show a high blastemal Ki-67 proliferation index.

Value of Histiocytes Detection in Papanicolaou Tests for Predicting Endometrial Pathology Revisited

Aziza Nassar, MD ([email protected])1; Seth R. Fleisher, CT(ASCP)2; Joseph F Nasuti, MD.21Department of Pathology, Emory University Hospital, Atlanta, Ga; 2Department of Pathology, University of Pennsylvania Medical Center, Philadelphia.

Context: This study was undertaken to determine the clinical implications of the finding of histiocytes in cervical/vaginal Papanicolaou (Pap) tests in our patient population.

Design: The medical records and Pap tests of patients in which the presence of histiocytes was mentioned in the diagnosis between August 1996 and August 2001 were reviewed in conjunction with follow-up surgical findings. The positive predictive value (PPV) for significant endometrial pathology for the isolated finding of histiocytes on Pap tests was determined.

Results: Of the 238 225 women screened during a 60-month period (1996–2001), 325 were reported to have histiocytes in their Pap tests. Of these, 238 patients (73.2%) had subsequent endometrial sampling, hysterectomy or both and follow-up Pap tests. Two hundred seven (87%) failed to disclose endometrial pathology. Thirty-one cases (13%) resulted in significant histopathologic findings, including 12 uterine malignancies, 8 endocervical polyps, 7 endometrial polyps, 2 submucosal leiomyomata, 1 simple hyperplasia without atypia, and 1 patient with tamoxifen-related changes. Upon review of the clinical records, 58% (18/31) of these patients had other significant clinical and/or cytologic findings. Five of the 18 patients (27.8%) had associated postmenopausal bleeding; 11 had additional abnormal Pap test findings (atypical glandular cells, 6/18 [33.3%]; endometrial cells, 5/18 [27.8%]); and another 2 had both postmenopausal bleeding and atypical glandular cells (2/18 [11.1%]). The PPV for significant uterine pathology for women with the isolated finding of histiocytes on Pap test is 5.5%, and 60% with additional clinical and/or Pap test findings. The PPV for endometrial cancer is 1.3% for women with the isolated finding of histiocytes on Pap test, but is 20% for women with histiocytes and additional clinical or Pap test findings.

Conclusions: Based on the findings of this study and recently published data, we conclude that the isolated finding of increased histiocytes in the absence of postmenopausal bleeding or endometrial cells or atypical glandular cells on Pap test is a poor indicator of uterine disease.

Training of Pathology Professionals From Developing Countries: Experience of the Los Angeles Society of Pathology

James F. Keefe, MD ([email protected])1; Victor W. Lee, MD, PhD2; Krishna P. Sharma, MBBS.31Los Angeles Society of Pathology, Los Angeles, Calif; 2Pathologists Overseas, Fullerton, Calif; 3Department of Pathology, Jigme Dorje Wangchuck National Hospital, Thimphu, Bhutan.

Context: The Los Angeles Society of Pathology has established a fund under the supervision of the College of American Pathologists Foundation. The purpose of the fund is to support the training of pathology professionals working in underdeveloped countries. We will use the resources of our local pathology residency programs to accomplish this task.

Design: Funds to underwrite this project have been donated by local pathologists and corporations. We have designed our program to fit the particular needs of our first recipient when he returns to his country of origin. The first pathologist is Krishna Sharma, a resident of the country of Bhutan. Up to now he has received only 2 years of formal pathology training in the country of Myanmar. He and his senior associate are the only pathologists providing services for nearly 800 000 residents. Dr Sharma will spend his time (15 months) under the supervision of the following institutions: USC, UCLA Medical Center, Cedars Sinai, and City of Hope Medical Centers. Dr Sharma is documenting his training as he progresses through his 15-month program. We will publish an article delineating the results of his sabbatical.

Results: The result we intend to accomplish is to provide Dr Sharma with the expertise needed to serve the needs of his nation in all areas of clinical and anatomic pathology. We will use the Internet to stay connected with him over time.

Conclusion(s): The members of the Los Angeles Society of Pathology think that now is the time to begin the process of transferring the abundance of scientific knowledge and resources that we possess to other deserving countries.

Immunohistochemical Expression of Human Chorionic Gonadotrophin in Metaplastic Breast Carcinoma

Shahnila Latif, MD ([email protected]); Cooper Heard, MD; Deepti Shukla, MD; Jorge Alberos Saavedra, MD. Department of Pathology, Louisiana State University Health Sciences Center, Shreveport.

Context: In 1963, McArthur first reported the production of human chorionic gonadotrophin (hCG) in conventional breast carcinoma (Ca). We recently had the opportunity to study the metaplastic breast Ca that gave rise to elevated serum b-hCG levels and the tumor expressed b-hCG by immunohistochemistry. To our knowledge, the expression of b-hCG in metaplastic breast Ca has not been reported.

Design: To ascertain the expression of b-hCG in metaplastic breast Ca patients and determine its clinical utility in the management of such patients. Eighteen patients with the diagnosis of metaplastic breast Ca were studied. All cases had features of either spindle cells, osteoclast-like giant cells, squamous or cartilaginous differentiation. Deparaffinized tissue sections were stained with DAKO (Carpinteria, Calif) N-Series b-hCG antibody, rabbit anti-human anti–Ki-67 proteins, p53 antigen, and cocktail cytokeratin. Two pathologists analyzed the stains manually.

Results:

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Conclusion(s): In this study, 33% of metaplastic breast Ca expressed b-hCG by immunohistochemistry. Elevated serum b-hCG levels were noted in one patient prior to surgery (21 ng/dL) which normalized (0.6 ng/dL) after excision and increased again with recurrence of malignancy (48 ng/dL). These features of metaplastic breast Ca may be helpful in the management and follow-up of such patients.

Identification of Cyclin-D1 Amplification in Cases of Atypical Ductal Hyperplasia Diagnosed on Breast Mammotome Core Biopsy: Correlation With Immunohistochemical Expression of Cyclin-D1 and Subsequent Excisional Biopsy Diagnosis

Omar Hameed, MD ([email protected]). Department of Pathology, Washington University School, St Louis, Mo.

Context: A diagnosis of atypical ductal hyperplasia (ADH) on breast mammotome core biopsy (CBX) is often followed by a diagnosis of ductal carcinoma in situ (DCIS) on subsequent excisional biopsy (EBX). This study was performed to determine whether cyclin-D1 (CyD1) gene amplification is associated with such an outcome, and whether it correlates with the immunohistochemical (IHC) expression of CyD1.

Design: Fluorescent in situ hybridization (FISH) using the LSI Cyclin-D1 (11q13/CeP11) Dual Color Probe (Vysis, Downers Grove, Ill) was performed blindly on 11 cases of ADH diagnosed by CBX. Cells with an orange (11q13, CyD1 region) to green (CEP11, 11p11.11-q11 control) signal ratio of 2 or more were considered to have CyD1 amplification. The proportion of cells in the duct space(s) involved by ADH showing amplification was correlated with the percentage of cells positive for CyD1 by immunohistochemistry (CyD1 index, calculated in a previous study), and subsequent EBX diagnoses.

Results:

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Correlation coefficient between CyD1 amplification status and CyD1 index = 0.9; P < .001

Conclusion(s): CyD1 amplification status strongly correlates with the immunohistochemical expression of CyD1; CyD1 amplification in less than 2 cells of ADH diagnosed on CBX is unlikely to be associated with a subsequent diagnosis of DCIS on EBX.

E-Cadherin and β-Catenin Expression Changes in Pancreatic Intraepithelial Neoplasia

Maamoun M. Al-Aynati, MD, FRCPC, FCAP ([email protected])1; Robert H. Riddell, MD, FRCPC2; Ming-Sound Tsao, MD, FRCPC.21Department of Pathology, University of Toronto, Mississauga, Ontario, Canada; 2Department of Pathology, University of Toronto, Toronto, Ontario, Canada.

Context: Cadherins and associated catenins are important mediators of epithelial cell-cell adhesion and signal transduction, and significant changes in their expression or structure have been implicated in aggressive behavior of tumors. Glycogen synthase kinase-3 β, involved in the cellular responses to Wnt-Wingless signaling pathway activation, is thought to down-regulate intracellular β-catenin levels, ultimately resulting in the activation of genes regulated by the T-cell factor/lymphoid enhancer factor family of architectural transcription factors. Pancreatic intraepithelial neoplasia (PanIN) is the newly used terminology for progressive pancreatic duct epithelial dysplasia. Genetic alterations related to cell-cell adhesion signaling, including E-cadherin and its downstream-related catenins, have not been investigated in these pancreatic ductal cancer precursor lesions.

Design: Ninety-four Whipple resection specimens were retrieved from the surgical pathology files of the Toronto General Hospital and Princess Margaret Hospital; tissue microarray blocks containing 36 pancreatic adenocarcinomas, 34 PanIN-1A lesions, 28 PanIN-1B lesions, 27 PanIN-2 lesions, 16 PanIN-3 lesions, and 32 normal ducts were constructed from the specimens. The E-cadherin–β-catenin and the glycogen synthase kinase-3 β part of the Wnt-Wingless/β-catenin pathways were immunohistochemically evaluated in the pancreatic lesions of interest.

Results: There was marked increase in cytoplasmic E-cadherin expression in PanIN lesions compared to normal pancreatic ducts (P < .001) and adenocarcinoma (P = .005). In pancreatic adenocarcinoma, there was a significantly reduced/absent E-cadherin membranous expression compared to PanIN lesions (P < .001) but not to normal ducts (P = .05). There was a significantly abnormal nuclear localization of β-catenin in higher grade PanIN lesions (PanIN2 in particular) and in adenocarcinoma compared to normal ducts and low-grade PanIN lesions (P < .001). No correlation was found between glycogen synthase kinase-3 β cytoplasmic expression and β-catenin aberrant nuclear expression (P = .07).

