Abha Khanna

Aldehyde dehydrogenase 1 is a tumor stem cell-Associated marker in lung cancer

Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features of CSCs, including capacities for proliferation, self-renewal, and differentiation, resistance to chemotherapy, and expressing CSC surface marker CD133. In vivo experiments show that the ALDH1-positive cells could generate tumors that recapitulate the heterogeneity of the parental cancer cells. Immunohistochemical analysis of 303 clinical specimens from three independent cohorts of lung cancer patients and controls show that expression of ALDH1 is positively correlated with the stage and grade of lung tumors and related to a poor prognosis for the patients with early-stage lung cancer. ALDH1 is therefore a lung tumor stem cell-associated marker. These findings offer an important new tool for the study of lung CSCs and provide a potential prognostic factor and therapeutic target for treatment of the patients with lung cancer. (Mol Cancer Res 2009;7(3):330–8)

PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer

PTEN haploinsufficiency is common in hormone‐sensitive prostate cancer, though the incidence of genomic deletion and its downstream effects have not been elucidated in clinical samples of hormone refractory prostate cancer (HRPC). Progression to androgen independence is pivotal in prostate cancer and mediated largely by the androgen receptor (AR). Since this process is distinct from metastatic progression, we examined alterations of the PTEN gene in locally advanced recurrent, non‐metastatic human HRPC tissues. Retrospective analyses of PTEN deletion status were correlated with activated downstream phospho‐Akt (p‐Akt) pathway proteins and with the androgen receptor. The prevalence of PTEN genomic deletions in transurethral resection samples of 59 HRPC patients with known clinical outcome was assessed by four‐colour FISH analyses. FISH was performed using six BAC clones spanning both flanking PTEN genomic regions and the PTEN gene locus, and a chromosome 10 centromeric probe. PTEN copy number was also evaluated in a subset of cases using single nucleotide polymorphism (SNP) arrays. In addition, the samples were immunostained with antibodies against p‐Akt, p‐mTOR, p‐70S6, and AR. The PTEN gene was deleted in 77% of cases, with 25% showing homozygous deletions, 18% homozygous and hemizygous deletions, and 34% hemizygous deletions only. In a subset of the study group, SNP array analysis confirmed the FISH findings. PTEN genomic deletion was significantly correlated to the expression of downstream p‐Akt (p < 0.0001), AR (p = 0.025), and to cancer‐specific mortality (p = 0.039). PTEN deletion is common in HRPC, with bi‐allelic loss correlating to disease‐specific mortality and associated with Akt and AR deregulation. Copyright

Gain of the 3q26 region in cervicovaginal liquid-based pap preparations is associated with squamous intraepithelial lesions and squamous cell carcinoma

BACKGROUND Chromosomal aberrations have been documented in cervical carcinomas, especially chromosome 3q. The human telomerase RNA gene (hTERC) is located in the chromosome 3q26 region, and its product, telomerase, is involved in the maintenance of chromosome length and stability. Upregulation of telomerase is in general associated with tumorigenesis. In this study, cervicovaginal specimens were analyzed by fluorescence in situ hybridization (FISH) for gain of chromosome 3q26 containing hTERC, and FISH findings were compared with the cytologic and histologic diagnoses. METHODS Slides prepared from 66 liquid-based preparations from cervical specimens with cytologic diagnoses of negative for squamous intraepithelial lesion or malignancy (NILM, n=4), atypical squamous cells of undetermined significance (ASC-US, n=15), low-grade squamous intraepithelial lesion (LSIL, n=20), high-grade squamous intraepithelial lesion (HSIL, n=24), or cervical squamous cell carcinoma (SCCA, n=3) were analyzed for aberrations of 3q26 using a commercially available two-color FISH probe. The results of the cytologic analysis and those of concurrent or subsequent biopsies, when available, were compared with the FISH-detected 3q26 abnormalities. The Wilcoxon rank-sum test was used to assess associations between 3q26 gains and diagnoses. RESULTS Gain of 3q26 was significantly associated with the cytologic diagnosis (p<0.0001). Patients with HSIL or SCCA cytology diagnoses had significantly higher percentages of cells with 3q26 gain than did patients with NILM or ASC-US cytologic diagnoses. CONCLUSIONS FISH can be performed on cervicovaginal liquid-based preparations to detect gain of 3q26. Gain of 3q26 is associated with HSIL and SCCA. This test may be an adjunct to cytology screening, especially high-risk patients.

