Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative.

AIMS: To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies. METHODS: Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry. RESULTS: MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues. CONCLUSIONS: Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.

Clusterin as a possible predictor for biochemical recurrence of prostate cancer following radical prostatectomy with intermediate Gleason scores: a preliminary report.

Disease recurrence following radical prostatectomy is a major concern in prostate cancer patients. Gleason scores are useful in predicting recurrence. Low Gleason scores are usually associated with long disease-free intervals, while high Gleason scores are suggestive of early recurrence. However, prediction of recurrence has been difficult with intermediate Gleason scores. Clusterin is a ubiquitous secretory sulfated glycoprotein. It is also an antiapoptotic mediator in prostate cancer. The objective of the present study is to determine if clusterin can serve as a predictive biomarker for recurrence of prostate cancer with intermediate Gleason scores in patients following radical prostatectomy. Prostatic specimens with Gleason score of 6 (3+3) or 7 (3+4) were obtained from the archival bank. Three groups of specimens were investigated. The first group was from nine patients who developed recurrent disease according to a persistent rise of serum PSA within 3 years following radical prostatectomy. Those in the second group and the third group were from patients who showed no evidence of disease recurrence for at least 5 y (11 patients) and 10 y (eight patients), respectively following the surgery. Histological sections were subjected to immunohistochemical staining using a monoclonal antibody specific for clusterin. The staining intensity was scored as 0, 1, 2, and 3, with 0 being no staining, 1 showing less than 25% positive staining, 2 being 25-50% positive, and 3 showing greater than 75% positive staining. One-way ANOVA with Bonferroni correction was used for statistical analysis. Evaluation of the scores of clusterin staining was carried out according to four specific areas in each specimen. They were (a) benign epithelial cells, (b) malignant epithelial cells (cancer epithelia), (c) stromal cells surrounding benign cells, and (d) stromal cells surrounding malignant cells (cancer stroma). Staining score in prostatic epithelial cells, benign as well as malignant, showed no significant relationship among the three patient groups. However, when staining scores in stromal cells were compared, there was a significant difference between patients with recurrent disease and those showed no evidence of disease recurrence for at least 10 y. Results of this preliminary study support the important role of clusterin in the stromal component for prostate cancer progression. Clusterin immunostaining may be useful to aid the prediction of chance of disease recurrence in patients with Gleason score 6 or 7 prostate cancer following radical prostatectomy. Further studies with a large number of cases are warranted to verify this preliminary finding.

Thrombospondin-1, vascular endothelial growth factor and fibroblast growth factor-2 are key functional regulators of angiogenesis in the prostate.

BACKGROUND: Prostate cells secrete many molecules capable of regulating angiogenesis; however, which of these actually function as essential regulators of neovascularization is not yet clear. METHODS: Functional angiogenic mediators secreted by normal and diseased prostate cells were identified using an in vitro angiogenesis assay. These factors were quantified by immunoblot or ELISA and localized in tissue by immunohistochemistry. RESULTS: Normal prostate epithelial cell secretions were anti-angiogenic due to inhibitory thrombospondin-1 (TSP-1) whereas this inhibitor was decreased in the pro-angiogenic secretions derived from benign prostatic hyperplasia (BPH) and cancer cells. This pro-angiogenic activity depended primarily on fibroblast growth factor-2 (FGF-2) and/or vascular endothelial growth factor (VEGF) whose secretion was increased. Immunolocalization studies confirmed that the changes detected in vitro also occurred in vivo. CONCLUSIONS: During disease progression in the prostate, production of TSP-1, the major inhibitor, is down-regulated while that of stimulatory FGF-2 and/or VEGF rise, leading to the induction of the new vessels necessary to support tumor growth.

Apoptotic activity of doxazosin on prostate stroma in vitro is mediated through an autocrine expression of TGF-beta1.