Conclusion(s): This is the first study to evaluate the expression of E-cadherin and β-catenin in PanIN lesions. Abnormal expression of E-cadherin and its associated β-catenin shows a stepwise progression in pancreatic carcinogenesis. The role of the Wnt-Wingless signaling pathway in β-catenin stabilization is probable but needs further investigation.

Expression of G1-S Cyclins in Multi-Stage Pancreatic Duct Carcinogenesis in Humans

Maamoun M. Al-Aynati, MD, FRCPC, FCAP ([email protected])1; Robert H. Riddell, MD, FRCPC2; Ming-Sound Tsao, MD, FRCPC.21Department of Pathology, University of Toronto, Mississauga, Ontario, Canada; 2Department of Pathology, University of Toronto, Toronto, Ontario, Canada.

Context: Several cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors have been shown to play important regulatory roles during cell cycle progression. Disruption of the G1-S checkpoint leads to uncontrolled cell growth, resulting in the development of cancers. Pancreatic intraepithelial neoplasia (PanIN) is the newly used terminology for progressive pancreatic duct epithelial dysplasia. The expression of the cyclins active in the G1-S phase of the division cycle in pancreas cancer and its premalignant intraductal lesions is far from established.

Design: Ninety-four Whipple resection specimens were retrieved from the surgical pathology files of the Toronto General Hospital and Princess Margaret Hospital; tissue microarray blocks containing 36 pancreatic adenocarcinomas, 34 PanIN-1A lesions, 28 PanIN-1B lesions, 27 PanIN-2 lesions, 16 PanIN-3 lesions, and 32 normal ducts were constructed from the specimens. We immunohistochemically analyzed the expression of the cell cycle–related proteins cyclin D1, cyclin D3, cyclin E, and cyclin A in the lesions of interest.

Results: None of the cyclins showed overexpression in normal ducts. Cyclin D3 protein expression occurred in PanIN-1A and increased significantly with progression of pancreatic carcinogenesis. It correlated strongly with cumulative cyclin expression and weakly with cyclins E and A. Cyclin E and A overexpression occured only in PanIN-2 lesions increasing progressively with a peak in adenocarcinoma. Cyclin D1 did not show significant differential expression between normal ducts, PanIN lesions and adenocarcinoma. All cyclins were correlated with each other with the exception of cyclin D1.

Conclusion(s): Cyclin D3 protein overexpression occurs early and cyclin E and A overexpression occurs at an intermediate stage in pancreatic intraepithelial neoplasia, suggesting a role in pancreatic carcinogenesis. Cyclin D3 overexpression correlates strongly with cumulative cyclins alterations and may represent an important molecular marker for subsequent invasive potential.

The Pathologist's Perspective on Webcast Medical Education

Deepak Mohan, MD ([email protected]); Ishtiaque Ahmed, BS; Michael J. Becich, MD, PhD; Yukako Yagi, BS. Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pa.

Context: We have more than 30 residents, more than 15 fellows, and more than 120 faculty members spread across 12 hospitals. As our hospital system has grown and encompasses several hospitals distributed over a great distance, there was a need for an asynchronous delivery system for continuing medical education conferences. Residents and pathologists were not able to attend the time-, location-, and space-constrained conferences. We explored the possibility of delivering content via webcast with the hope of maintaining the educational value of the original on-site course.

Design: “Lectures on-demand” methodology was achieved using multimedia-streaming technologies. These technologies deliver real-time and asynchronous content via the Internet with a hybrid synchronous-asynchronous Learning Network (S-ALN) model. Currently the lectures are stored behind a firewall and are available only to University of Pittsburgh Medical Center physicians in the UPMC Network.

Results: Media Server Usage Statistics—one day: 108 hits; Distinct User Requests: 21.

Conclusion(s): The University of Pittsburgh approach to technology-enhanced continuing medical education, delivery of webcasts of conferences and didactics, represents a revolution in the way pathologists learn. This channel of teaching represents a new horizon to working pathologists, offering them a chance to enhance their knowledge and accrue CME credit. This system has tremendous utility for resident pathologists, who can access these lectures to create ad-hoc board examination review courses or primers for an upcoming anatomic pathology or clinical pathology rotation. This system is optimal for the hybrid synchronous and asynchronous learning environment of pathology training programs. The content can be adapted into several convenient forms, such as DVD/CD-ROM or WWW-based modules coupled with virtual slides. These technologies have certainly overcome geographical, logistical, and time constraints associated with the current learning paradigm.

Enhanced Susceptibility of PHOX−/−/NOS2−/− Knockout Mice to Systemic Infection by Candida albicans

Suaad H Traif, MD ([email protected])1; Thomas F. Warner, MD1; Edward Balish, PhD.21Department of Pathology, University of Wisconsin, Madison; 2Department of Pathology, Medical University of South Carolina, Charleston.

Context: Macrophages and neutrophils kill ingested Candida albicans by several mechanisms, including superoxide anion and reactive nitrogen intermediates. Knockout of NADPH oxidase (GP 91 PHOX) renders mice susceptible to intraperitoneal infection by C albicans. Macrophages from mice with induced nitric oxide (NOS2) synthase deficiency are unable to kill C albicans.

Design: Germfree GP 91 PHOX−/−/NOS2−/−, C57BL6, and Balb/c control mice were colonized orally with C albicans at 8 weeks of age. Mice sacrificed after 10 days colonization were autopsied. Intestinal contents and tissue homogenates were cultured on Sabouraud's medium. Gastric mucosal infection was graded from I–IV.

Results: GP 91 PHOX−/−/NOS2−/− mice were moribund within 2 weeks of colonization. Lungs, liver, spleen, mesenteric lymph nodes, and Peyer patches contained multiple abscesses. Gastric mucosal invasion by C albicans was grade II–III; esophageal invasion was absent. Rare blastoconidia and hyphae were found in abscesses. C albicans was not cultured from liver or spleen. Immunocompetent control mice were free of abscesses and disseminated candidiasis.

Conclusion(s): Severe endogenous systemic infection with abscess formation is rare in gnotobiotic mice after oral colonization by C albicans. We have not encountered it in beige, nude, beige-nude, Tge 26 mice, NO-deficient, SCID, 1L–2,−/− IL10,−/− IL8R,−/− TCR|Á,−/− and INF|Ã−/− germ-free mice orally infected with C albicans. Mesenteric lymph nodes are infected via Peyer patches. Peroxynitrite production from NO and SO2•− by phagocytes is essential for defense against transmucosal invasion by C albicans in mice.

Coronary Artery Fibrointimal Hyperplasia Is Related to Mean Acute Rejection Score in the Cardiac Allograft; Coronary Artery Atherosclerosis and Systemic Malignancy Are Not

Russell T. Alexander, MD ([email protected]); Charles Steenbergen, MD, PhD. Department of Pathology, Duke University Medical Center, Durham, NC.

Context: We present the relation between mean acute rejection score and the development of systemic malignancy and autopsy-determined coronary artery disease after heart transplant.

Design: This study included heart transplant patients who had an autopsy and at least one antemortem endomyocardial biopsy to evaluate for rejection. Histologic examination was used to divide coronary artery disease into the typical atherosclerosis seen in the general population and the fibrointimal hyperplasia characteristic of graft vascular disease. The occurrence of malignancy after transplant was determined by a review of the patient's records and autopsy findings. A mean rejection score for each patient was calculated as the average of all the rejection episodes determined by endomyocardial biopsy. The International Society of Heart and Lung Transplantation grading scheme of 0 to 4 was used with the A or B designation dropped for this analysis. Mean rejection scores were calculated for all patients and for those surviving greater than 1 year, because fibrointimal hyperplasia and malignancy did not develop until after 1 year. Mean rejection scores were compared with a 2-tailed t test. P values less than .05 were considered significant.

Results: The 28 patients in this study had a total of 560 endomyocardial biopsies. The average number of biopsies per patient was 20. The mean rejection scores for each of the different study populations are shown in the table.

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Conclusion(s): Coronary artery fibrointimal hyperplasia was significantly related to mean rejection score when examined in all patients (P = .005) and in patients surviving greater than 1 year (P = .03). Neither coronary artery atherosclerosis nor systemic malignancy was significantly related to mean rejection score.

Anion Gap Acidosis in Postmenopausal Women Is Reversed by Estrogen Treatment

Deepak Mohan MD ([email protected]); Charles Williams, BS; Mohamed A. Virji, MD, PhD; Giuliana Trucco, MD; Harry C. Blair, MD. Department of Pathology, VA Healthcare System, University of Pittsburgh, Pittsburgh, Pa.

Context: One of the well-known changes seen during menopause is an increase in the rate of bone resorption associated with low estrogen levels. Up to one third of women older than age 50 experience a vertebral crush fracture, and more than 250 000 osteoporosis-related hip fractures occur annually. Bone loss is driven by hypocalcemia, acidosis, and changes in calciuric hormones. Postmenopausal women have normal calcium activity, and it is unclear why they should lose bone mass, which does not respond well to calcium and vitamin D supplementation but does respond to estradiol replacement. One of the unexplained associations between osteoporosis and menopause is that the anion gap increases about 30% with menopause, suggesting that acids, such as lactate, sulfate, or phosphate, are retained in postmenopausal women, which may in part cause bone loss. We studied whether this change was reversed by estrogen, which also reduces bone loss.

