Gefitinib in definitive management of esophageal or gastroesophageal junction cancer: a retrospective analysis of two clinical trials.

The role of epidermal growth factor receptor inhibition in resectable esophageal/gastroesophageal junction (E/GEJ) cancer is uncertain. Results from two Cleveland Clinic trials of concurrent chemoradiotherapy (CCRT) and surgery are updated and retrospectively compared, the second study differing only by the addition of gefitinib (G) to the treatment regimen. Eligibility required a diagnosis of E/GEJ squamous cell or adenocarcinoma, with an endoscopic ultrasound stage of at least T3, N1, or M1a (American Joint Committee on Cancer 6th). Patients in both trials received 5-fluorouracil (1000 mg/m(2) /day) and cisplatin (20 mg/m(2) /day) as continuous infusions over days 1-4 along with 30 Gy radiation at 1.5 Gy bid. Surgery followed in 4-6 weeks; identical CCRT was given 6-10 weeks later. The second trial added G, 250 mg/day, on day 1 for 4 weeks, and again with postoperative CCRT for 2 years. Preliminary results and comparisons have been previously published. Clinical characteristics were similar between the 80 patients on the G trial (2003-2006) and the 93 patients on the no-G trial (1999-2003). Minimum follow-up for all patients was 5 years. Multivariable analyses comparing the G versus no-G patients and adjusting for statistically significant covariates demonstrated improved overall survival (hazard ratio [HR] 0.64, 95% confidence interval [CI] = 0.45-0.91, P = 0.012), recurrence-free survival (HR 0.61, 95% CI = 0.43-0.86, P = 0.006), and distant recurrence (HR 0.68, 95% CI = 0.45-1.00, P = 0.05), but not locoregional recurrence. Although this retrospective comparison can only be considered exploratory, it suggests that G may improve clinical outcomes when combined with CCRT and surgery in the definitive treatment of E/GEJ cancer.

Clinicopathologic features and treatment outcomes of patients with human epidermal growth factor receptor 2-positive adenocarcinoma of the esophagus and gastroesophageal junction.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 21% of gastric and 33% of gastroesophageal junction (GEJ) adenocarcinomas. Trastuzumab has been approved for metastatic HER2-positive gastric/GEJ cancer in combination with chemotherapy. This retrospective analysis was undertaken to better define the clinicopathologic features, treatment outcomes, and prognosis in patients with HER2-positive adenocarcinoma of the esophagus/GEJ. Pathologic specimens from 156 patients with adenocarcinoma of the esophagus/GEJ treated on clinical trials with chemoradiation and surgery were tested for HER2. Seventy-six patients also received 2 years of gefitinib. Baseline characteristics and treatment outcomes of the HER2-positive and negative patients were compared both in aggregate and separately for each of the two trials. Of 156 patients, 135 had sufficient pathologic material available for HER2 assessment. HER2 positivity was found in 23%; 28% with GEJ primaries and 15% with esophageal primaries (P= 0.10). There was no statistical difference in clinicopathologic features between HER2-positive and negative patients except HER2-negative tumors were more likely to be poorly differentiated (P < 0.001). Locoregional recurrence, distant metastatic recurrence, any recurrence, and overall survival were also statistically similar between the HER2-positive and the HER2-negative groups, in both the entire cohort and in the gefitinib-treated subset. Except for tumor differentiation, HER2-positive and negative patients with adenocarcinoma of the esophagus and GEJ do not differ in clinicopathologic characteristics and treatment outcomes. Given the demonstrated benefit of trastuzumab in HER2-positive gastric cancer and the similar incidence of HER2 overexpression in esophageal/GEJ adenocarcinoma, further evaluation of HER2-directed therapy in this disease seems indicated.

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.

