Tanweer M. Zaidi

Evaluation of interphase fluorescence in situ hybridization for the t(14;18)(q32;q21) translocation in the diagnosis of follicular lymphoma on fine-needle aspirates: a comparison with flow cytometry immunophenotyping.

Diagnosing lymphoproliferative disorders on fine‐needle aspiration (FNA) can be challenging due to variable cellularity and lack of architecture. Ancillary studies often are required for diagnosis. Follicular lymphoma (FL) is characterized by a monoclonal B‐cell proliferation with coexpression of CD19/CD10 and a t(14;18)(q32;q21) reciprocal translocation, resulting in the immunoglobulin heavy chain/BCL‐2 fusion gene. These features also can be found, with much lower frequency, in diffuse large B‐cell lymphoma (DLBCL) of follicle center cell origin. The objective of the current study was to compare the accuracy in detecting FL and DLBCL of follicle center cell origin by interphase fluorescence in situ hybridization (I‐FISH) versus flow cytometry immunophenotyping (FCM) on FNAs.

Use of Fluorescence In Situ Hybridization to Predict Response to Bacillus Calmette-Guérin Therapy for Bladder Cancer: Results of a Prospective Trial

PURPOSE No reliable methods currently exist to predict patient response to intravesical immunotherapy with bacillus Calmette-Guérin given after transurethral resection for high risk nonmuscle invasive bladder cancer. We initiated a prospective clinical trial to determine whether fluorescence in situ hybridization results during bacillus Calmette-Guérin immunotherapy can predict therapy failure. MATERIALS AND METHODS Candidates for standard of care bacillus Calmette-Guérin were offered participation in a clinical trial. Fluorescence in situ hybridization was performed before bacillus Calmette-Guérin, and at 6 weeks, 3 months and 6 months during bacillus Calmette-Guérin therapy with maintenance. Cox proportional hazards regression was used to assess the relationship between fluorescence in situ hybridization results and tumor recurrence or progression. The Kaplan-Meier product limit method was used to estimate recurrence-free and progression-free survival. RESULTS A total of 126 patients participated in the study. At a median followup of 24 months 31% of patients had recurrent tumors and 14% experienced disease progression. Patients who had positive fluorescence in situ hybridization results during bacillus Calmette-Guérin therapy were 3 to 5 times more likely than those who had negative fluorescence in situ hybridization results to experience recurrent tumors and 5 to 13 times more likely to have disease progression (p <0.01). The timing of positive fluorescence in situ hybridization results also affected outcomes. For example, patients with a negative fluorescence in situ hybridization result at baseline, 6 weeks and 3 months demonstrated an 8.3% recurrence rate compared to 48.1% for those with a positive result at all 3 points. CONCLUSIONS Fluorescence in situ hybridization results can identify patients at risk for tumor recurrence and progression during bacillus Calmette-Guérin immunotherapy. This information may be used to counsel patients about alternative treatment strategies.

Automated detection of genetic abnormalities combined with cytology in sputum is a sensitive predictor of lung cancer

Detection of lung cancer by sputum cytology has low sensitivity but is noninvasive and, if improved, could be a powerful tool for early lung cancer detection. To evaluate whether the accuracy of diagnosing lung cancer by evaluating sputa for cytologic atypia and genetic abnormalities is greater than that of conventional cytology alone, automated scoring of genetic abnormalities for 3p22.1 and 10q22.3 (SP-A) by fluorescence in situ hybridization (FISH) and conventional cytology was done on sputa from 35 subjects with lung cancer, 25 high-risk smokers, and 6 healthy control subjects. Multivariate analysis was performed to select variables that most accurately predicted lung cancer. A model of probability for the presence of lung cancer was derived for each subject. Cells exfoliated from patients with lung cancer contained genetic aberrations and cytologic atypias at significantly higher levels than in those from control subjects. When combined with cytologic atypia, a model of risk for lung cancer was derived that had 74% sensitivity and 82% specificity to predict the presence of lung cancer, whereas conventional cytology achieved only 37% sensitivity and 87% specificity. For diagnosing lung cancer in sputum, a combination of molecular and cytologic variables was superior to using conventional cytology alone.