Design: We studied 40 samples of postmenopausal women 53 to 58 years of age with and without estrogen replacement, measuring follicle-stimulating hormone by enzyme-linked immunosorbent assay to determine estrogen status. Serum keto acids, inorganic acids, and organic acids were estimated from b-hydroxybutyrate, phosphate, and lactate concentrations.

Results: There was no difference between keto acids in the estrogen replacement patients (FSH <25 mIu/mL) and unreplaced patients. There were differences in both lactate and phosphate, with lactate in estrogen-replaced patients declining 15% from 2.76 to 2.33 mg/dL, while phosphate was reduced 5% from 3.9 to 3.7 mg/dL. The change in lactate was significant (P = .04) while that in phosphate was marginal (P = .07).

Conclusion(s): The results suggest that both inorganic and organic acids are retained in estrogen deficiency. Keto acids are unlikely to be related to changes with menopause. Retained organic and inorganic acids may contribute significantly to bone loss in postmenopausal women, and this abnormality is reversed in the age-matched population by administration of estrogen.

Comparison of Serum Quantitative Free Light Chains (Freelite) to Serum Quantitative Total Light Chains in Recurrent Multiple Myeloma

Brent Staggs, MD ([email protected]); Alex Pappas, MD. Department of Pathology, University of Arkansas for Medical Sciences, Little Rock.

Context: The Freelite (The Binding Site, Inc, San Diego, Calif) immunoassay quantitatively measures excess serum-free light chains in multiple myeloma. Quantitative serum-free light chains (LCs) may better detect recurrent multiple myeloma than serum total LCs.

Design: Sera and bone marrow biopsies were obtained at follow-up from 90 patients previously diagnosed with multiple myeloma, including involvement on bone marrow biopsy, and after treatment were found to have no evidence of disease. Morphologic criteria was considered to place the current bone marrow examination in one of the following 3 categories: 30 negative, 30 positive, and 30 inconclusive for recurrent MM. Simultaneously, serum from each patient was examined for free LCs (reference range: κ, <1.94 mg/dL; λ, <2.63 mg/dL) and total LCs (reference range: κ, <490 mg/dL; λ, <230 mg/dL).

Results: Ten of 30 negative patients by morphology had elevated free LCs (κ, range (R) = 0.07–7.62 mg/dL; λ, R = 0.36–9.96 mg/dL) and all had normal total LCs. Seventeen of the 30 inconclusive patients had elevated free LCs (κ, R = 0.22–127 mg/dL; λ, R = 0.29–23.2 mg/dL) and 12 had elevated total LCs (κ, R = 50–1030 mg/dL; λ, R = 19–400 mg/dL). Twenty-one of 30 positive patients had elevated free LCs (κ, R = 0.01–1390 mg/dL; λ, R = 0.04–257 mg/dL) and 11 had elevated total LCs (κ, R = 1.0–1130 mg/dL; λ, R = 19–1020 mg/dL). The clinical sensitivity and specificity for free LCs were 70% and 67%, respectively, and for total LCs, 36% and 100%, respectively.

Conclusion(s): In this preliminary study, serum-free LCs appear more sensitive but less specific than serum total LCs for recurrent multiple myeloma based on morphologic criteria alone and may be useful in detecting recurrence.

Aberrant Expression of T-Catenin During Colorectal Tumorigenesis: Lack of Nuclear Localization

Mingfu Yu, MD ([email protected]); Jon R. Ritter, MD; Hanlin L. Wang, MD. Department of Anatomic Pathology, Washington University, St Louis, Mo.

Context: Membranous protein γ-catenin, a β-catenin homologue, has been recently reported to be regulated by the adenomatous polyposis coli (APC) tumor suppressor. Like β-catenin, the oncogenic activity of γ-catenin demonstrated in cultured cells is thought to involve Tcf function to upregulate the expression of c-myc proto-oncogene. Whether γ-catenin is indeed dysregulated and its relationship with c-Myc protein expression during human colorectal tumorigenesis have not been studied.

Design: Twenty-four cases of sporadic colorectal adenocarcinoma and 27 cases of sporadic colorectal adenoma were examined immunohistochemically for γ-catenin expression. The staining pattern (membranous, cytoplasmic, or nuclear) and intensity were compared with adjacent nonneoplastic colonic epithelium. Immunostains for c-Myc and the carboxyl terminus of the APC were also performed for correlation.

Results: A membranous staining pattern for γ-catenin was universally present in nonneoplastic colonic epithelium. However, a significant loss of the immunoreactivity with a patchy preservation of the membranous positivity was seen in all adenomas. A predominantly cytoplasmic staining was observed in all adenocarcinomas. Compared to nonneoplastic colonic epithelium, the staining intensity for γ-catenin was increased in 18 cases (75%) of adenocarcinomas. In the remaining 6 cases, the expression levels of γ-catenin in tumor cells were either comparable or decreased when compared to nonneoplastic epithelium. No nuclear staining for γ-catenin was noted in adenomas or adenocarcinomas. Fifteen of the 18 adenocarcinomas (83%) with elevated γ-catenin protein levels stained negative for the carboxyl terminus of the APC, presumably secondary to truncation mutations of the APC gene. Among them, only 7 cases (38%) exhibited elevated levels of c-Myc expression when compared to nonneoplastic colonic epithelium.

Conclusion(s): There is a lack of nuclear localization of γ-catenin and a lack of correlation with c-Myc expression during colorectal tumorigenesis. These data thus do not appear to support the notion that γ-catenin functions as a transcription factor for c-Myc upregulation. The observed changes in the staining pattern and intensity, as well as a frequent association with APC mutations, suggest that dysregulation of γ-catenin may serve an important role during colorectal tumorigenesis.

Comparison of Laboratory Costs for Medical Diagnoses in Academic and Nonacademic Hospitals for Patients in the Same Severity-Adjusted Medical Diagnosis–Related Groups

Brad B. Brimhall, MD, MPH ([email protected]); Ronald B. Lepoff, MD. Department of Pathology, University of Colorado, Denver.

Context: Studies evaluating laboratory costs have been limited to academic facilities. These studies did not adjust patient groups for comorbid conditions or complications and reviewed only total costs. A quantitative determination of laboratory cost differences would be of great use in medical reimbursement policy making.

Design: Discharge abstracts from 7 academic (N = 426 509) and 10 large nonacademic (N = 279 927) hospitals were extracted from hospital case mix data sets (1999–2000) obtained from the Massachusetts Division of Health Care Finance and Policy. In-hospital deaths, interhospital transfers, children (<18 years of age), and records with missing cost data (<1%) were excluded. Costs were compared for the 20 most common medical diagnosis-related group (DRG) clusters. Laboratory charges were converted to direct laboratory costs (LDC) using the ratio of direct laboratory cost to charges and adjusted for inflation. All DRGs were severity-adjusted using the refined-DRG (RDRG) method. Median LDC differences between hospital groups was compared. Statistical significance was determined using the Mann-Whitney U test.

Results: At the DRG level, median LDC was statistically higher at academic hospitals in all but 1 of 20 DRG comparisons (430; organic disturbances and mental retardation). Academic LDC ranged from $5 to $180 (4–46%) higher. After severity adjustment, academic LDC was $3 to $279 (3%–54%) higher in 57 of 60 comparisons. More than 95% of comparisons were statistically significant (P < .001). If patients at nonacademic hospitals were treated at academic hospitals, the LDC for these diagnoses would be $5 095 878 higher.

Conclusion(s): LDC for common medical conditions is significantly higher at academic hospitals, even after adjustment for patient comorbid conditions and complications.

Hospital Costs and Length of Stay for Patients of Different Socioeconomic Groups

Katherine G. McLucas, MD ([email protected]); Bradley B. Brimhall, MD, MPH. Department of Pathology, University of Colorado, Denver.

Context: Previous studies have suggested that patients from lower socioeconomic groups have greater hospital costs. Data describing differences in resource utilization from different socioeconomic groups would be useful for health care policy makers.

Design: Discharge abstracts from 13 academic and 10 community hospitals (N = 918 859) were extracted from hospital case mix data sets (1999–2000) obtained from the Massachusetts Division of Health Care Finance and Policy. Exclusionary criteria were in-hospital deaths, patients younger than 18 years of age, interhospital transfers, and records with missing case data (<1%). Four surgical and 4 medical severity-adjusted DRG (diagnosis-related group) clusters involving different organ systems were used for comparison. Laboratory direct cost (LDC), total direct cost (TDC), and hospital length of stay (LOS) were compared between two groups: patients with median household income <$55 000/year (“poor”) and ≥55 000/year (“rich”). Median household income based on patient zip codes was derived from U.S. Census Bureau statistics for the year 2000.

Results: Within each DRG, LOS was higher for “poor” patients than for “rich” patients (percent difference, 0.9–16.4; mean, 0.346 days longer). The difference was statistically significant (P < .05) for all groups except DRG 209 (P = .19). TDC was similar for both groups. LDC and LDC/day were higher in the “poor” group as compared to the “rich” group.

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Conclusion(s): Hospital LOS was longer in patients coming from poor areas as compared to patients coming from rich areas. LDC and LDC per hospital day tended to be higher among patients from poor areas.

Potential Role of β-Catenin in the Differential Diagnosis of Follicular Adenomas, Follicular Carcinomas

Sherif A. Rezk, MD ([email protected])1; Viera Nelson, MD2; Russell K. Brynes, MD2; Ashraf Khan, MD1; Maung Thein, MD.21Department of Pathology, University of Massachusetts Medical Center, Worcester, Mass; 2Department of Pathology, USC Medical Center, Los Angeles, Calif.