BACKGROUND: Doxazosin, an alpha-adrenergic antagonist, has been shown to induce apoptosis in prostatic stromal cells. The mechanism of this apoptotic action by Doxazosin remains undefined. The present study was carried out to demonstrate that the effect of Doxazosin on apoptosis of prostate stromal cells is mediated through an autocrine action of TGF-beta1. METHODS: Primary cultures of human prostate cells were treated with varying concentrations of Doxazosin (0, 0.1, 1, 10, and 100 microM) for a period up to 3 days. At the end of the 3-day culture, cell numbers were counted. Apoptosis was assessed by a colorimetric terminal deoxyribonucleotide transferase labeling technique. TGF-beta1 was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to control cultures, cell numbers were significantly decreased as much as 68.4% in cultures treated with 10 microM of Doxazosin after 3 days incubation, while apoptosis increased by 64.7% in cultures treated with the same concentration of Doxazosin after 24 h. This decrease in cell number was reversed when antibody to TGF-beta1 was added to these cultures. Addition of TGF-beta1 (0, 1.0, and 10 ng/mL) to the cultures also decreased the cell numbers. Quantitation of TGF-beta1 in lysates of cells by ELISA revealed that the cells treated with Doxazosin (10 microM) produced as much as 62.5% more TGF-beta1 than in that of untreated cells. CONCLUSIONS: These results demonstrate that the apoptotic effect of Doxazosin on human prostatic stromal cells is mediated through an autocrine production of TGF-beta1.

Solitary fibrous tumor of the prostate a report of 2 cases and review of the literature.

We report 2 cases of solitary fibrous tumor of the prostate. Histologically, both tumors demonstrated a multipatterned architecture with varying degrees of collagenization and hemangiopericytoma-like foci, and both were composed of CD34-immunopositive spindled cells that insinuated themselves between strips of collagen. The tumor in case 1 was well circumscribed and showed minimal mitotic activity or pleomorphism, whereas the tumor in case 2 was more cellular, less collagenous, had a more diffuse growth pattern, and exhibited cytologic atypia and high mitotic activity. Prostatic solitary fibrous tumor must be distinguished from other spindle cell tumors reported to occur in the prostate. To our knowledge, these cases represent only the fifth and sixth reported cases of prostatic solitary fibrous tumor.

Isochromosome 7q in adult Wilms' tumors: diagnostic and pathogenetic implications.

Wilms' tumors affecting adults are rare and are thought to have a worse prognosis than similar stage tumors in the pediatric population. To understand these tumors better, the authors reviewed their multi-institutional experience in a series of nine lesions diagnosed as Wilms' tumors in adults. In addition to histologic and immunohistochemical examination, they performed cytogenetic analysis and fluorescence in situ hybridization. On review, four cases were reclassified: two "blastema only" as Ewing's sarcoma/primitive neuroectodermal tumor and the other two as clear cell sarcoma of soft parts and sarcoma not otherwise specified (NOS). Of the remaining five cases, three exhibited biphasic histology and two were triphasic. In this group, there were three women and two men, and patient age ranged from 17 to 37 years (median age, 26 years). Tumor size was large and ranged from 10 to 31 cm (median tumor size, 12.5 cm). Histologically, the tumors showed the typical features of Wilms' tumors with varying amounts of blastema (n = 5), epithelium (n = 5), and stroma (n = 2). No tumors contained anaplasia, and persistent renal blastema was not identified in the non-neoplastic kidney in any specimen. All tumors were positive for cytokeratins (CK7, n = 3; pankeratin, n = 5), and one tumor was weakly positive for CD99 (0-13). Molecular analysis including dual color fluorescence in situ hybridization (all tumors), and cytogenetic analysis (n = 2) disclosed the presence of isochromosome 7q in three of five tumors whereas all tumors were diploid with respect to chromosome 12. Follow-up data ranged from 6 to 133 months (median follow-up, 82 months) with progression in only one patient who had stage IV disease with lymph node and lung metastases at presentation. The authors conclude that adult Wilms' tumor has been overdiagnosed. Most "blastema-only" tumors in adults are not Wilms' tumors, and in an adult, biphasic morphology should be the minimum criteria for their diagnosis. Using strict diagnostic criteria, adult Wilms' tumors have a relatively favorable prognosis. The characteristic findings of isochromosome 7q, lack of trisomy or tetrasomy for chromosome 12, and absence of persistent renal blastema suggest that the pathogenesis of Wilms' tumors in adults may be different than in the pediatric population. These genetic features may be helpful in distinguishing adult Wilms' tumors from other primary renal tumors.