Resistance to anti-HER2 (human epithelial growth factor receptor 2) trastuzumab therapy occurs commonly in HER2-positive breast cancer and involves overactivation of HER2 and/or AKT1. Using the model of trastuzumab-sensitive or trastuzumab-resistant HER2-positive cells with wild-type PTEN, negative regulator of AKT1, we explore the involvement of cysteine protease calpain in mechanisms of trastuzumab resistance. Overexpression of calpain1 or activation of endogenous calpain during adhesion or trastuzumab treatment of trastuzumab-sensitive cells induces cleavage of cytoplasmic domains of HER2/phospho-HER2; cleavage occurs in HER2-positive tumors. Expression of the catalytically inactive mutant of calpain1 reduces the cleavage to enhance the activity of HER2, inactivates PTEN to enhance the activation of AKT1, induces desensitization to trastuzumab and promotes survival of trastuzumab-sensitive cells. In the model of trastuzumab resistance, constitutive overactivation of HER2 and AKT1 correlates with reduced activation of calpain. Moreover, inhibitors of the catalytic site of calpain reduce the increase in constitutive activity of AKT1 and survival of trastuzumab-resistant cells selectively. Together, by regulating the activation of HER2 and PTEN/AKT1, calpain regulates trastuzumab sensitivity and survival, and the deregulation of the activation of calpain promotes trastuzumab resistance. Trastuzumab-resistant cells activate AKT1 in a mechanism dependent on the residual calpain activity, inhibition of which restores trastuzumab sensitivity and rescues resistance. These data identify calpain as a new therapeutic target in HER2-positive breast cancer.

Prognostic value of regulatory T cells, lymphoma-associated macrophages, and MUM-1 expression in follicular lymphoma treated before and after the introduction of monoclonal antibody therapy: a Southwest Oncology Group Study.

BACKGROUND: The purpose was to examine the prognostic impact of features of tumor cells and immune microenvironment in patients with follicular lymphoma treated with and without anti-CD20 monoclonal antibody therapy. PATIENTS AND METHODS: Tissue microarrays were constructed from archived tissue obtained from patients on three sequential Southwest Oncology Group (SWOG) trials for FL. All three trials included anthracycline-based chemotherapy. Anti-CD20 monoclonal antibodies were included for patients in the latter two trials. Immunohistochemistry was used to study the number and distribution of cells staining for forkhead box protein P3 (FOXP3) and lymphoma-associated macrophages (LAMs) and the number of lymphoma cells staining for myeloma-associated antigen-1 (MUM-1). Cox proportional hazards regression was used to evaluate the association between marker expression and overall survival (OS). RESULTS: The number or pattern of infiltrating FOXP3 cells and LAMs did not correlate with OS in sequential SWOG studies for FL. The presence of MUM-1 correlated with lower OS for patients who received monoclonal antibody but not for those treated with chemotherapy alone. CONCLUSIONS: Immune cell composition of lymph nodes did not correlate with OS in this analysis of trials in FL. The mechanism of the observed correlation between MUM-1 expression and adverse prognosis in patients receiving monoclonal antibody therapy requires confirmation.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

Posttransplant lymphoproliferative disorders in lung transplant patients: the Cleveland Clinic experience.

PTLD is a well-recognized complication of organ transplantation. Large series of heart, renal, and liver transplants have been examined for the incidence and behavior of PTLD. However, reports of the incidence and characteristics of PTLDs in lung transplant (LTx) patients are few. We report our experience with PTLDs in a large series of LTx recipients at a single institution and compare them to other solid organ transplant recipient PTLDs seen at our institution. Twenty-eight patients were found to have PTLD, of whom 8 were lung transplant recipients. We evaluated nine PTLD specimens from these 8 patients for their histology, immunophenotype (CD20, CD3, EBV-LMP1), EBER status by in situ hybridization, and clinical features. The incidence of PTLD was 3.3% (8/244 patients). The time to development of PTLD, after transplant, was short (median time, 7 mo). All were of B-cell lineage. Overall, EBV was demonstrated in 77.7% (7 of 9 specimens) of PTLDs. All specimens tested for clonality were found to be monoclonal. Five patients died, with a median time to death of only 4.6 months. PTLDs in LTx patients are EBV-associated B-cell, predominantly monoclonal lymphoid lesions similar to other solid organ transplant PTLDs. Compared with other solid organ transplant recipients with PTLD at our institution, PTLDs in LTx patients have a propensity to involve the transplanted organ (P =.001, Fisher's exact test), occur earlier after transplant (P =.003, Wilcoxon test), and have a shorter survival (P =.002, log rank test). Reasons for this may include the relatively higher level of immunosuppression required in these patients and limited options in decreasing it. Although the incidence is low, careful early monitoring of lung transplantation patients is warranted because of the poor prognosis of patients developing this complication.