Context: The morphologic distinction of thyroid follicular lesions including follicular adenoma (FA), minimally invasive encapsulated follicular carcinoma (FC), and follicular variant of papillary carcinoma (FVPC) can sometimes be challenging; therefore, an immunohistochemical marker which may aid in making this distinction would be useful. β-Catenin is one such potential marker. It is part of a cytoplasmic membrane-bound cell growth–signaling complex that plays a role in cell adhesions, as well as in promotion of neoplastic growth through activation of the Wnt signaling pathway. Oncogenic signaling occurs when β-catenin is released, accumulates in the cytoplasm, is translocated into the nucleus, and promotes transcription of genes including bcl-1 and c-myc that induce cell proliferation.

Design: Twenty-four FAs, 25 FVPCs, 10 FCs, and 25 papillary thyroid carcinomas (PTCs) were obtained from the surgical pathology files of UMASS Medical Center and USC Medical Center. A monoclonal antibody reactive with β-catenin (C19220, 1:100; BD Transduction Lab, San Diego, Calif) was used. Normal thyroid tissue was used as a control group.

Results: Eight of 10 (80%) FCs showed strong cytoplasmic staining and minimal membranous staining. One case was negative and the other had stronger membranous staining than cytoplasmic. Nineteen of 25 (76%) FVPCs expressed strong cytoplasmic staining, while the other 6 cases had minimal cytoplasmic expression. Nineteen of 24 (79%) FAs showed strong membranous staining with minimal cytoplasmic staining. Two cases showed strong cytoplasmic staining and 3 cases were negative. Twenty-one of 25 PTCs (84%) showed strong cytoplasmic reactivity. Interestingly, in 22 PTCs (88%) and 5 FVPCs (20%), the intranuclear inclusions were positive. The intensity and number of positive inclusions varied from one case to the other without a relation to the staining intensity of the rest of the cell. The normal thyroid control group showed membranous staining with complete absence of any cytoplasmic reactivity.

Conclusion(s): β-Catenin can be of diagnostic utility for thyroid lesions because it shifts from a membranous localization (normal and benign) to a cytoplasmic/nuclear localization in follicular, as well as in papillary thyroid carcinomas. It also highlights the intranuclear inclusions in PTC and FVPC. We speculate that the localization of β-catenin in intranuclear inclusions may reflect a cytoskeletal remodeling activity of β-catenin that is functionally significant for both PTC and FVPC pathways.

Do Cytogenetic Changes Precede Morphologic Changes in Developing Malignant Conditions?

Swarupa A Gadre, MD ([email protected]); Yemin Ge, MD; Gopalrao V. N. Velagaleti, PhD; Jill K. Northup, CLSp(CG). Department of Pathology, University of Texas Medical Branch, Galveston.

Context: Cytogenetic evaluation of bone marrow and neoplastic tissues plays a critical role in determining patient management and prognosis. Here we highlight 2 cases in which the cytogenetic studies challenge the common practice of using hematologic and morphologic changes as key factors in malignant disease management.

Design: The first case is that of a lymph node sample from a 40-year-old non-Hodgkin lymphoma patient sent for determination of disease progress. The second case is of a patient with a history of human immunodeficiency virus and blastic non-killer leukemia/lymphoma.

Results: In the first case, hematologic studies showed no evidence of tranformation to high grade (less than 15% blasts with rare mitotic figures). Cytogenetic studies from lymph node showed multiple clonal abnormalities, most notably a der(18) from a t(14;18) which is associated with high-grade NHL. After 2 cycles of chemotherapy with fludarabine, the patient has not shown any clinical response suggesting possible progression to high-grade lymphoma. In the second case, hematologic studies of ascitic fluid classified the patient as having primary effusion lymphoma while bone marrow analysis showed no malignancy. Bone marrow cytogenetic studies showed multiple clonal abnormalities including a t(8;14), which is commonly associated with Burkitt lymphoma. To our knowledge, this is the first case wherein a morphologically normal bone marrow showed presence of clonal abnormalities consistent with Burkitt or primary effusion lymphoma. After 2 cycles of CHOP chemotherapy, the patient's general condition and ascitis improved, and she was discharged.

Conclusion(s): These studies clearly demonstrate that genetic changes often precede morphologic changes in a developing malignant condition. Therefore, the critical information needed for patient care of malignant disorders may be incomplete or inaccurate if cytogenetic evaluation is overlooked.

Identification of Crystalloids of Reinke by Intraoperative Cytology

Kambridge P. Hribar, BS ([email protected]); Andy E. Sherrod, MD; Nancy E. Warner, MD. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles.

Context: Leydig cell tumors are the most common of the sex-cord tumors in males. Although not required for diagnosis, crystalloids of Reinke are pathognomonic for Leydig cell tumor. However, intraoperative examination by conventional frozen section rarely reveals their presence.

Design: Upon receiving the fresh gross specimen, the tumor was bivalved. A scalpel was used to scrape the surface and smear the product on a glass slide. Imprints of the freshly cut tissue also were taken by directly applying a glass slide to the cut surface of the tissue. The slides were placed immediately in 95% alcohol for 30 to 60 seconds. Finally, the slides were stained with hematoxylin and eosin for microscopic evaluation.

Results: We present 2 cases in which intraoperative cytology yielded abundant crystalloids of Reinke, permitting a rapid, definitive diagnosis of Leydig cell tumor. The crystalloids have the usual rectangular- or rhomboid-shaped hexagonal appearance. They are most numerous lying between the cells in the scrape preparations, in contrast to the sparse number visible in the cytoplasm in the H&E sections.

Conclusion(s): It appears that disruption of the membranes of the Leydig cell caused by the scraping, and to a lesser extent the process of imprinting, disperses the crystalloids to an extracellular location, thus aiding their identification. The useful role of crystalloids in diagnosing Leydig cell tumor justifies a need for a more efficient and effective method of identifying them. These 2 cases demonstrate that intraoperative cytology is a superior technique for this purpose.

Introduction of Case-Based, Virtual Slide Microscopy Into Medical School Curriculum—A Preliminary Assessment

Deepak Mohan, MD ([email protected]); Michael Becich, MD, PhD; Rebecca Crowley, MD, MS; Katsura Fujita, MS. Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pa.

Context: Medical schools have significantly reduced the amount of time for learning microscopic histology and pathology. In most curricula, static digitized microscopic images have entirely replaced glass slide microscopy. This practice limits the opportunity for students to learn important skills in locating and interpreting microscopic abnormalities. To bridge this gap, we have introduced the use of whole slide virtual microscopy into the first- and second-year medical curriculum, using a Web-based virtual slide system of our own design. We present early results of student evaluation of the virtual slide system.

Design: Participants included ∼120 MS-I students already enrolled in the cancer biology course. Using a standard, Java-enabled Web browser, students viewed virtual slide cases composed of clinical history, gross photographs, virtual slides, and annotations, and then worked through case-based questions and answers on 5 cases (http://virtualslide.upmc.edu). Virtual slides allowed students to pan in any direction and change from low- (0.5×) to high-power (∼35×) magnification. Rich text-to-image annotations provided guidance to students who needed help finding the important histopathologic features. Students had control over the level of this guidance. Following the session, all students were asked to complete a 55-question standardized survey that included components for evaluating student response to this technology, as well as a standard set of items designed to rank students by level of computer skill and level of knowledge about computers and technology. Seventy-seven surveys were returned.

Results: Students rated the virtual microscopy laboratory highly on several parameters including resolution of the virtual slide images, ability to find the pathologic features with ease, and ease of navigating the slide in the interface. One hundred percent of the respondents, or 65% of all the students, agreed either moderately or strongly that the virtual slide system should also be used in other pathology courses in the curriculum. Favorable responses did not correlate with aggregate, standardized measures of computer experience or knowledge.

Conclusion(s): Virtual slide technology was well accepted by students across a wide range of experience and knowledge about computers and technology. Further work is needed to determine optimum methods for instruction using virtual slide microscopy.

Localization of p16INK4A Staining in Cervical Intraepithelial and Invasive Neoplasms

Amy C. Baruch, MS, MD ([email protected]); John R. Davis, MD; Thomas M. Grogan, MD. Department of Pathology, Arizona Health Sciences Center, Tucson.

Context: p16INK4A is a cyclin-dependent kinase inhibitor that may be overexpressed in cervical intraepithelial neoplasia (CIN) and carcinoma due to inactivation of the tumor suppressor gene pRB by the human papillomavirus oncogene E7. Detection of p16INK4A in cervical specimens may increase sensitivity and decrease inter-observer variability in grading cervical dysplasia.

Design: Forty-six cases of archived formalin-fixed, paraffin-embedded cervical biopsies were stained using a mouse monoclonal anti-p16INK4A (clone 6H12, Novocastra Laboratories Ltd, Newcastle upon Tyne, United Kingdom), then reviewed by 2 independent observers. The cervical biopsies included 5 normals, 16 cases of CIN1, 7 cases of CIN2, 9 cases of CIN3, and 9 cases of invasive squamous cell carcinoma. p16INK4A staining was scored based on intensity and diffuseness of staining, as well as localization within the squamous epithelial membrane.

Results: While p16INK4A uniformly stained all squamous epithelial cells in CIN3 lesions and invasive carcinomas, staining was focal or absent in CIN1 and CIN2 lesions, and limited to the lower third of the epithelial membrane when present. Koilocytes with obvious viral effect, present in the more superficial epithelium, failed to stain in all 16 cases of CIN1 and in 5 of 7 cases of CIN2. No reactivity was observed in normal controls, but reactivity was occasionally present in reserve cell hyperplasia.