Epithelioid and spindle-celled leiomyosarcoma of the heart. Report of 2 cases and review of the literature.

BACKGROUND: Primary cardiac leiomyosarcomas are rare. Isolated reported cases and small series generally describe spindle-celled, high-grade tumors with poor short-term survival; however, the pathologic features of many of these tumors are incompletely documented. The authors report in detail the clinicopathologic features of 2 relatively low-grade epithelioid and spindle-celled primary cardiac leiomyosarcomas. METHODS: Cases 1 and 2 were studied using standard histochemical and immunohistochemical techniques, and case 1 was examined by electron microscopy. The literature was reviewed with regard to primary cardiac leiomyosarcomas. RESULTS: Both tumors showed epithelioid and spindle-celled areas. The tumor in case 1 was low grade, and the tumor in case 2 was predominately low grade with a high-grade focus. A review of 28 reported cases revealed a wide age range (mean, 43 years), equal male-to-female ratio, and a predilection for the left atrium (48%). Follow-up of reported cases with fewer than 5 mitoses per 10 high-power fields showed a mean survival of 22 months compared with a 9-month mean survival for all others. CONCLUSIONS: Short-term follow-up of reported cases of high-grade cardiac leiomyosarcoma suggests a poor prognosis. Long-term follow-up in our case 2, along with follow-up of reported cases that were histologically similar to our cases, suggests that cardiac leiomyosarcomas with low-grade features or mixed low- and high-grade features also have a poor overall long-term survival, with a high rate of local recurrence and systemic spread.

Metanephric adenoma-like tumors of the kidney: report of 3 malignancies with emphasis on discriminating features.

BACKGROUND: Metanephric adenoma is a very rare benign renal tumor; only 80 well-documented cases have been reported to date. We have seen several renal tumors that were originally incorrectly diagnosed as metanephric adenoma. DESIGN: We present 3 unusual renal tumors (2 primary and 1 metastatic), each of which illustrates important pathologic features useful in discriminating metanephric adenoma from malignant mimics. RESULTS: Case 1 involved a 46-year-old man with multiple small, cortical, solid, papillary (chromophil) renal cell carcinomas in his right kidney; the patient developed multiple, histologically identical, solid, papillary (chromophil) carcinomas in the opposite kidney 17 months later. Case 2 involved a 32-year-old woman with a 14-cm right renal tumor who developed soft tissue and bone metastases over a 17-year period. Case 3 involved a 52-year-old woman who presented with a 1.8-cm corticomedullary renal nodule, which eventually proved to represent a metastasis from a poorly differentiated (insular) carcinoma of the thyroid. All 3 tumors superficially resembled metanephric adenoma and consisted of primitive, dark-staining cells arranged in tubules or sheets. Each tumor, however, also had features inconsistent with the diagnosis of metanephric adenoma, including multifocal lesions with a variable nuclear-cytoplasmic ratio and diffuse cytokeratin 7 and epithelial membrane antigen immunopositivity in case 1, a 14-cm-diameter tumor with occasional mitoses in case 2, and a distinct fibrous capsule with capsular and vascular invasion in case 3. In addition, all 3 tumors lacked the cytologic features of bland overlapping nuclei with imperceptible cytoplasm consistently seen in metanephric adenoma. CONCLUSION: Adherence to strict histopathologic criteria will discourage misdiagnosis of a malignant or potentially malignant renal neoplasm as the rare and always benign metanephric adenoma.

Transplantation tolerance prevents cardiac allograft vasculopathy in major histocompatibility complex class I-disparate miniature swine.

