[Show abstract][Hide abstract] ABSTRACT: There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity.
We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications.
We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%-100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription- polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis.
An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer.
Breast cancer 06/2015; 20159:31-40. DOI:10.4137/BCBCr.s23577
[Show abstract][Hide abstract] ABSTRACT: The benefit of intrathecal therapy and systemic rituximab on the outcome of diffuse large B-cell lymphoma at risk of central nervous system disease is controversial. Furthermore, the effect of intrathecal treatment and rituximab in diffuse large B-cell and Burkitt lymphoma with occult leptomeningeal disease detected by flow cytometry at diagnosis is unknown. Untreated diffuse large B-cell (n=246) and Burkitt (n=80) lymphoma at clinical risk of central nervous system disease and had pretreatment cerebrospinal fluid analyzed by flow cytometry and and cytology. Spinal fluid involvement was detected by flow cytometry alone (occult) in 33 (13%) diffuse large B-cell and 9 (11%) Burkitt lymphoma patients, and detected by cytology in 11 (4.5%) and 5 (6%) patients, respectively. Diffuse large B-cell lymphoma with occult spinal fluid involvement had poorer survival (p=0.0001) and freedom from central nervous system relapse (p<0.0001) compared to negative cases. Burkitt lymphoma with occult spinal fluid involvement had an inferior freedom from central nervous system relapse (p=0.026) but not survival. The amount of intrathecal chemotherapy was quantitatively associated with survival in diffuse large B-cell lymphoma with (p=0.02) and without (p=0.001) occult spinal fluid involvement. However, progression of systemic disease and not control of central nervous system disease was the principal cause of treatment failure. In diffuse large B-cell lymphoma, systemic rituximab was associated with improved freedom from central nervous system relapse (p=0.003) but not survival. Our results suggest that patients at risk of central nervous system disease should be evaluated by flow cytometry and that intrathecal prophylaxis/therapy is beneficial.
[Show abstract][Hide abstract] ABSTRACT: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies.
From a cohort of 1514 patients with metastatic melanoma, biopsies were procured over a 17 year period from 3086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we utilized established immunohistochemical methods to perform semi-quantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDAs) including gp100, MART-1, and tyrosinase (TYR). To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4+ and CD8+ T cell infiltrate.
Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Further, TYR expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8+ and CD4+ T cell infiltrate.
Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen specific immunotherapies.
Clinical Cancer Research 03/2014; 20(10). DOI:10.1158/1078-0432.CCR-13-2690 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes.
IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation.
We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis.
Our findings support the central role of IRF-1 in influencing different tumour phenotypes.
British Journal of Cancer 06/2013; 109(1). DOI:10.1038/bjc.2013.335 · 4.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The diagnosis of melanoma can be challenging, especially in metastatic lesions, due to the ability of melanoma cells to morphologically mimic carcinoma, sarcoma and even lymphoma cells. Moreover, melanomas can exhibit negative immunostaining for the melanoma markers HMB-45 and MART-1/Melan-A, often used in the diagnosis of this tumor. KBA.62 is a recently described antibody that reacts with benign and malignant melanocytic proliferations. In this study, we report our experience with KBA.62 and S100 protein immunostaining in the diagnosis of metastatic melanoma on fine-needle aspiration and effusion samples. We reviewed 60 cytology samples from 58 patients with metastatic melanoma. Our results showed that KBA.62 stained 75% of the cases and S100 protein 87% of the cases. KBA.62 and S100 protein stained the majority of metastatic melanomas that were negative for HMB-45 and MART-1; KBA.62 stained 73% of the cases and S100 protein 73% of the cases. The majority (85%) of the cases negative for HMB-45 and MART-1 were positive for KBA.62 and/or S100 protein. Additionally, we also observed that KBA.62 staining was positive in the majority of epithelioid and spindle cell type melanoma cells. In conclusion, the performances of KBA.62 and S100 protein were similar and both markers are useful in the diagnosis of metastatic melanoma in cytology material, especially when the tumor cells lack expression of HMB-45 and MART-1.
[Show abstract][Hide abstract] ABSTRACT: Background/Aim: To determine if early passage tumor cells obtained from patients with mesothelioma continue to express the tumor differentiation antigen mesothelin and their sensitivity to the anti-mesothelin immunotoxin SS1P.
Cell cultures were established from ascites or pleural effusion of 6 peritoneal and 3 pleural mesothelioma patients, respectively. These cells were evaluated for mesothelin expression by immunohistochemistry and flow cytometry.