Surfactant Protein A Gene Deletion and Prognostics for Patients with Stage I Non–Small Cell Lung Cancer

Purpose: The present study was conducted to determine clinical relevance of surfactant protein A (SP-A) genetic aberrations in early-stage non–small cell lung cancer (NSCLC). Experimental Design: To determine whether SP-A aberrations are lung cancer–specific and indicate smoking-related damage, tricolor fluorescence in situ hybridization with SP-A and PTEN probes was done on touch imprints from the lung tumors obtained prospectively from 28 patients with primary NSCLC. To further define the clinical relevance of SP-A aberrations, fluorescence in situ hybridization was done on both tumor cells and adjacent bronchial tissue cells from paraffin-embedded tissue blocks from 130 patients NSCLC for whom we had follow-up information. Results:SP-A was deleted from 89% of cancer tissues and the deletion was related to the smoking status of patients (P < 0.001). PTEN was deleted from 16% in the cancer tissues and the deletion was not related to the smoking status of patients (P > 0.05). In the cells isolated from paraffin-embedded tissue blocks, SP-A was deleted from 87% of the carcinoma tissues and 32% of the adjacent normal-appearing bronchial tissues. SP-A deletions in tumors and adjacent normal-appearing bronchial tissues were associated with increases in the risk of disease relapse (P = 0.0035 and P < 0.001, respectively). SP-A deletions in the bronchial epithelium were the strongest prognostic indicators of disease-specific survival (P = 0.025). Conclusions: Deletions of the SP-A gene are specific genomic aberrations in bronchial epithelial cells adjacent to and within NSCLC, and are associated with tumor progression and a history of smoking. SP-A deletions might be a useful biomarker to identify poor prognoses in patients with NSCLC who might therefore benefit from adjuvant treatment.

Genetically Abnormal Circulating Cells in Lung Cancer Patients: An Antigen-Independent Fluorescence In situ Hybridization–Based Case-Control Study

Purpose: We performed a study to determine if a fluorescence in situ hybridization (FISH)–based assay using isolated peripheral blood mononuclear cells (PBMCs) with DNA probes targeting specific sites on chromosomes known to have abnormalities in non–small cell lung cancer (NSCLC) cases could detect circulating genetically abnormal cells (CACs). Experimental Design: We evaluated 59 NSCLC cases with stage I through IV disease and 24 controls. PBMCs and matched tumors were hybridized with 2 two-color [3p22.1/CEP3 and 10q22.3 (SP-A)/CEP10) and 2 four-color [CEP3, CEP7, CEP17, and 9p21.3 (URO); and EGFR, c-MYC, 6p11-q11, and 5p15.2 (LAV)] FISH probes. Percentages of cytogenetically abnormal cells (CACs) in peripheral blood and in matched tumor specimens were quantified by using an automated fluorescent scanner. Numbers of CACs were calculated based on the percentage of CACs (defined as PBMCs with genetic abnormalities) per milliliter of blood and expressed per microliter of blood. Results: Patients with NSCLC had significantly higher numbers of CACs than controls. Mean number of CACs ranged from 7.23 ± 1.32/μL for deletions of 10q22.3/CEP10 to 45.52 ± 7.49/μL for deletions of 3p22.1/CEP3. Numbers of CACs with deletions of 3p22.1, 10q22.3, and 9p21.3, and gains of URO, increased significantly from early to advanced stage of disease. Conclusions: We have developed a sensitive and quantitative antigen-independent FISH-based test for detecting CACs in peripheral blood of patients with NSCLC, which showed a significant correlation with the presence of cancer. If this pilot study can be validated in a larger study, CACs may have a role in the management of patients with NSCLC. Clin Cancer Res; 16(15); 3976–87. ©2010 AACR.