BACKGROUND: The mechanisms and treatment of cardiac allograft vasculopathy (CAV) remain elusive. We have used partially inbred miniature swine to determine the role of class I MHC antigens in the pathogenesis of CAV and to determine whether acquired tolerance to donor antigen can prevent the development of CAV in large animals. METHODS: Previous studies demonstrated that miniature swine treated with 12 days of cyclosporine (CsA) after the transplantation of MHC class I-disparate kidney allografts all became tolerant to the donor kidneys and survived indefinitely. In the present study, heart allografts were transplanted across the same MHC class I disparity in CsA-treated swine. RESULTS: Unlike kidney allografts, heart allografts were rejected in 33-55 days. By postoperative day 28, all cardiac allografts had developed the intimal proliferation characteristic of CAV. When hearts and kidneys from the same donors were transplanted simultaneously into class I-disparate, CsA-treated recipients, the hosts became tolerant to their cardiac allografts and survived long-term. Furthermore, none of the hearts from the combined heart/kidney recipients developed evidence of CAV. Thus, this report demonstrates that: (1) MHC class I antigens play an important role in the pathogenesis of CAV, (2) the specific unresponsiveness to donor class I antigen induced by a class I-disparate kidney protects a heart transplanted from the same organ donor, and (3) the induction of acquired tolerance prevents the development of CAV. CONCLUSION: These findings in a preclinical system establish the significance of antigen-dependent mechanisms in the pathogenesis of CAV and underscore the importance of achieving tolerance in clinical transplantation.

Cardiac allograft vasculopathy is abrogated by anti-CD8 monoclonal antibody therapy.

BACKGROUND: Cardiac allograft vasculopathy, a diffuse and accelerated form of arteriosclerosis, is a major cause of graft loss or heart transplant recipient death after the first transplant year. This study examined the effects of depleting host CD8 + T lymphocytes on the development of cardiac allograft vasculopathy in miniature swine. METHODS: Cardiac allografts were heterotopically transplanted across a major histocompatibility complex class I barrier in partially inbred miniature swine and monitored for rejection by serial biopsies, electrocardiograms, and echocardiograms. Four control animals received cyclosporine on postoperative days 0 to 11. Another four miniswine were given 14.5 mg/kg of 76-2-11 (a mouse anti-swine CD8 monoclonal antibody) on postoperative day 0, in addition to a 12-day course of cyclosporine. Host CD8+ T cells and circulating 76-2-11 monoclonal antibodies were monitored by flow cytometry. RESULTS: As compared with cyclosporine-treated control animals, swine receiving 76-2-11 demonstrated near-complete depletion of peripheral CD8+ T cells by postoperative day 2, which persisted for 14 to 18 days. Mean allograft survival of the antibody-treated group and the control group was not statistically different (33 days versus 39 days, respectively) and both groups demonstrated severe interstitial rejection at necropsy. Control animals demonstrated florid intimal thickening of large and small arteries at necropsy. However, swine treated with 76-2-11 showed no intimal proliferation. CONCLUSIONS: Depletion of host CD8+ T cells prevents or delays the development of intimal proliferation in miniature swine. CD8+ lymphocytes play an important role in the early development of cardiac allograft vasculopathy in large animals.

Solid variants of papillary (chromophil) renal cell carcinoma: clinicopathologic and genetic features.

Papillary renal cell carcinomas (RCCs) traditionally have been defined histologically as tumors with at least 50% true papillae. However, these tumors also have characteristic immunohistochemical and genetic features that separate them from other RCCs. We identified six tumors composed of solid sheets of cells without true papillae but that otherwise resembled papillary RCCs, which we feel represent solid variants of papillary RCCs. All six tumors were primary lesions of the kidney, all were strongly reactive for epithelial membrane antigen, cytokeratin 7, and callus keratin, and all were negative for the high molecular weight keratin antibody 34BE12. Four of four tumors tested showed trisomies for chromosome 7, chromosome 17, or both by either cytogenetic analysis or fluorescence in situ hybridization. Four cases were composed of solid sheets of cells containing distinct micronodules that in some cases resembled abortive papillae. The cells composing the micronodules had abundant eosinophilic cytoplasm, open chromatin, and in some cases prominent nucleoli. The intervening cells had similar nuclei, but the amount of cytoplasm was variable. In three of these micronodular cases, multiple tumors diffusely replaced the kidney; in the fourth case two typical clear cell RCCs and a typical papillary RCC were also present in the same kidney, but the micronodular tumor was unifocal. The two remaining cases were solitary tumors consisting of solid sheets of cells forming ill-defined tubules. These cells had scant clear cytoplasm and small round to elongate nuclei with occasional nuclear grooves but only rare small nucleoli. Limited follow-up has shown no evidence of disease in any patient thus far. The differential diagnosis includes "renal adenoma," renal adenomatosis, metanephric adenoma, and clear/granular cell RCC. We conclude that solid variants of papillary RCCs lack true papillae but have characteristic histologic, immunohistochemical, and genetic features.