Conclusion(s): The inconsistent staining observed in CIN1 and CIN2 lesions with this anti-p16INK4A clone limits its utility in detecting HPV-associated lesions. Similarly, the lack of staining of koilocytes in the superficial cervical epithelium in CIN1 and CIN2 cases precludes an effective application in pap cytologic specimens.

Smear Preparation: An Accurate and Fast Tool for the Diagnosis of Ependymomas During Frozen Section

Aly Gh Saad, MD ([email protected])1; Gabrielle DeCourten-Myers, MD1; Hassan W. Nakhla, MD2; Robert Shelper, MD.21Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio; 2Department of Pathology, Upstate Medical University, Syracuse, NY.

Context: The cytologic evaluation of smear preparation (SP) of central nervous system tumors has become a routine supplement to frozen section diagnosis. This reliable and rapid method has greatly increased intraoperative diagnostic accuracy, even sometimes replacing the traditional frozen section. It also has the advantage of using only a small amount of tissue, an essential requirement when dealing with stereotactic brain biopsies. Although it is a widely accepted and performed technique, SPs have rarely been used to determine cytologic features of ependymomas.

Design: Twenty-two SPs from central nervous system ependymomas were studied for cytologic and architectural features. One smear per case was obtained. They included 19 cases of grade II ependymomas and 3 cases of myxopapillary ependymomas.

Results: The cellularity of each SP was variable. A moderately cellular SP showed clusters of tightly packed polygonal cells with abundant ill-defined eosinophillic cytoplasm reminiscent of ependymal cells. They typically displayed ovoid dark staining nuclei with small nucleoli and fine chromatin. Two architectural features were characteristically present: true rosettes and perivascular rosettes. In myxopapillary ependymomas, these rosettes were frequently separated by abundant extracellular mucin-like material, which sometimes separated the tumor cells from their attachment to the blood vessels, producing balls of tumor cells surrounding collections of mucin. Focal fibrillary areas were present in all cases.

Conclusion(s): Ependymomas are particularly suitable for SP diagnosis due to a high prevalence of diagnostic histologic features being present in cohesive cell clusters. Thus, SP permits accurate and rapid diagnosis of ependymomas especially when combined with clinical and radiologic findings.

Age-Related Inpatient Laboratory Costs for Patients in the Most Common Severity-Adjusted Medical and Surgical Diagnostic Groups

Bradley B. Brimhall, MD, MPH ([email protected]); William Reiquam, MD. Department of Pathology, University of Colorado School of Medicine, Denver.

Context: Differences in laboratory costs between older and younger hospitalized patients have been evaluated for a small number of medical diagnosis related group (DRG) clusters. Age-related cost differences have not been evaluated for surgical DRGs.

Design: Discharge abstracts from 7 academic (N = 426 509) hospitals were extracted from hospital case mix data sets (1999–2000) obtained from the Massachusetts Division of Health Care Finance and Policy. In-hospital deaths, inter-hospital transfers, and records with missing cost data (<1%) were excluded. Laboratory charges were converted to direct laboratory costs (LDC) using the ratio of direct laboratory cost to charges and adjusted for inflation. All DRGs were severity-adjusted using the refined-DRG (RDRG) method. Costs were compared for the 10 most common surgical DRGs (40 RDRGs) and the 10 most common medical DRGs (30 RDRGs). Median LDC was compared for older (≥65 years old) and younger (18–64 years old) patients. Statistical significance was determined using the Mann-Whitney U test.

Results: For medical diagnoses, the older patient group had a lower LDC in 24 of 30 (80%) RDRG comparisons; they also had a lower LDC per day in 25 of 30 (83%) of comparisons. For surgical diagnoses, the older patient group had a lower LDC in only 11 of 40 (28%) and a lower per diem LDC in 16 of 40 (40%) severity-adjusted comparisons. Almost all comparisons of LDC per day were statistically significant (P < .05).

Conclusion(s): At academic hospitals, older patients tend to have lower laboratory costs for medical diagnostic groups. Laboratory costs for older surgical patients, however, are higher than that of younger patients for most diagnostic groups.

Brain Natriuretic Peptide: Impact of Age, Gender, Smoking Status, and β-Blocker Use

Vassil Kaimaktchiev, MD ([email protected]); Steven C. Kazmierczak, PhD; Veselina Korcheva, MD. Department of Pathology, Oregon Health and Science University, Portland.

Context: Brain natriuretic peptide (BNP) is an accurate indicator of left ventricular dysfunction. Information regarding the effect of age, gender, smoking status, and therapy with β-blockers on BNP concentrations is insufficient or lacking. We investigated the effect of these variables on BNP concentrations in patients presenting with dyspnea subsequently diagnosed with congestive heart failure (CHF).

Design: We evaluated BNP in patients presenting with suspected heart failure. Those patients with a diagnosis of myocardial infarction and no CHF and renal insufficiency, and patients with pulmonary etiology of dyspnea were excluded. BNP was measured in whole blood (Biosite Diagnostics, San Diego, Calif). Clinical data were reviewed and summarized. An investigator, blinded to each patient's BNP concentration, determined which patients met the inclusion criteria.

Results: Of 70 patients, 44 (24 females and 20 males; age range, 39–94 years) met the inclusion criteria. Regression analysis of age (x) versus BNP concentration (y) revealed: BNP (pg/ml) = 2.20 (age)(pg/ml) + 293 pg/ml. Effect of age, gender, smoking status, and β-blocker use on BNP is shown below.

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Conclusion(s): Age has previously been shown to be a significant factor affecting interpretation of BNP. We also found age to significantly affect interpretation of BNP. In addition, use of β-blockers may impact BNP interpretation. Gender and smoking status may also be important variables affecting BNP concentration, although our study did not find any significant effect. Further studies are warranted to evaluate the effect of these factors.

Lymphoepithelial-Like Ca and Undifferentiated Nasopharyngeal Ca Are Immunophenotypically Similar

Xiaohong Zhang, MD ([email protected]); Carmen Gomez-Fernandez, MD; Francisco Civantos, MD; Mehrdad Nadji, MD. Department of Pathology, University of Miami, Jackson Memorial Medical Center, Miami, Fla.

Context: Lymphoepithelial-like carcinomas (LELCs) are a group of epithelial neoplasms that arise from a number of different tissues and organs, and closely resemble undifferentiated nasopharyngeal carcinomas (UNCs). We evaluated LELCs for the expression of HLA-DR and Bcl-2, and compared the staining results with that of morphologically similar non-LELCs of the same organs.

Design: Fourteen cases of LELC (urinary bladder, lung, skin, parotid, stomach, and uterine cervix) as well as 10 examples of non-LELC tumors from the same organs were included in this study. Seventeen cases of UNC and 13 examples of squamous cell carcinomas of the head and neck were similarly evaluated. Immunohistochemistry was performed using antibodies to HLA-DR (BioGenex, San Ramon, Calif, clone LN3) and Bcl-2 (DAKO Corporation, Carpinteria, Calif, clone 124) and a streptavidin-biotin peroxidase system.

Results: In positive cells, HLA-DR was localized on the cell membranes whereas Bcl-2 was intracytoplasmic. All 17 UNCs and 14 LELCs were diffusely positive for HLA-DR (100%). Bcl-2 was present in 16 UNCs (94%) and 12 LELCs (86%). Only one squamous cell carcinoma of head and neck reacted positively for HLA-DR. Focal Bcl-2 staining was observed in 3 squamous cell carcinomas, 2 from head and neck and 1 from the uterine cervix.

Conclusion(s): Regardless of the organ of origin, LELCs share antigenic similarities with UNCs, including the expression of HLA-DR and Bcl-2. These 2 immunohistochemical markers can also reliably distinguish LELCs from morphologically similar neoplasms of the same organs.

Utility of p16INK4a and Ki-67 Staining of Cell Blocks Prepared From Residual ThinPrep Cervicovaginal Material

Ilkser Akpolat, MD ([email protected])1; Dina R. Mody, MD1; Ibrahim Ramzy, MD2; Debora A. Smith, CT(ASCP).31Department of Pathology, Baylor College of Medicine, Houston, Tex; 2Department of Pathology, University of California–Irvine, Orange, Calif; 3Department of Pathology, The Methodist Hospital, Houston, Tex.

Context: Cell blocks (CB), prepared from residual cervicovaginal ThinPrep material, were used for immunohistochemical staining for p16INK4a and Ki-67, antigens related to human papilloma viral infection and cervical neoplasia. The purpose of this study was to assess the reliability of CBs in the diagnosis of epithelial abnormalities, as compared with those of ThinPrep, and to elucidate the role of p16INK4a and Ki-67 in identifying significant preneoplastic lesions.

Design: Cervicovaginal cytology specimens from 85 patients were studied. Based on ThinPrep cytologic diagnoses, the study group included: 3 squamous cell carcinomas (SCC), 27 high-grade squamous intraepithelial lesions (HSIL), 20 low-grade squamous intraepithelial lesions (LSIL), 11 cases of atypical squamous cells of uncertain significance (ASCUS), and 24 negative for intraepithelial lesion or malignancy (NILM). Cell blocks were prepared from residual ThinPrep fluid in all 85 cases, and sections were stained with hematoxylin-eosin and antibodies for p16INK4a and Ki-67.