3p22.1 and 10q22.3 deletions detected by fluorescence in situ hybridization (FISH): a potential new tool for early detection of non-small cell lung Cancer (NSCLC).

Background: Our objective was to study the feasibility of detecting chromosomal deletions at 3p22.1 and 10q22.3 by fluorescent in situ hybridization (FISH) and to examine their distribution in different areas of the airway in patients with non-small cell lung cancer. Methods: Brush biopsies from the mainstem bronchus on the normal side contralateral to the tumor (NBB) and mainstem bronchus on the tumor side (TBB) were obtained from 122 patients who underwent surgical resection. Touch preparations from the tumor (TTP), normal lung parenchyma, and bronchi adjacent to the tumor were also obtained. Two FISH assays using probes complementary to 3p22.1 and 10q22.3 were used to detect deletions. Results: NBB showed a relatively low deletion rate of 3p22.1 and 10q22.3 compared with TTP (p < 0.0001). TBB showed a significantly higher rate of deletions compared with NBB but lower than TTP from the tumor (p < 0.05) for both 3p22.1 and 10q22.3. A significantly higher deletion rate was seen at TTP compared with normal lung parenchyma at both the 3p22.1 and 10 q22.3 (p < 0.0001). Correlations were seen between the deletion rates of TTP and TBB at 3p22.1 (&rgr; = 0.61, p < 0.0001) and between TTP and bronchi adjacent to the tumor at 10q22.3 (&rgr; = 0.64, p < 0.0001). Conclusion: Deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISH. As one progresses from the contralateral normal bronchus to the bronchus on the side of tumor and the tumor itself, the percentage of chromosomal deletions increases in a statistically significant fashion. This suggests that, FISH analysis of bronchoscopic brushes may be useful for identifying patients at high risk for developing non-small cell lung cancer.

Evaluation of peripheral blood involvement of mantle cell lymphoma by fluorescence in situ hybridization in comparison with immunophenotypic and morphologic findings.

Mantle cell lymphoma is non-Hodgkins B-cell lymphoma characterized by the t(11;14)(q13;q32) translocation. Peripheral blood involvement of mantle cell lymphoma is usually associated with a poor prognosis and therefore, its identification is clinically important. In this study, we performed cyclin D1/IgH-probe fusion fluorescence in situ hybridization analysis on 223 peripheral blood samples: 185 from 125 mantle cell lymphoma patients, and 38 normal controls. The cutoff values for the test were established using normal controls. Flow cytometry on peripheral blood and corresponding bone marrow samples was used to evaluate this test. In all, 26% of the 185 peripheral blood samples and 27% of the 161 corresponding bone marrow samples were flow cytometry positive for mantle cell lymphoma. The mean numbers of single and- double-fusion signals and the mean number of CD5/CD19-positive cells, absolute blood lymphocyte count, and white blood cell count were significantly higher in peripheral blood and corresponding bone marrow samples with mantle cell lymphoma-positive flow cytometry. Double-fusion signals were more specific than single-fusion ones. Fluorescence in situ hybridization was far more likely to be positive for mantle cell lymphoma when the peripheral blood and the corresponding bone marrow samples had positive flow cytometry results or morphology (P<0.01). Our study indicates that cyclin D1/IgH-fusion fluorescence in situ hybridization analysis could be used to determine the presence and character of circulating mantle cell lymphoma cells in peripheral blood, thus enhancing our ability to evaluate leukemic mantle cell lymphoma and minimum residual disease.

Abstract 287: Deletion of 3p22.1 by FISH in non-small cell lung cancer (NSCLC) is significantly correlated with loss of β-catenin membrane expression and dysregulated cell adhesion.