Cervical squamous cell carcinoma in situ with intraepithelial extension to the upper genital tract and invasion of tubes and ovaries: report of a case with human papilloma virus analysis.

A 55-year-old woman, who was found to have malignant squamous cells on a routine cervical smear, underwent a conization biopsy, followed by hysterectomy with bilateral salpingo-oophorectomy. No gross tumor was present in the uterus, but both ovaries, which were of normal size, contained multiple cysts filled with light brown, soft material. Microscopic examination showed squamous cell carcinoma in situ of the cervix with contiguous spread to the endometrium, fallopian tubes, and ovaries; squamous cell carcinoma extensively replaced the endometrial and tubal epithelium, focally invaded the wall of the fallopian tubes, and involved the parenchyma of both ovaries. Although an invasive cervical carcinoma occasionally spreads to the ovary, this case illustrates that exceptionally an in situ tumor spreads along the epithelium of the upper genital tract and the ovarian surface and invades the ovary and tubes. The detection of human papillomavirus DNA in the cervical, endometrial, tubal, and ovarian tumors by the polymerase chain reaction suggests a role for human papilloma virus infection in this case.

Accelerated acute rejection of an apparent A2 renal allograft in an O recipient: report of a case with flow cytometric analysis.

We report a case of accelerated acute rejection of a renal allograft from a presumed ABO histo-blood group A2 donor in an O recipient, in which all of the published criteria for compatibility had been met. Flow cytometric analysis of the A and H antigen expression on the kidney donor's erythrocytes suggested that this donor did not have an A2 phenotype, but rather another subgroup of A. Some of the reported cases of accelerated acute rejection of A2 renal allografts in O recipients may have resulted from misapplication of the results of standard lectin agglutination to the transplant setting. The current case suggests that a more sophisticated method of ABO phenotyping, such as erythrocyte flow cytometric analysis, may be necessary in the transplant setting.

Primary squamous cell carcinoma of the ovary. Report of 37 cases.

A total of 37 cases of ovarian primary squamous cell carcinoma (SCC)-19 associated with a dermoid cyst (SCCD), seven associated with endometriosis (SCCE), and 11 pure (SCCP)-are described. The last 18 cases belong within the new World Health Organization category of SCC in the surface epithelial-stromal category. The 19 patients with SCCD were 21-75 (mean, 52) years old; three of the carcinomas were in situ and seven, six, and three tumors were stages I, II, and III, respectively. The tumors and associated dermoid cysts were 6-35 cm in greatest dimension, usually forming mural nodules with intracavitary protrusion and focal necrosis and hemorrhage; two, seven, and seven tumors were grades 1, 2, and 3, respectively. SCCD was focally associated with a columnar epithelial cyst lining in 13 cases, suggesting an origin therein. One patient with stage I, grade 1 SCCD also had squamous cell carcinoma in situ (CIS) of the cervix. The seven patients with SCCE were 29-70 (mean, 49) years old, and one, three, one, and two tumors were stages I, II, III, and IV, respectively; all of the tumors were grade 3. One was associated with squamous cell carcinoma in situ of the cervix. The 11 patients with SCCP were 27-73 (mean, 56) years old, and one, four, five, and one tumors were stages I, II, III, and IV, respectively. The tumors were 6-26 cm in greatest diameter, usually solid with focal necrosis; one and 10 tumors were grades 2 and 3, respectively. Three patients with SCCP also had cervical squamous cell carcinoma in situ. The patients with SCCE had a poorer overall survival than those with SCCD. Five of the six patients with SCCE for whom adequate follow-up information was available died of their disease (mean survival, 5 months); also, in all five cases of SCCE reported in the literature, the patients died of their disease (mean survival, 4 months). The stage of the tumor and its grade correlated best with overall survival for all three types of SCC.

Isolation of presumptive Streptobacillus moniliformis from abscesses associated with the female genital tract.