Typical and atypical chronic lymphocytic leukemia differ clinically and immunophenotypically.

We compared the features of 17 cases of atypical chronic lymphocytic leukemia (aCLL) with those of a clinical control group of 24 cases of CLL. Quantitative flow cytometric data, available for 12 cases, were compared with an immunophenotypic control group of 58 cases using a relative fluorescence indexfor CD5, CD23, CD79b, and surface immunoglobulin light chain (sIg). Compared with the clinical control group, patients with aCLL had a higher mean WBC count and a lower platelet count. Patients with aCLL had a significantly higher probability of disease progression. Compared with an immunophenotypic control group of 58 CLL cases, 12 cases of aCLL demonstrated significantly higher expression of CD23. There was no significant difference in expression of sIg, CD79b, or CD5 between the groups. CD38 expression was noted in only 1 (9%) of 11 tested cases; 2 (18%) of 11 cases had trisomy 12. aCLL can be distinguished from typical CLL morphologically, clinically, and immunophenotypically. Atypical morphologic features in CLL seem to be a marker of aggressive clinical behavior.

Angiotropic lymphoma: an immunophenotypically and clinically heterogeneous lymphoma.

Angiotropic lymphoma (AL) is an uncommon lymphoma often presenting with nonspecific clinical features and having a high mortality rate. Although not specifically recognized by the Revised European-American Classification of Lymphoid Neoplasms, it likely will appear as a subtype of diffuse large B-cell lymphoma in the upcoming WHO classification. Some authors may also consider it to be a subtype of cutaneous lymphomas. Recent studies have reported an immunophenotypic heterogeneity of AL, and in rare instances, an association with other NHL. To further characterize AL, we studied the immunophenotype by immunohistochemistry for CD5, CD10, CD20, bcl-2, and bcl-6 in 18 cases of B-cell AL identified at three medical centers in North America. Bcl-2 gene rearrangement status by polymerase chain reaction and Epstein Barr virus status by in situ hybridization also were evaluated. Eight men and 10 women were identified with AL (median age 71 years). Eleven patients were diagnosed in life and seven were diagnosed at autopsy. Neurologic symptoms were the most common presentation, seen in six patients. Skin was the most commonly biopsied site. All showed classic intravascular localization; in two cases, there was also a minor diffuse large cell lymphoma component observed in some organs. Most (89%) of the cases expressed bcl-2 protein; CD10, bcl-6 and CD5 were each expressed in 22% of cases. Based on CD5 and CD10 expression, three major groups were evident: CD5-, CD10- (11 cases); CD5+, CD10- (3 cases), and CD5-, CD10+ (3 cases). Even though a follicle center lymphoma preceded the AL in one patient, we did not detect bcl-2 gene rearrangement in any of these cases. All cases were negative for Epstein Barr virus. Of the five treated with chemotherapy, two achieved a complete remission. Based on these findings, we conclude that ALs are clinically and immunophenotypically heterogeneous and may represent more than one pathogenetic entity. In some instances AL may be preceded by another lymphoproliferative disorder, raising the possibility that some cases of AL may represent a transformation from another type of lymphoma. Cutaneous manifestations of AL are common; however, it appears to be a systemic lymphoma. Although often fatal, patients with AL who are diagnosed early and treated with chemotherapy may achieve remission.