The role of WNT signaling is well established in lung cancer and numerous studies have shown that loss/reduction in expression of β catenin, a key regulator of this pathway, is related to dysregulated cell adhesion, epithelial-mesenchymal transformation (EMT) and poor survival. A recent genome-wide association study (GWAS) in patients with advanced NSCLC and receiving platinum-based therapy showed that a SNP involving 3p22.1 (rs7629386) situated close to β catenin (CTNNB1) was associated with worse survival. In prior studies we have shown that deletion 3p22.1 (del 3p) was frequently involved in early events of lung cancer pathogenesis. The fluorescent in situ hybridization (FISH) BAC probe for 3p22.1 used in our studies is closely adjacent to CTNNB1. We therefore hypothesized that del 3p might involve a concurrent deletion of CTNNB1, and hence would be associated with reduced β catenin expression. Tumor microarrays (TMAs) from 145 NSCLC patients, 107 early stage (I and II) and 38 advanced stage (III and IV), were reacted with anti-β catenin antibody and scored in triplicate. Normal staining was defined as complete membranous or partial membranous with/ without cytoplasmic staining in >90% of tumor cells, and all others were classified as reduced. FISH performed on bronchoscopic brush biopsies: ipsilateral to tumor (TBB), contralateral to tumor (NBB), normal lung (NTP), tissue adjacent to tumor (TAB), and tumor touch preparations (TTP) were evaluated for del 3p by comparing to an internal centromeric probe. A logistic regression model was used to assess the relationship between del 3p at the different sites and β catenin expression. TMAs were also evaluated for Ki-67 by immunohistochemistry. A Cox proportional hazard model was used to assess for overall survival by del 3p at the different sites and Ki-67 proliferation index. Of the 145 cases, 113 stained normal and 32 had reduced staining. Statistical analysis showed there was no significant effect of del 3p on reduced β catenin staining for sites TBB (p=0.14), NBB (p=0.39), NTP (p=0.24), and TAB (p=0.24); however there was a significant correlation of del 3p from tumor touch preparations (TTP) on the percentage of cases with reduced staining (p=0.015).We did not observe any significant correlation of reduced staining when compared to stage of disease, histologic subtype, overall survival (OS) or recurrence-free survival (RS). There was also no significant correlation observed between Ki-67 proliferation index and β catenin expression or Ki-67 and OS/ RS. In conclusion, there is a significant correlation between del 3p22.1 and loss of β catenin staining in primary NSCLC, and reduced β catenin expression likely relates to tumor initiation and progression, secondary to dysregulated cell adhesion and EMT. (Funding: Specialized Program of Research Excellence grant P50CA70907) Citation Format: Sinchita Roy Chowdhuri, Tanweer M. Zaidi, Abha Khanna, Feng Jiang, Margaret R. Spitz, Ara A. Vaporciyan, Jack A. Roth, Joe Ensor, Ruth L. Katz. Deletion of 3p22.1 by FISH in non-small cell lung cancer (NSCLC) is significantly correlated with loss of β-catenin membrane expression and dysregulated cell adhesion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2013-287