Although mesothelin was highly expressed in tumor biopsies of all patients, only 3 out of 9 malignant effusions from these patients when grown in short-term culture showed strong mesothelin positivity by IHC. By flow cytometry, the number of mesothelin sites per cell was variable ranging from 580 to 210,000 sites/cell. Cells with strong mesothelin expression by IHC and increased number of mesothelin sites/cell were sensitive to SS1P.
Most mesothelioma tumors loose mesothelin when grown in vitro and the sensitivity of these cells to SS1P is dependent on the number of mesothelin sites/cell.
Anticancer research 12/2012; 32(12):5151-8. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies have definitively determined that flow cytometry (FC) is significantly more sensitive than cytomorphology (CM) in detection of hematolymphoid neoplasms (HLNs). However, its utility in paucicellular bronchoalveolar lavage (BAL) specimens has not been established.
FC was performed on BAL specimens submitted from 44 patients with a prior diagnosis of HLN. Panels chosen were based upon cellularity of specimen and patient history. FC results were compared with concurrent CM evaluations.
All 44 BALs were deemed satisfactory for FC and yielded informative results that assisted in diagnosis. Diagnoses included 22/44 B-cell neoplasms, 16/44 T-cell neoplasms, four/44 myeloid neoplasms, and two/44 plasma cell neoplasms. Overall concordance was demonstrated between FC and CM in 77% (34/44) of cases. In nine/44 cases (20%), one technique (FC or CM) clearly detected malignant cells when the other did not. FC was more sensitive than CM in detecting a HLN in eight/nine discordant cases. In only one case (one/44, 2%) were malignant HLN cells suspected by CM, but not identified by FC (one/44, 2%).
We demonstrate, in the largest series published to date, that FC can be performed on BAL specimens. FC is indicated in evaluation of BAL for HLN and improves sensitivity of detection of HLN over CM alone. An integrated FC and CM approach is superior to either technique alone in diagnostic evaluation of BAL. Published 2012 Wiley Periodicals, Inc.
Cytometry Part B Clinical Cytometry 09/2012; 82(5):305-12. DOI:10.1002/cyto.b.21033 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flow cytometric T-cell receptor V(β) repertoire analysis (TCR-V(β)-R) is a sensitive method to detect T-cell clonality; however, its implementation in low-cellularity specimens has not been established. We developed a strategy to use TCR-V(β)-R in cerebrospinal fluid (CSF) and fine-needle aspirate (FNA) specimens. Initially, full TCR-V(β)-R was evaluated in diagnostic/screening specimens from 8 patients with T-cell neoplasia to determine tumor-specific TCR-V(β) protein expression. Subsequently, an abbreviated, patient-specific TCR-V(β)-R evaluation was performed in 17 paucicellular specimens from the patients (8 CSF, 9 FNA) for staging and monitoring of minimal residual disease (MRD). A single cocktail containing 3 anti-V(β) antibodies (1 tumor-specific and 2 negative controls) in combination with other antibodies chosen to help gate on atypical T cells is highly sensitive and specific for detecting low-level neoplastic T-cell involvement in paucicellular specimens. This TCR-V(β)-R strategy is valuable in staging and evaluating MRD in patients with T-cell non-Hodgkin lymphoma.
American Journal of Clinical Pathology 02/2012; 137(2):220-6. DOI:10.1309/AJCPPT93VZMAREHK · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: In the past decade molecular diagnostics has changed the clinical management of lung adenocarcinoma patients. Molecular diagnostics, however, is largely dependent on the quantity and quality of the tumor DNA that is retrieved from the tissue or cytology samples. Frequently, patients are diagnosed on cytology specimens where the tumor cells are scattered within the cell block, making selecting for tumor enrichment difficult. In the past we have used laser capture microdissection (LCM) to select for pure populations of tumor cells to increase the sensitivity of molecular assays. This study explores several methods for semiautomated computer-guided LCM. Study Design: Hematoxylin and eosin- or TTF-1-immunostained slides from a pleural effusion cell block with metastatic lung adenocarcinoma were used for LCM with either AutoScan or a recently described pattern-matching algorithm, spatially invariant vector quantization (SIVQ), to define morphologic predicates (vectors) to select cells of interest. Results: We retrieved pure populations of tumor cells using both algorithm-guided LCM approaches with slight variations in cellular retrievals. Both methods were semiautomated, requiring minimum technical supervision. Conclusion: In this study we demonstrate the first semiautomated, computer-guided LCM of a cytology specimen using SIVQ and AutoScan, a first step towards the long-term goal of integrating LCM into the clinical cytology-molecular workflow.