Evaluation of a new paraffin-reactive CD7 T-cell deletion marker and a polymerase chain reaction-based T-cell receptor gene rearrangement assay: implications for diagnosis of mycosis fungoides in community clinical practice.

BACKGROUND: T-cell deletion and T-cell receptor (TCR) gene rearrangement studies are helpful in the early diagnosis and subsequent management of mycosis fungoides (MF). However, this often requires fresh-frozen tissue that can be difficult to obtain and evaluate in community clinical practice. A new CD7 antibody, the most sensitive and specific T-cell deletion marker, and a new TCR-gamma gene rearrangement polymerase chain reaction (PCR) assay (TCR-gamma PCR) are now available on routine paraffin-embedded biopsy specimens. OBJECTIVE: Our purpose was to assess the utility of CD7 deletion and TCR-gamma PCR in the diagnosis of MF using routine paraffin-embedded biopsy material. METHODS: Cases of MF (n = 17) with matching frozen tissue immunohistochemistry and benign reactive dermatoses (lichen planus; n = 27) were assessed for CD7 (Clone: CD7-272) deletion and TCR-gamma PCR using paraffin-embedded biopsy specimens. RESULTS: Excellent concordance comparing frozen and paraffin embedded CD7 immunostaining (88%) was observed. CD7 deletion and TCR-gamma PCR was sensitive (94%) and specific (96%) for a diagnosis of MF using paraffin-embedded biopsy specimens. CONCLUSION: In the diagnosis of MF, detection of CD7 deletion and monoclonal TCR rearrangements can be successfully performed in a cost-effective, timely fashion using routine formalin-fixed paraffin-embedded biopsy specimens.

Breast tumor immunophenotype of BRCA1-mutation carriers is influenced by age at diagnosis.

PURPOSE: Breast tumors of BRCA1 mutation carriers and those of early onset breast cancer cases share similar histological features, being generally high-grade, highly proliferative, aneuploid tumors that are predominantly estrogen- and progesterone-receptor negative. Because histological features of tumors of premenopausal women differ from those of tumors of older women, we sought to determine whether the immunophenotype of breast tumors of BRCA1 mutation carriers was influenced by age at diagnosis. EXPERIMENTAL DESIGN: We examined 31 breast tumors from BRCA1 mutation carriers and compared them with 81 tumors of age-matched (plus or minus 5 years) breast cancer patients unselected for family history. Tumors were further matched for histology, grade, and size. Paraffin-embedded tumor tissues were examined for protein expression of estrogen receptor (ER), PR, Ki-67, cyclin D1, TP53, HER2, beta-catenin, and cyclin E using immunohistochemical approaches. RESULTS: ER (P = 0.01), PR (P = 0.06), and cyclin D1 (P = 0.002) were less frequently expressed and Ki-67 (P = 0.01) and beta-catenin (P = 0.04) were more frequently expressed in tumors of BRCA1 mutation carriers than controls. After age stratification, we found a significant difference in the frequency of tumors of BRCA1 mutation carriers diagnosed before 50 years of age compared with age-matched controls that stained positive for ER (P = 0.01), PR (P = 0.03), Ki-67 (P = 0.008), cyclin D1 (P < 0.001), HER2 (P = 0.04), and beta-catenin (P = 0.05). However, no significant differences were observed in tumors of BRCA1 mutation carriers diagnosed at age 50 or older compared with age-matched controls. CONCLUSIONS: These data suggest that age at diagnosis, possibly related to menopausal status, may be an important factor in the expression of specific proteins in breast tumors of BRCA1 mutation carriers.

Absence of CCND1 gene amplification in breast tumours of BRCA1 mutation carriers.