We report three cases in which Streptobacillus moniliformis was isolated from abscesses. Abscess material in each case contained small, pleomorphic, gram-negative to gram-variable bacilli. Anaerobic blood agar cultures yielded pinpoint colonies adjacent to small gray-white colonies. The pinpoint colonies did not gram stain, and the gray-white colonies varied from gram-variable coccobacilli to long, curly, gram-variable rods. The pinpoint colonies microscopically resembled L-forms on Dienes-stained agar preparation. Subculture to serum-supplemented thioglycolate broth demonstrated "puff ball" colonies. Fatty acid profiles obtained with use of gas chromatography coupled with mass spectrometry showed major peaks for C16:0, C18:2, C18:1, and C18:0 fatty acids, a profile characteristic of S. moniliformis. Results of biochemical testing of each isolate were equivocal. S. moniliformis, bacterial L-forms, and common isolates from genital tract abscesses are discussed.

Cyst fluid NB/70K concentration and leukocyte esterase: two new markers for differentiating pancreatic serous tumors from pseudocysts.

Cystic lesions of the pancreas include inflammatory pseudocysts, serous cystadenomas, and mucinous tumors, some of which are malignant. Preoperative clinical and radiological parameters are unreliable and may result in incorrect diagnosis and inappropriate treatment. Cyst fluid analysis for cytology, viscosity, carcino-embryonic antigen, CA 72-4, and CA 15-3 will distinguish mucinous from nonmucinous lesions and usually help in determining malignancy. Currently, there is no reliable method to differentiate inflammatory pseudocysts from serous cystadenomas. This distinction is important because the treatment of these two lesions is different; pseudocysts are either observed or drained, whereas serous tumors are usually resected. The tumor marker NB/70K was measured in aspirated cyst fluid from 13 inflammatory pseudocysts and 11 serous cystadenomas by a commercial immunoassay. Leukocyte esterase was measured using Chemstrip SG urine test strips and amylase and lipase on a routine chemistry analyzer. The cyst fluid NB/70K concentration was significantly higher in pseudocysts (mean, 555 U/ml; range, 42-1,926 U/ml) than in serous cystadenomas (mean, 12 U/ml; range 0-130 U/ml) and this difference was significant (p < 0.0002). Leukocyte esterase was detected in 7 of 11 pseudocysts but was absent in 10 of 10 serous tumors (p = 0.002). Amylase and lipase values were generally higher in pseudocysts but these markers were unreliable due to marked outliers. Cyst fluid NB/70K and leukocyte esterase are promising markers to help differentiate pseudocysts from serous tumors on percutaneous aspirates. When combined with previously reported cyst fluid parameters (amylase, lipase, cytology, and amylase isoenzymes), these two cystic lesions can be reliably distinguished.

Variability in cyst fluid carcinoembryonic antigen level, fluid viscosity, amylase content, and cytologic findings among multiple loculi of a pancreatic mucinous cystic neoplasm.

The procedure of percutaneous aspiration and analysis of cyst contents has been advocated to provide a preoperative diagnosis of pancreatic cystic lesions (pseudocysts and cystic tumors), but it is not known whether variation in the contents of separate loculi of a multilocular neoplasm might misrepresent the identity of the tumor. The authors measured the cyst fluid carcinoembryonic antigen (CEA) level, fluid viscosity, and amylase content and performed cytologic analysis on aspirates rates from ten different loculi of a single mucinous cystic neoplasm of the pancreas. The CEA levels were highly variable (median, 6,326 ng/mL; range, 962-64,670 ng/mL) but in all cases were diagnostic of a mucinous tumor. Fluid relative viscosity values were also variable (median, 2.4; range, 1.3-10+) but diagnostic in eight of nine aspirates. The amylase content in all of the loculi was low (< 91 U/L), and values were consistent with a cystic tumor. Cytologic analysis showed diagnostic mucin-secreting epithelial cells in nine of ten loculi. Although cytologic examination was nondiagnostic in one loculus, there were no false-positive results for malignancy. The combination of all four tests would not have resulted in misclassification of any of the tumors. The authors conclude that the characteristics of the contents of different loculi of pancreatic cystic neoplasms may be variable, but the use of a combination of tests still ensures accurate diagnosis.