Results: The diagnoses of 85 CB preparations were SCC = 2, HSIL = 20, LSIL = 30, NILM = 32, and nondiagnostic = 1. The diagnoses in 62 of 85 CBs (73%) were in agreement with the ThinPrep diagnosis. Immunostaining of CBs for p16INK4a and Ki-67 showed statistically significant association (P < .05) with the presence of significant epithelial lesions, as diagnosed by either CB or ThinPrep test.

Conclusion(s): Our results indicate that CB offers an additional diagnostic tool in the evaluation of cervical samples, and suggests their use in immunohistochemical studies. Immunoreactivity to p16INK4a and Ki-67 may be helpful in identifying significant neoplastic or preneoplastic epithelial lesions and differentiating them from other lesions, such as atrophy.

Osteolysis of the Distal Clavicle: An Investigation of Histopathology, Pathophysiology, and Etiology

Faripour A. Forouhar, MD ([email protected])1; Kevin Shea, MD2; Nikoletta Sidiropoulos, BS.11Department of Pathology and 2Department of Orthopedic Surgery, University of Connecticut Health Center, Farmington.

Context: Osteolysis of the distal end of the clavicle is a clinical and radiologic entity characterized by persistent pain and gradual disappearance of the distal end of the clavicle, radiographically. The cause is acromioclavicular joint injury secondary to repetitive high-pressure stress such as weightlifting (atraumatic) or blunt trauma (traumatic). The pathology is complex and has not been adequately described.

Design: Seventeen patients (10 posttraumatic, 7 atraumatic) were studied. Various histologic findings were graded in each specimen. The pathologist (F.A.F.) was blinded to etiology, duration of symptoms, and radiographic stage of disease. Graded characteristics between the traumatic and atraumatic groups were statistically analyzed.

Results: Significant pathologic findings included erosion of articular cartilage and underlying bone, necrosis, granulation tissue formation and fibrocartilage ingrowth within the eroded pockets, and “fibrocartilaginous metaplasia” whereby bone is remodeled into a mixture of bone and cartilage in an abnormal pattern for adults. Furthermore, no statistically significant differences were observed in age, duration of symptoms, radiographic stage of disease, or graded histologic features between the 2 groups.

Conclusion(s): The pathology of osteolysis is described in detail. Excess fibrocartilage and cartilaginous metaplasia of bone are unusual findings. We speculate that the unique physiology of this joint in the context of repetitive trauma may explain its unusual pathologic findings. In addition, we conclude that etiology does not have a significant effect on pathology.

Clinicopathologic Profile Comparisons Between Patients With HER-2/neu Gene Amplification, HER-2/neu Protein Overexpression, and Both Amplification and Overexpression of HER-2/neu

Deepti Shukla, MD ([email protected]); Fleurette Abreo, MD; Mary Nordberg, PhD; Runhua Shi, MD, PhD. Department of Pathology, Louisiana State University, Shreveport.

Context: HER-2/neu is a prognostic and predictive indicator in breast cancer. Some patients show discordant results in HER-2 gene amplification and overexpression. Though there are studies showing overexpression of HER-2/neu is associated with poor prognosis, studies comparing clinical outcome in patients with variations in HER-2 overexpression and amplification are limited. Comparisons to other prognostic predictors (p53, Ki-67) and lymph node (LN) metastasis are needed.

Design: Paraffin-embedded tissue from 113 invasive breast cancer patients was stained for immunohistochemical (IHC) analyses with anti-p53 and -Ki-67 and -HER-2/neu (DAKO, Carpinteria, Calif). IHC slides were analyzed using an automated cellular image analysis system (ACIS) (ChromaVision, Inc, San Juan Capistrano, Calif); values >2 were positive. Fluorescence in situ hybridization (FISH) for HER-2/neu amplification was performed on all cases (PathVysion, Vysis/Abbott, Inc, Downers Grove, Ill) and evaluated per established criteria (values >2 were considered amplified). Quantitative antigenic expression levels for p53 and Ki-67 were compared between the 4 groups. Statistics were performed using the Fisher exact test.

Results:

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Conclusion(s): Ki-67 expression was increased in patients with HER-2/neu amplification, regardless of IHC HER-2 status (groups II and IV) (P = .01). p53 expression was significant (P = .02) in the IHC(+) group (group III) (75%) versus the FISH(+) group (group II) (33.3%). LN metastasis was significant in the FISH(+), IHC(−) group (group II) compared to the FISH(−), IHC(+) group (group III).

Amphicrine Carcinomas of the Pancreas

Choladda Vejabhuti, MD ([email protected]); Paul H. Jordan, Jr, MD; Mary R. Schwartz, MD; Mamoun Younes, MD. Department of Pathology, Baylor College of Medicine, Houston, Tex.

Context: Amphicrine neoplasms, tumors expressing both endocrine and exocrine features within the same cells, are rare. They are distinguished from the more common composite and collision mixed exocrine-endocrine neoplasms. We studied the clinical and pathologic features of 5 amphicrine neoplasms of the pancreas.

Design: The cases were studied using standard histochemical and immunohistochemical techniques. Two cases were examined by electron microscopy and 3 had flow cytometric DNA and cell cycle analysis.

Results: The 3 men and 2 women in this study ranged in age from 45 to 79 years. Four of the patients underwent Whipple resection and one underwent core needle biopsy only. Survival after diagnosis for those with known follow-up was 2 to 17 months. All of the tumors had high-grade nuclear pleomorphism and most had a solid architectural pattern. Lymphovascular, perineural, and/or duodenal invasion was identified in all of the resected neoplasms. All 5 showed coexpression of carcinoembryonic antigen, synaptophysin, and chromogranin in the same tumor cells. No consistent pattern of CA19-9 or hormone expression was found. Four of the cases were mucicarmine positive and most were argyrophilic. Ultrastructural examination of 2 cases showed mucin and neurosecretory granules in the same cells. Two cases were diploid and one was aneuploid. The Ki-67 proliferative fraction using the MIB-1 antibody ranged from 2% to 60% (mean, 27.8%). There was overexpression of p53 in 2 of the cases and absence of overexpression in 2.

Conclusion(s): All of the amphicrine neoplasms in this series were high-grade carcinomas with aggressive behavior, thus their designation, amphicrine carcinomas. Despite their endocrine component, the natural history more closely resembles that of ductal adenocarcinoma than islet cell tumors of the pancreas.

Assessment of Angiogenesis in Chronic Lymphocytic Leukemia (CLL)

John L. Frater, MD ([email protected])1; Susan E. Crawford, DO1; Charles L. Goolsby, PhD1; Neil E. Kay, MD2; LoAnn C. Peterson, MD.11Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Ill; 2Division of Hematology Research, Mayo Clinic, Rochester, Minn.

Context: Enhanced bone marrow (BM) angiogenesis has been documented in chronic lymphocytic leukemia (CLL). However, its extent and relationship to proangiogenic factors and known prognostic indicators are largely unexplored.

Design: Forty-two adults with CLL were studied. Immunohistochemistry (IHC) (n = 21 pts) with antibodies to CD20, CD3, CD34, VEGF (proangiogenic factor), TSP-1 (antiangiogenic factor), and HIF-1 α (transcriptional regulator enhanced by hypoxia) was performed to examine pattern and extent of BM involvement by CLL, microvessel density (MVD) (number of microvessels/high-power field), location of microvessels relative to CLL infiltrates, and cell types producing factors. Flow cytometry (FC) with specific gating of blood/BM CD5+ clonal B cells was performed to examine an additional 21 cases for cytoplasmic VEGF and TSP-1.

Results: Compared to 10 controls, CLL patients had higher MVD (23.8 vs 14.6, P < .001) in BM sections. MVD was highest at the periphery of focal infiltrates, was not enhanced in proliferation centers, and increased irrespective of the presence or absence of cytogenetic/immunophenotypic markers of aggressive disease. By IHC, CLL cells were VEGF(+), HIF-1 α(+), and TSP-1(−). By FC immunophenotyping, CD19+ CLL cells had 1.4 to 2.0× brighter VEGF staining than CD19CD5+ T cells and were TSP-1(−).

Conclusion(s): (1) MVD is increased in CLL, but regionally localized. (2) CLL cells demonstrate a phenotypic shift in favor of increased angiogenesis, with upregulated VEGF (proangiogenic), and downregulated TSP-1 (antiangiogenic). (3) Hypoxia, as assessed by HIF-1 α upregulation, may be of importance. (4) Differences in MVD did not correlate with +12, 17p−, IgV status, or CD38 expression in this patient population.

Analysis of Telithromycin Resistance in Clinical Streptococcus pneumoniae and Streptococcus pyogenes

Chad D. Galderisi, DO ([email protected]); Peter C. Appelbaum, MD; Fredrick A. Browne, MD; Bulent Bozdogan, MD, PhD. Department of Pathology, Penn State/Hershey Medical Center, Hershey, Pa.

Context: Presence of erm(B) gene confers different levels of macrolide and telithromycin resistance in S pneumoniae and S pyogenes. The correlation of macrolide and telithromycin resistance level with the sequence of erm(B) genes was tested.

Design: Twelve S pneumoniae and 4 S pyogenes strains with erm(B) gene were studied. The erm(B) gene including its regulatory region from each strain was sequenced and compared to plasmid pAM77. One inducibly expressed erm(B) gene from S pyogenes was cloned in a shuttle vector and the recombinant plasmid, pCA4, was used for transformation of S pyogenes NZ strain.