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.

Fluorescence In Situ Hybridization for Detecting Urothelial Carcinoma A Clinicopathologic Study

Because urothelial carcinoma (UC) is associated with a significantly high risk of disease recurrence and progression, patients with UC require long‐term surveillance. Fluorescence in situ hybridization (FISH) has been shown to be more sensitive than cytology in the detection of UC. The current study evaluated the use of FISH for detecting UC.

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.

Grading follicular lymphoma on fine needle aspiration specimens: Comparison with proliferative index by DNA image analysis and Ki-67 labeling index

OBJECTIVE To determine whether follicular lymphoma (FL) can be graded on fine needle aspiration (FNA) biopsies by determining the percentage of centroblasts in the neoplastic follicles on the smears. STUDY DESIGN Eighty-nine cases of histologically confirmed cases of FL, including 31 grade 1, 46 grade 2 and 12 grade 3, were evaluated. Proliferative index (PI) by DNA image analysis (DIA) and Ki-67 labeling index (LI) were obtained on all cases. A minimum of 200 cells were counted per case (range, 200-800 cells) at 40x magnification, and the number of large cells (centroblasts) was expressed as a percentage of the total number of cells counted within the follicles. RESULTS The percentage of centroblasts in the follicular aggregates was 9.7 +/- 2.9% in grade 1 FLs, 24.7 +/- 5.6% in grade 2 and 48.4 +/- 7.5% in grade 3. These differences were significant (P < .05). DNA image analysis of PI and Ki-67 LI differed significantly between grade 1 FLs and grade 2 and 3 FLs (P < .05), but there were no significant differences between grade 2 and 3 FLs. CONCLUSION Determining the percentage of centroblasts in the follicular aggregates on FNA specimens is a good method of grading FLs. Using the percentage of centroblasts per follicular structure, FL grades 1, 2 and 3 were adequately distinguished. PI by DIA and Ki-67 LI clearly distinguished FL grade 1 from FL grades 2 and 3; however, it did not clearly distinguish between grades 2 and 3.

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.

Cervista HPV assays for fine-needle aspiration specimens are a valid option for human papillomavirus testing in patients with oropharyngeal carcinoma

The objectives of this study were to evaluate the validity of Cervista human papillomavirus (HPV) assays in head and neck fine‐needle aspiration (FNA) specimens from patients with head and neck squamous carcinomas and to verify that the Cervista assay in FNA specimens is a valid option for determining HPV status in patients with oropharyngeal carcinomas.

Chromosomal Abnormalities Detected by Multicolor Fluorescence In Situ Hybridization in Fine-Needle Aspirates From Patients With Small Lymphocytic Lymphoma Are Useful for Predicting Survival

Fine‐needle aspiration (FNA) of lymph nodes is commonly used to assess disease progression in patients with small lymphocytic lymphoma (SLL). Although cytologic features are helpful for diagnosing typical SLL and transformed large‐cell lymphoma (tLCL), SLL in accelerated phase (SLLacc) is more difficult to diagnose. Additional tests are needed to identify those patients who are transforming to a higher‐grade lymphoma. This study evaluated the use of a multicolor fluorescence in situ hybridization (FISH) probe panel specifically designed for chronic lymphocytic leukemia (CLL)/SLL and assessed the association between FISH findings and cytologic diagnosis, proliferation index, and risk of death.