BACKGROUND/AIMS: It was recently reported that significantly fewer breast tumours of BRCA1 mutation carriers overexpressed cyclin D1 and HER2 protein than tumours of age matched breast cancer cases unselected for family history. This study aimed to examine the genetic basis of this reduction by determining the frequency of tumours within this cohort showing DNA amplification of these genes. METHODS: Paraffin wax embedded sections of breast tumours from BRCA1 mutation carriers and age, grade, histological type, and tumour size matched non-familial controls that had previously been stained for cyclin D1 and HER2 protein overexpression were analysed for CCND1 and HER2 gene amplification using fluorescence in situ hybridisation. RESULTS: CCND1 amplification was detected in none of the 30 tumours of the BRCA1 mutation carriers and in 19 of 74 tumours of the matched controls. Of those samples previously determined to overexpress the HER2 protein, HER2 amplification was detected in one of three tumours from BRCA1 mutation carriers and in 13 of 17 tumours of the age matched non-familial cases. CONCLUSION: None of the tumours of BRCA1 mutation carriers showed CCND1 amplification and only one tumour showed HER2 amplification. In contrast, a large proportion of cyclin D1 and HER2 overexpression in tumours of non-familial breast cancer cases could be accounted for by amplification of these genes. These data suggest that breast tumorigenesis in BRCA1 mutation carriers occurs by a molecular mechanism distinct from that of age matched non-familial cases.

Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.

BACKGROUND: Several studies have reported what seem to be false-positive results using the Food and Drug Administration (FDA)-approved HercepTest (Dako Corp, Carpinteria, CA) to profile Her-2/neu amplification and overproduction in breast carcinoma. False-positive status has been based on comparisons with gene copy enumeration by fluorescence in situ hybridization (FISH) and with comparisons to immunohistochemistry (IMH) results using a monoclonal antibody. However, simple overexpression by tumor cells that have normal gene copy has not been evaluated by profiling mRNA expression, ie, such cases could simply represent true-positive, transcriptionally upregulated overexpression. MATERIALS AND METHODS: Four hundred infiltrating ductal carcinomas of breast were evaluated by IMH using monoclonal (CB11; Ventana Medical Systems, Inc, Tucson, AZ) and polyclonal (HercepTest; Dako) antibodies after antigen retrieval (AR). A polyclonal antibody sans AR (PCA/SAR) was also used. All IMH stains were evaluated and scored according to the guidelines for the FDA-approved HercepTest. A total of 145 of 400 carcinomas were subsequently evaluated by direct and digoxigenin-labeled (Dig) FISH, and 144 of 400 were evaluated by detection of mRNA overexpression via autoradiographic RNA:RNA in situ hybridization. RESULTS: Overall HercepTest/CB11 IMH discordance was 12%. Expression of mRNA was highly concordant with FISH and DigFISH amplification and with CB11 and PCA/SAR immunohistology. IMH false-positive cases (no Her-2/neu gene amplification) occurred with both HercepTest (23%) and CB11 (17%), and the majority of false-positive results (34 of 44) were scored as 2+. All 2+ false-positive cases were mRNA-negative. Combined results of HercepTest and CB11 showed that 79% (38 of 48) of 3+ cases were Her-2/neu gene amplified, but only 17% (seven of 41) of 2+ cases had increased gene copy. CONCLUSION: Discordant HercepTest/FISH results, and to a lesser extent discordance with CB11 IMH, are most commonly false-positive results with a score of 2+. The 2+ score as defined in the guidelines for the FDA-approved HercepTest should not be used as a criterion for trastuzumab therapy unless confirmed by FISH. Determination of Her-2 gene copy number by FISH may be a more accurate and reliable method for selecting patients eligible for trastuzumab therapy.

Aneusomy of chromosomes 7, 8, and 17 and amplification of HER-2/neu and epidermal growth factor receptor in Gleason score 7 prostate carcinoma: a differential fluorescent in situ hybridization study of Gleason pattern 3 and 4 using tissue microarray.