Results: Induction with erythromycin increased the minimal inhibitory concentrations (in mg/mL) of 7 S pneumoniae from 0.25–1 to >64 for erythromycin and from 0.06–0.5 to >64 for clindamycin. However, strains remained susceptible to telithromycin. In 4 S pneumoniae strains, resistance was expressed constitutively but only 2 of these strains were resistant to telithromycin. All S pyogenes strains were resistant to telithromycin with or without induction. Analysis of sequences showed presence of point mutations (pAM77 numbering) G134A, C164T, G286A, T291C, T311A, G312A, or GT insertion after position 52, in strains with different phenotypes, and all had insertion of AATA, (after 197) and A (after 298). After transformation with erm(B), susceptible S pyogenes NZ became resistant to telithromycin.

Conclusion(s): No correlation was found between phenotype of macrolide resistance and genotype. Structural differences of the target in the bacterial host may explain different level of telithromycin resistance in S pyogenes and S pneumoniae in presence of the same gene, erm(B).

p53, Ki-67, HER-2/neu, Estrogen Receptor, and Progesterone Expression Do Not Predict Lymph Node Metastasis in African American Breast Cancer Patients

Deepti Shukla, MD ([email protected]); Mary L. Nordberg, PhD; Fleurette Abreo, MD; Runhua Shi, MD, PhD. Department of Pathology, Louisiana State University Health Science Center, Shreveport.

Context: African American (AA) women are at lower risk of developing breast cancer than white American women, but they frequently have worse prognosis if they develop the disease. Apart from the histopathologic findings associated with breast carcinoma, important prognostic parameters have emerged that facilitate stratification of low-risk and high-risk patients. Ki-67, p53, HER-2/neu, estrogen receptor (ER), and progesterone receptor (PR) are some of the prognostic and predictive indicators used in breast cancer. In this study, all the markers are correlated with lymph node (LN) metastasis, still the most important prognostic indicator of disease.

Design: Paraffin-embedded tissue from 65 invasive AA breast cancer patients were immunostained with anti-ER, -PR, -Ki-67, and -HER-2/neu (DAKO, Carpinteria, Calif). Immunohistochemical (IHC) analysis of all the stains was performed using an automated cellular image analysis system (ACIS, ChromaVision, Inc, San Juan Capistrano, Calif). Antigenic expression was correlated with LN metastasis. Statistics were performed using the Fisher exact test.

Results:

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Conclusion(s): p53, Ki-67, HER-2/neu, ER, and PR are frequently expressed in AA females, but none of these are useful in predicting LN metastasis in AA breast cancer patients.

Cytokeratin Profiles of Breast Cancers in Young Women

Monica E. de Baca, MD ([email protected]); Federico A. Monzon-Bordonaba, MD; Juan P. Palazzo, MD. Department of Pathology, Thomas Jefferson University, Philadelphia, Pa.

Context: Breast cancer in young women constitutes a group of tumors associated with aggressive disease and variable response to therapy. Here, the expression of an extended panel of cytokeratins in breast cancer patients less than age 50 was studied.

Design: The study included 34 cases of women younger than 50 years of age. Of these 34 cases, 26 (2 ductal carcinoma in situ [DCIS], 10 invasive ductal carcinomas with in situ component, 12 ductal carcinomas without in situ component, 2 lobular carcinomas) were reviewed in this study. Tumors were stratified by size, nuclear grade, histologic grade, and receptor expressions. The following cytokeratins were analyzed by immunohistochemistry: CK 5–6, CK 8, CK 14, CK 17, CK 18, CK 19. Vimentin was also studied.

Results: CK 8, CK 18, and CK 19 demonstrated the highest expression in all tumors (in situ and invasive components). The lowest expression level was seen in CK 14. Intermediate between these 2 groups was CK 17 and CK 5–6 expression. No significant expression differences were appreciated between DCIS and invasive components. Small (<2 cm) ER-positive tumors showed the highest expression with all CK subtypes. CK 14 expression was consistently low in DCIS and invasive components of high nuclear and histologic grade tumors.

Conclusion(s): The expression of cytokeratins is maintained in most cases of DCIS, with or without invasive components. CK 14 expression may be lost in high-grade tumors. The progression from DCIS to invasive cancer does not seem to affect the CK expression profile of the breast. High-grade cancers lose expression of CK 14, which may be related to differentiation.

DNA Ploidy in Breast Cancer: Comparison of Blue Feulgen Staining of Fine-Needle Aspiration and Tissue Using the Automated Cellular Imaging System

Ravindra Veeramachaneni, MD ([email protected]); Sarada Gummadi, MD; Mary L. Nordberg, PhD; Fleurette Abreo, MD. Department of Pathology, Louisiana State University Health Sciences Center, Shreveport.

Context: Cellular DNA content is used for determination of DNA ploidy in normal and abnormal cellular populations. Improved detection and patient awareness strategies have allowed clinicians to detect and biopsy tumor specimens much earlier, resulting in specimens too small for optimal analysis by flow cytometric techniques. The Automated Cellular Imaging System (ACIS, ChromaVision, Inc, San Juan Capistrano, Calif) measures DNA content in cell nuclei stained with the Feulgen stain.

Design: In a series of 25 cases of invasive breast cancer, comparisons of DNA ploidy between fine-needle aspiration (FNA) specimens and excised tissue evulated by the ACIS were performed. The ChromaVision Blue Feulgen stain kit was used and ACIS protocols were followed. DNA analyses were interpreted using ChromaVision DNA Ploidy/ACIS software.

Results: DNA content analysis was initially successful on 24 cases of breast cancer. Analyses of FNA slides showed that 12 tumors (50%) were aneuploid and 12 tumors (50%) were diploid. ACIS results were obtained by analyzing an average of 62 cells per FNA slide (range, 33–126) while an average of 112 cells per tissue section specimen (range, 49–198) were analyzed. ACIS analyses of tissue sections on all tumors were concordant for both diploid and aneuploid tumors. Twelve cases had only FNA, and of these, 8 were aneuploid and 4 were diploid.

Conclusion(s): DNA ploidy analysis on FNA slides using ACIS offers significant advantages, particularly when populations of tumor cells were small and/or particular cells of interest were interrogated. It offers significant information to clinicians in cases where excision of tumor is not warranted. ACIS offers semi-automated analysis of DNA ploidy and direct visualization of the data in a standard DNA ploidy software application. ACIS analysis can be performed on cytology specimens of low cellularity to verify ambiguous results or visually confirm abnormal cells.

Cytokeratin Profiles in Male Breast Cancer

Vincenzo Ciocca, DO ([email protected])1; Alessandro Bombonati, MD2; Zoran Gatalica, MD3; Federico Monzon, MD4; Juan P. Palazzo, MD1; Marcello Di Pasquale, MD, PhD.21Department of Pathology, Thomas Jefferson University, Philadelphia, Pa; 2Department of Pathology, University of Tor Vergata, Rome, Italy; 3Department of Pathology, Creighton University, Omaha, Neb; 4Department of Pathology, University of Pittsburgh, Pittsburgh, Pa.

Context: The prognostic factors and expression of molecular markers of male breast carcinomas have been found to be similar to female cancers. The identification of distinct cytokeratin (CK) profiles (basal as opposed to luminal cells) helps to identify subsets of tumors with different clinical behavior.

Design: Twenty cases of male breast cancer were studied. The panel of CKc studied by immunohistochemistry included: 5/6, 14, 17, 18, and 19 (DAKO, Carpinteria, Calif). A correlation between pathologic findings, stage, and CK expression was analyzed in all the cases.

Results: The tumors represented either in situ or invasive ductal carcinomas (majority). Focal and heterogeneous positivity with CK 5/6 and 14 was identified in 5 cases. CK 17 was negative in all the cases but one, which was negative for CK 5/6. All cases expressing either CK 5/6 or 14 were invasive carcinomas, and were larger, had higher nuclear and histologic grades and advanced stage compared to the tumors not expressing CK 5/6 or 14. All tumors except one (also negative for CK 5/6) expressed CK 18 and 19. No association was seen between CK 5/6, 14 and estrogen receptor or HER-2 expression.

Conclusion(s): The expression of CK 5/6 identifies a subset of pathologically and clinically aggressive male breast cancers. This profile appears to be independent of estrogen receptor status and HER-2 activation. Because expression is heterogeneous, the results should be interpreted with caution when limited tissue is available.

Role of p63 in Assessment of Bronchioloalveolar Proliferations of the Lung

Hina A. Sheikh, MD ([email protected]); Kimberly Fuhrer, HT; Kathleen Cieply, BS; Samuel Yousem, MD. Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pa.

Context: Discrimination of well-differentiated adenocarcinoma from reactive bronchioloalveolar epithelium is difficult on routine histology, especially on small biopsies. Ancillary studies to help in this distinction are desirable. p63, a p53-homologous nuclear protein, is a marker of reserve cells of the bronchus and terminal lobular unit, and may be helpful in this regard.

Design: Twenty-four cases of adenocarcinoma (19 open-lung and 5 transbronchial biopsies) and 24 cases of benign fibrotic lungs with metaplasia (20 open-lung and 4 transbronchial biopsies) were studied for p63 expression by immunohistochemistry (Dako, Carpinteria, Calif). The diagnostic categories of benign lung conditions included interstitial pneumonias and pulmonary scars. Only nuclear staining was considered positive. In cases of adenocarcinoma, p63 positivity was calculated as a percentage of all tumor cells examined.

Results: In areas of normal lung histology, p63 decorated the reserve cells of large and small airways and occasional cells of the distal lobular unit. In the fibrotic/reactive processes, an interrupted but distinct pattern of nuclear staining was present in all cases, with staining of basal cells of the airways as well as areas of bronchiolar and squamous metaplasia (24/24, 100%). Adenocarcinomas showed rare nuclear staining in 2 of 24 cases (8.3%) with one case showing <1% and the other 10% positivity of malignant cells. Areas of potential confusion included the junction between normal lung and lepidic growth of adenocarcinoma and retrograde spread of adenocarcinoma into small airways.