Abstract LB-279: Quantitative assessment of tumor infiltrating neutrophils in primary renal cell carcinoma correlates with presence of metastases.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: An emerging hallmark of cancer is tumor promoting inflammation. Cancer associated myeloproliferation is a new emerging therapeutic target. Cancers may produce cytokines like G-CSF that recruit neutrophils. The recruitment of neutrophils promotes tumor angiogenesis and suppresses NK cells and cytotoxic T-cells. Cancer patients with leukemoid reactions have higher rates of metastasis. Lung, gastric, ovarian, hepatic, pancreatic, renal and colon cancer are cancers that have been shown to have increased tumor infiltrating neutrophils (TINs). Effectors released from neutrophils, include matrix metalloproteases and elastase, which may support the metastatic dissemination of tumor cells. Elastase degrades E-cadherin, promotes tumor cell dissociation and promotes epithelial to mesenchymal transition in pancreatic tumor cell lines In-vitro . To date there have been no reported studies in humans that have found a role for TINs and associated elastase in promoting tumor progression. Objectives: To study whether the number of TINs and released elastase associated with conventional renal cell carcinoma (RCC) can be used as predictors of tumor progression and patient prognosis. We predicted that RCCs with increased numbers of TINs and extracellular elastase would be associated with advanced tumor stage and decreased overall survival. We wished to evaluate if increased TINs and elastase can be used as a predictor of metastasis and patient survival. Methods : 37 randomly selected cases of primary RCC diagnosed by fine needle aspiration (FNA) from 2002-2005 were analyzed for TINs. TINS were scored on aspirated tissue according to numbers of neutrophils infiltrating a minimum of 50 malignant cells/hpf and scored on a scale of 0-4 (0-1=Low, 2-4=High TIN count). TIN count was correlated with tumor characteristics including size of tumor, Fuhrmans nuclear grade (FNG), presence of metastasis and patient survival data using student t-tests calculations for statistical significance. Results: Of 37 RCCs, 20 cases had high TIN count versus 17 cases that demonstrated a low TIN count. At time of initial FNA, 100% of cases with high TINS had distant metastasis (lungs, liver, bone) whereas only 19% of cases with low TINs showed metastases (p=0.0003). There was a positive linear correlation with the number of distant metastatic sites and numbers of TINs in the primary tumor. Moreover, the mean patient survival time was lower in cases with high TIN counts (32.6 weeks), versus cases with low TIN counts (143 weeks) (p=0.0003). There was no association between numbers of TINS and FNG (p=0.526), or size of tumor (p=0.3) . Neutrophil- associated elastase immunostaining of tumors with high TINs was strongly positive. Conclusion: Our preliminary results indicate that high TIN counts and elastase in primary RCCs appear to be associated with metastases and poor prognosis. TINs may be an independent prognostic factor that can be immediately assessed at time of first biopsy/FNA. Citation Format: Davide Salina, Tanweer Zaidi, Ruth L. Katz. Quantitative assessment of tumor infiltrating neutrophils in primary renal cell carcinoma correlates with presence of metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-279. doi:10.1158/1538-7445.AM2013-LB-279

Abstract 4311: Identification of a subset of peripheral blood cells expressing ALDH1 with chromosomal abnormalities in lung cancer patients suggests these are putative cancer stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The concept that cancers originate from cytogenetically abnormal stem cells (SC) is becoming accepted. Mechanisms by which cancer stem cells (CSC) contribute to tumor initiation and progression are unknown, however CSCs are resistant to chemotherapy and have metastatic potential. ALDH1 is a marker for identifying SCs. Increased ALDH1 expression in lung cancer (LC) was shown to correlate with disease stage and poor survival. Recently we showed that cytogenetically abnormal cells (CACs) similar to those in the primary tumor are present in the peripheral blood (PB) of patients with LC and numbers of CACs correlated with disease stage. We wished to discover if there were PB cells that expressed ALDH1 and showed chromosomal abnormalities (CA), representing CSCs, and if their presence correlated with tumor subtype and survival. Materials and Methods: Prior to resection of lung cancers, peripheral blood mononuclear cells (PBMNCs) were isolated from 5 controls and 11 cases of LC. Patients ranged in age from 41-83 years. There were 4 low stage (I-II) and 7 high stage (III-IV) patients. Patients were followed prospectively over a 20 month period. Immunostaining for ALDH1 (BD Pharmingen) was performed, following which FISH for CEP 3, CEP 7, CEP 17 and LSI 9p21 probe set (Abbott Molecular) was performed on relocated ALDH1+ cells. FISH signals were scored for any chromosomal abnormality; deletion or gain. Results: A low percentage of ALDH1+ cells were present in PB of 7/7 adenocarcinoma (ADC), 2/2 non-small cell lung carcinoma (NSCLC) 1/2 squamous carcinoma (SQC) patients and in controls. In LC, 53 ALDH1+ cells were detected of which 18 (34%) showed CAs. Controls were negative for CAs. Conclusions: The numbers of ALDH1+ cells with CA tended to increase with stage. Genetically abnormal ALDH1+ cells may represent CSCs and provide a therapeutic target for efficient treatment of lung cancer. View this table: Demographic data and chromosomal abnormalities in ALDH1 + cells Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4311.