Recent evidence shows that the proportion of poorly differentiated prostate carcinoma (Gleason pattern [GP] 4/5) is a surrogate factor for biochemical failure after radical prostatectomy (RP). However, little is known about specific molecular and cytogenetic changes in this aggressive component of localized prostate cancer. We constructed a tissue microarray containing areas of GP 3 and 4 from formalin-fixed radical prostatectomy specimens of 39 patients with Gleason score 7 carcinoma (>or=50% GP 4), known pathologic staging parameters (stage < T3b), and biochemical failure data (mean follow-up, 30 months; range, 5 to 74 months). Interphase fluorescent in situ hybridization (FISH) was performed on 5-microm microarray sections using pericentromeric probes to chromosomes 7, 8, and 17 and probes for the HER-2/neu and epidermal growth factor receptor (EGFR) genes. Low-level amplification of HER-2/neu was found in 26% of cases (3 to 5 signals per nucleus, corrected for chromosome 17 aneusomy). Aneusomy of chromosomes 7, 8, and 17 was identified in 21%, 15%, and 5% of cases, respectively. All aberrations occurred almost exclusively in GP 4 carcinoma (8 of 8 aneusomies 7, 2 of 2 trisomies 17, 9 of 10 HER-2/neu amplifications, and 5 of 6 aneusomies 8; P < .001). The presence of HER-2/neu amplification was associated with high tumor volume (>2.0 cm(3), P = 0.004). Among patients with negative surgical margins, gain of chromosome 7 was associated with biochemical failure after RP (P =.004, log-rank). Amplification of the EGFR gene occurred in only 1 case (3%). Significant differences in HER-2/neu amplification and gain of chromosomes 7, 8, and 17 were detected between GP 4 prostate carcinoma and GP 3. The frequency of aberrations increased with tumor volume. Chromosome 7 abnormalities may play an important role in cancer progression in margin-negative patients. EGFR amplification was rare, suggesting that this oncogene is not altered at the gene copy number level.

Use of novel t(11;14) and t(14;18) dual-fusion fluorescence in situ hybridization probes in the differential diagnosis of lymphomas of small lymphocytes.

Increasingly, molecular biologic techniques have become important in the diagnosis of non-Hodgkin lymphomas. In the differential diagnosis of lymphoma(s) of small lymphocytes (LSL), reliable detection of t(11;14) or t(14;18) would confirm the diagnosis of mantle cell lymphoma (MCL) or follicle center lymphoma (FCL), respectively. A total of 87 LSL cases (27 MCL, 39 FCL, 17 small lymphocytic lymphoma [SLL], 3 marginal zone lymphomas, and 1 paraimmunoblastic variant of SLL) were diagnosed by a combination of light microscopy, immunohistochemistry, and flow cytometric immunophenotyping. Interphase fluorescence in situ hybridization (FISH) for t(11;14) and t( 14;18) using dual-fusion probes (Vysis, Downers Grove, IL) was performed on touch (n = 69) or gravity (n = 18) preparations from these cases. Of 27 MCL cases tested, 25 (93%) had demonstrable t(11;14), none had t(14;18), and 2 were negative for t(11;14) and t(14;18). Twenty-five of 39 (64%) FCL cases had t(14;18), none had t(11;14), and the remaining FCL cases (14 cases [35%]) had neither t(11;14) nor t(14;18). All 17 (100%) SLL cases had neither t(11;14) nor t(14;18). All 3 (100%) marginal zone lymphoma cases had neither t(11;14) nor t(14;18). The case of paraimmunoblastic variant of SLL had t(11;14) and was negative for t(14;18). No discrepant [i.e., positive for both t(11;14) and t(14;18)] or false-positive cases were noted. Interphase FISH using these commercially available probes is a useful adjunct to light microscopy, immunohistochemistry, and flow cytometric immunophenotyping in the diagnosis of LSL. FISH can be performed successfully on archival single-cell preparations (touch preparations or gravity preparations) when fresh tissue is unavailable. No discordant or false-positive cases were identified.

Validation of a multicolor interphase fluorescence in situ hybridization assay for detection of transitional cell carcinoma on fresh and archival thin-layer, liquid-based cytology slides.