Conclusion(s): p63 immunostaining may be very helpful in distinguishing reactive from neoplastic epithelial proliferations in the lung.

Evaluation of Clostridium difficile Isolates for Clonality and Toxin Production

Manjiri S. Lele, MD ([email protected]); Sue B. Overman, SM (AAM); Julie A. Ribes, MD, PhD; John P. Seabolt, MT(ASCP)SM, EdD. Department of Pathology, University of Kentucky, Lexington.

Context: To investigate a high number of rapid enzyme immunoassay (EIA) toxin-A-negative, cytotoxin-B-positive specimens, we evaluated toxin production and bacterial clonality in C. difficile isolates.

Design: Stool specimens were tested using the VIDAS C. difficile Toxin-A EIA (bioMerieux, Hazelwood, Mo) and bacterial culture on C difficile Selective Agar (BBL, Cockeysville, Md). Cultures were grown anaerobically for 4 days and yellow colonies were confirmed using RapID ANA II (Remel, Lenexa, Kan). Isolates were grown for 48 to 72 hours in chopped meat broth (CMB) and the supernatants tested using the VIDAS and cytotoxin assays. Cytotoxin B production was identified by cytopathic effect on MRC-5 cells with confirmation using toxin-B-specific antitoxin (Bartels, Farmsworth, Conn). Fatty acid cell wall composition analysis (MIDI System, Newark, Del) was used to determine relatedness of bacterial isolates.

Results: Of the 3263 analyzable specimens screened by the VIDAS EIA, 244 were positive for toxin-A and 3019 were negative. Only 164 specimens yielded positive cultures. Cytotoxin B was detected in 120 isolates. Seventy of these were initially toxin-A-positive, while 50 demonstrated detectable toxin-A only on testing of the CMB supernatant. Eight isolates produced toxin-A only. Thirty-six isolates represented “normal flora,” producing no toxins. A total of 66 bacterial strains were detected in the 101 isolates analyzed by MIDI. No more that 5 patients shared the same strain.

Conclusion(s): Both the VIDAS assay and bacterial culture missed significant proportions of the positive specimens. No clonal population of toxin-A-negative C difficile was identified.

Clinical Evaluation of the AtheNA System for Autoimmune Multi-Analyte Profiling

Stanley J. Podlasek, MD ([email protected]); Elizabeth T. Rankin, MT(ASCP), SCC. Department of Pathology, INOVA Alexandria Hospital, Alexandria, Va.

Context: Multi-analyte arrays helpful in proteonomics and molecular genetics have reached the clinical laboratory. One example is the AtheNA Multi-Lyte ANA Test System (Wampole Laboratories/Zeus Scientific, Inc, Princeton, NJ). Autoantibodies found in autoimmune conditions are traditionally tested independently by immunofluorescence assays or enzyme immunoassays. On the AtheNA system, the screening antinuclear antibody and 9 specific autoantibodies are each measured 50 separate times simultaneously. Polystyrene microspheres are coded as sets with fluorescent fluorophores embedded within the core of each particle. Microparticles containing a mixture of 10 sets (each uniquely coated with 1 of 10 antigens in our profile) are sequentially incubated with patient serum, incubated with reporter fluorophore, and passed through a flow cytometer detector. Separate lasers excite label and reporter fluorophores.

Design: We ran samples on the AtheNA system and sent duplicate samples to Quest Diagnostics (Teterboro, NJ) for screening by indirect immunofluorescence and to Mayo Medical Laboratories (Rochester, Minn) for specific autoantibodies done by enzyme immunoassay.

Results: Correlation percent and proportion of concordance are given for each autoantibody:

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Discordant results for the ANA and anti-dsDNA were often positive by the reference labs but negative by AtheNA. Clinical reviews of 7 discordant patients revealed 6 had non-immune disorders and one had systemic lupus erythematosus.

Conclusion(s): This technology is a cost-effective tool that will eliminate turnaround time delays in evaluation of a positive ANA. Further, the system appears to eliminate some biological false positive interferents caused by sepsis and other severe inflammatory disorders such as acute hepatitis.

Expression of Vascular Endothelial Growth Factor in Prostate

Suman Setty, MD, PhD ([email protected])1; Stefan E. Pambuccian, MD1; Michael Wilson, PhD.21Department of Pathology, University of Minnesota Medical Center, Minneapolis; 2Department of Pathology, Veterans Administration Medical Center, Minneapolis, Minn.

Context: Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis with a role in prostatic carcinoma (CaP) growth and metastasis. A diffusible endothelial-cell–specific mitogen which induces endothelial cell proliferation, it is reported to be produced in high levels by various tumors. We studied protein expression levels in CaP, the data on the role and level of VEGF expression in prostatic tissues being controversial.

Design: Nine selected surgical specimens exhibiting areas with CaP, prostatic intraepithelial and benign prostatic hyperplasia histology were evaluated for VEGF protein expression by immunohistochemistry (VEGF, Ab-7, Neomarkers, Calif). Results were compared with tumor differentiation (Gleason Grade), age, and prognosis.

Results: VEGF specific antisera revealed significant staining of small and medium sized vessel endothelial cells with increased peritumoral expression. All tumors expressed high levels of VEGF protein with highest expression in grade 3 (3+). In contrast to normal glands and BPH, which showed weak positivity in the abluminal perinuclear cytoplasm, tumor cells showed strong membrane accentuation or concentration in the luminal cytoplasm. Lower levels (<2+) in grade 4 and 5 tumors were associated with increased peri-tumoral stromal expression. Adjacent normal glands and BPH areas were weakly positive.

Conclusion(s): VEGF expression was shown to correlate with tumor grade, with maximal luminal and membrane expression in Gleason grade 3 and 4 CaP. Grade 5 CaP was associated with higher stromal expression of VEGF.

Gelsolin Expression Correlates With Clinical Outcome of Patients With Papillary Renal Cell Carcinoma

Sony Wiriosuparto, MD ([email protected]); Henry Tsai, MD; Jian Yu Rao, MD; Jonathan Said, MD. Department of Pathology, UCLA Medical Center, Los Angeles, Calif.

Context: The actin cytoskeleton is deranged during carcinogenesis. Gelsolin, an actin-binding protein, is a putative tumor suppressor gene and decreased expression has been reported with poor outcome in breast, prostate, and other carcinomas. The expression of gelsolin in papillary renal cell carcinoma (PRCC) has not been previously studied. This study evaluates gelsolin as a prognostic marker in PRCC and compares it with proliferative rate as determined by staining for Ki-67.

Design: Immunohistochemical staining for gelsolin and Ki-67 were performed in 32 patients with papillary renal cell carcinoma. Gelsolin staining intensity and percentage of tumor cell staining were studied. Tumor was considered weak for gelsolin staining if less than half of the cells had weak intensity or <25% of the tumor cells exhibited normal staining intensity. The distal renal tubules served as a positively staining internal control. For Ki-67, percentage of cells with nuclear staining was recorded; tumor was considered highly proliferative if more than 10% of the tumor cells showed nuclear staining.

Results: Decreased gelsolin expressions correlated with poor survival in PRCC (P < .001). Multivariate analysis shows that expression of gelsolin was associated with poor patient prognosis independent of tumor grade and stage. Patients with decreased gelsolin expression had much worse prognosis than would be predicted by proliferative rate as determined by Ki-67. There is no correlation between gelsolin and Ki-67.

Conclusion(s): Gelsolin is a prognostic biomarker for PRCC. Further investigations will extend to subtypes of PRCC.

Micrometastasis and Prognosis in Dukes B Colonic Carcinoma

Christina A. Samathanam, MD, PhD ([email protected]); John Z. Zhang, MD; Chad DeFrain, MD; Joanne K. L. Rutgers, MD. Department of Pathology, Long Beach Memorial Medical Center, Long Beach, Calif.

Context: Colon cancer is a significant cause of mortality in the world and the third most common cause of cancer death in the United States. The presence of pericolic lymph node metastasis is one of the most important prognostic factors in colon carcinoma and guides the use of adjuvant therapy. Patients with nodal metastasis (Dukes C) have a drop in survival rate when compared to negative lymph node metastasis (Dukes B). However, within Dukes B patients, the survival rate varies. Identification of reasons for variation and guidance of adjuvant therapy have prompted the search for micrometastasis.

Design: Slides and blocks from 64 cases of Dukes B adenocarcinoma of the colon treated at one institution were retrieved. The study obtained approval of the institution's research board and follow-up from the tumor registry. Immunohistochemical (IHC) stains for keratin (AE1-AE3) were performed on pericolic lymph node blocks. Micrometastasis were defined as IHC-positive tumor cells not seen on hematoxylin-eosin (H&E) slides. The slides were evaluated while blinded to patient outcome. H&E slides of the primary tumor were reviewed for lymphovascular space invasion (LVSI) and individual malignant cells at the advancing edge of the primary tumor, “tumor budding.” Kaplan Meyer survival curves relating survival to micrometastasis and LVSI were performed.

Results: Micrometastases were found in 53% of patients. The 5-year survival rate was not statistically different between patients with and without micrometastases (P = .63). Neither LVSI nor “tumor budding” correlated with micrometastasis.

Conclusion(s): This study found no correlation between IHC-detected micrometastases and survival. Larger, prospective studies to confirm this finding would be of interest. At this time, the current guidelines as to careful H&E examination of pericolic nodes is adequate for accurate staging.

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