OBJECTIVE: To evaluate the feasibility of performing multicolor interphase fluorescence in situ hybridization (FISH) on ThinPrep slides of transitional cell carcinoma (TCC). STUDY DESIGN: Slides from 20 voided urine specimens were prepared by the ThinPrep technique (Cytyc, Boxborough, Massachusetts, U.S.A.), pretreated using a pretreatment kit and subjected to hybridization with the multicolor FISH probe UroVysion (Vysis, Downers Grove, Illinois, U.S.A.). Archival slides were placed in xylene, destained in alcohol and washed prior to pretreatment. Urines from patients with cytology-positive, biopsy-proven grade 1 (n = 5), 2 (n = 7) and 3 (n = 5) TCC and negative cytology and biopsy (n = 3) were selected. Freshly prepared (n = 10) and archival (n = 10) slides were used. RESULTS: All carcinoma cases were FISH positive (> 5 cells with complex abnormalities of > or = 2 studied chromosomes per slide). None of the normal samples were aneusomic. Gain of chromosomes 3, 7 and 17 constituted the majority of positive cases. Proper destaining and slight decrease in stringency wash conditions enabled reliable detection of signals in archival cases. CONCLUSION: Routine ThinPrep slides can be used for multicolor interphase FISH analysis of urine cytology specimens. Archival slides provide the opportunity to analyze by FISH the nature of atypical cells identified by cytology. This revised method allows FISH technology more accessibility for routine use in cytology laboratories.

Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma.

Three different in situ hybridization (ISH) methods were compared for their clinical relevance and suitability in detecting human papillomavirus (HPV) 16/18 in 55 cases of squamous cell carcinoma (SCC) of the uterine cervix. After the initial biopsy, surgery, and/or radiation therapy, patients were followed for 5 to 8 years. A biotinylated cDNA probe for HPV 16/18 was applied to serial sections in combination with conventional streptavidin-biotin-peroxidase ISH (a widely applied routine procedure), streptavidin-Nanogold-silver ISH, and tyramide-signal amplified (TSA) streptavidin-Nanogold-gold ISH. The TSA principle is also known as catalyzed reporter deposition and is, apart from in situ PCR, probably today's most sensitive technique for detecting papillomavirus infection by microscopic means. Nearly 65.5% of the cases showed specific HPV 16/18 detection with TSA ISH, whereas 43.6% were positive with streptavidin-Nanogold-silver-ISH, and only 40.0% with peroxidase-based ISH. Statistical analyses comparing early and advanced stages in both HPV-positive and -negative groups revealed a significantly better outcome for early disease patients; statistical significance was most pronounced with TSA ISH. In a subgroup of patients who had received radiation therapy without prior surgery (n = 35), those with advanced disease were significantly less likely to have HPV 16/18 infection than those with early disease. A significantly better overall survival was observed in those women with HPV 16/18-positive carcinomas who had undergone surgery before radiation therapy (seen with all three methods). We conclude that TSA, in addition to being the most sensitive HPV in situ method applied in this study, gave the most significant and clinically relevant statistical results.

The nature of the myenteric infiltrate in achalasia: an immunohistochemical analysis.

Achalasia is an esophageal motor disorder characterized by abnormal relaxation of the lower esophageal sphincter and absence of progressive peristalsis in the esophageal body. Previous studies evaluating esophagomyotomy and esophageal resection specimens have shown the presence of myenteric inflammation to be a consistent and early pathologic change in patients with achalasia. Thus, the goal of this study was to determine the immunohistochemical characteristics of the inflammatory infiltrate within the myenteric plexus in patients with clinically early and end-stage achalasia. Using formalin-fixed tissue, we analyzed the immunohistochemical features of the myenteric lymphocytes using antibodies that recognize B cells (CD20), T cells (CD3), T cell subsets (CD8), and the activation state of T cell subpopulations (TIA-1 and granzyme B) in nine patients with clinically early achalasia who underwent esophagomyotomy and 13 patients with clinically endstage achalasia who underwent esophageal resection. The myenteric infiltrate in all nine esophagomyotomy specimens was composed predominantly of T cells (CD3-positive), the majority of which also stained for CD8. In five of nine specimens, the majority of CD8-positive cells stained for TIA-1. In the esophageal resection specimens, the myenteric infiltrate was composed predominantly of CD3-positive T cells in seven of 13 cases. In three cases, there was a predominance of CD20-positive B cells, and in the remaining three cases there were relatively equal numbers of T and B cells. In eight of 13 cases, the majority of T cells stained for CD8. TIA-1 immunoreactivity was found in the majority of CD8-positive cells in nine of 13 cases. In all esophagomyotomy and esophageal resection specimens studied, rare granzyme B-positive cells were detected. In conclusion, the majority of myenteric inflammatory cells in patients with achalasia are CD3-positive T cells, most of which are also CD8-positive, although the relative percentage of such cells appears to decrease with disease progression. Furthermore, many of the CD3-positive/CD8-positive myenteric lymphocytes also express TIA-1, suggesting they are resting or activated cytotoxic T cells. The immunohistochemical demonstration of granzyme B in a subpopulation of these cells supports the contention that achalasia is an immune-mediated disease, although the inciting antigen remains an enigma.

Biclonal chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is well characterized clinically and immunophenotypically. Demonstration of a monotypic CD19+, CD5+ B-cell population is central to the diagnosis. We report 2 cases of biclonal CLL. Two elderly men were encountered with an absolute lymphocytosis consisting of the typical CD5+, CD19+, CD23+ B-cell population seen in CLL; however, immunoglobulin light chain restriction by flow cytometry was not apparent as B cells expressed kappa or lambda light chains without a clear monotypic population. Molecular genetic analysis of flow cytometry-sorted cells (kappa and lambda populations) revealed in both cases 2 monoclonal B-cell populations. The characterization of these cases and a review of the issues surrounding biclonal CLL are presented.

Clinicopathologic reassessment of primary cutaneous B-cell lymphomas with immunophenotypic and molecular genetic characterization.

Primary cutaneous B-cell lymphomas (PCBLs) may have particular clinicopathologic characteristics distinct from their lymph node-based counterparts. It has been suggested that PCBLs should have a separate classification system. The aim of this study was to determine whether the Revised European-American Lymphoid Neoplasms (REAL) classification is applicable to PCBL. Thirty-nine cases of PCBL from 36 patients, consisting of 20 men and 16 women (median age 66 yrs), were included in this study. Paraffin-section immunohistochemistry for CD3, CD5, CD10, CD20, CD43, Bcl-2, Bcl-6, and cyclin D1 was performed in all cases. Immunostaining for immunoglobulin light chains was also performed on cases histologically diagnosed as extranodal marginal zone lymphoma (MZL) and primary cutaneous B-cell lymphoma unclassifiable (PCBLu). Polymerase chain reaction (PCR) analysis of t(14;18) was performed in all cases. Immunoglobulin heavy chain gene rearrangement (VDJ) was tested by PCR on all follicle center lymphoma (FCL), MZL, and PCBLu cases. The 39 cases consisted of 15 (39%) FCLs, 13 (33%) diffuse large B-cell lymphomas (DLCL), 9 (23%) extranodal MZL, and 2 cases of PCBLu. Anatomically, 59% of PCBLs occurred in the head and neck, of which approximately 57% were FCL. Five of six cases presenting on the lower extremity were DLCL. Follow-up data was available from all 39 patients with a mean of 50.8 months. All but two patients are alive with or without disease at last contact. One patient with DLCL died of lung metastases and the other DLCL patient died of sepsis as a complication of therapy. In all 15 cases of FCL, CD10 and/or Bcl-6 expression supported the follicle center origin of the neoplastic cells. In contrast to previous reports, we found that 53% (8 of 15) of primary cutaneous FCL had either Bcl-2 protein expression or t(14;18). Our data indicate that many cases of primary cutaneous FCL have Bcl-2 alterations similar to their nodal counterpart. We found that 95% (37 of 39) of PCBLs could be classified according to the REAL classification, supporting its applicability in cutaneous lymphomas.