[Show abstract][Hide abstract] ABSTRACT: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM).
Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep.
EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment.
Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy.
Clinical Cancer Research 01/2012; 18(1):77-90. DOI:10.1158/1078-0432.CCR-11-0962 · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The discovery of activating mutations in EGFR and KRAS in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Fine-needle aspirates and other cytologic procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used laser capture microdissection to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from cell block sections of 19 cytology samples from patients with known lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR or KRAS mutation that was present in the patient's prior molecular assay in every case, but we were also able to consistently detect the mutation from as few as 50 microdissected tumor cells. Furthermore, isolating a more pure population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from laser capture microdissection-enriched cases where the tumor load was low and traditional methods of whole slide scraping failed. Therefore, this method can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR and KRAS mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.
Modern Pathology 12/2011; 25(4):548-55. DOI:10.1038/modpathol.2011.184 · 6.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The increasing availability of whole slide imaging (WSI) data sets (digital slides) from glass slides offers new opportunities for the development of computer-aided diagnostic (CAD) algorithms. With the all-digital pathology workflow that these data sets will enable in the near future, literally millions of digital slides will be generated and stored. Consequently, the field in general and pathologists, specifically, will need tools to help extract actionable information from this new and vast collective repository.
To address this limitation, we designed and implemented a tool (dCORE) to enable the systematic capture of image tiles with constrained size and resolution that contain desired histopathologic features.
In this communication, we describe a user-friendly tool that will enable pathologists to mine digital slides archives to create image microarrays (IMAs). IMAs are to digital slides as tissue microarrays (TMAs) are to cell blocks. Thus, a single digital slide could be transformed into an array of hundreds to thousands of high quality digital images, with each containing key diagnostic morphologies and appropriate controls. Current manual digital image cut-and-paste methods that allow for the creation of a grid of images (such as an IMA) of matching resolutions are tedious.
The ability to create IMAs representing hundreds to thousands of vetted morphologic features has numerous applications in education, proficiency testing, consensus case review, and research. Lastly, in a manner analogous to the way conventional TMA technology has significantly accelerated in situ studies of tissue specimens use of IMAs has similar potential to significantly accelerate CAD algorithm development.
[Show abstract][Hide abstract] ABSTRACT: We present the cytological features along with histologic and imaging findings of a melanocytic bronchopulmonary carcinoid tumor in a patient with multiple endocrine neoplasia syndrome type 1 (MEN-1). Intraoperative touch preparations of the lung tumor showed single spindle cells and loosely cohesive aggregates of spindle cells with oval to elongated nuclei, "salt and pepper" chromatin pattern and inconspicuous nucleoli. The spindle cells occasionally contained cytoplasmic pigment, which revealed to be melanin by Fontana Masson stain on permanent processed material. Immunohistochemical stains for both synaptophysin and chromogranin were strongly positive in the spindle cells. The findings were consistent with melanocytic bronchopulmonary carcinoid tumor, which is relatively uncommon in MEN-1.
[Show abstract][Hide abstract] ABSTRACT: Ductal lavage has been used for risk stratification and biomarker development and to identify intermediate endpoints for risk-reducing intervention trials. Little is known about patient characteristics associated with obtaining nipple aspirate fluid (NAF) and adequate cell counts (> or =10 cells) in ductal lavage specimens from BRCA mutation carriers.
We evaluated patient characteristics associated with obtaining NAF and adequate cell counts in ductal lavage specimens from the largest cohort of women from BRCA families yet studied (BRCA1/2 = 146, mutation-negative = 23, untested = 2). Fisher's exact test was used to evaluate categorical variables; Wilcoxon nonparametric test was used to evaluate continuous variables associated with NAF or ductal lavage cell count adequacy. Logistic regression was used to identify independent correlates of NAF and ductal lavage cell count adequacy.
From 171 women, 45 (26%) women had NAF and 70 (41%) women had ductal lavage samples with > or =10 cells. Postmenopausal women with intact ovaries compared with premenopausal women [odds ratio (OR), 4.8; P = 0.03] and women without a prior breast cancer history (OR, 5.2; P = 0.04) had an increased likelihood of yielding NAF. Having breast-fed (OR, 3.4; P = 0.001), the presence of NAF before ductal lavage (OR, 3.2; P = 0.003), and being premenopausal (OR, 3.0; P = 0.003) increased the likelihood of ductal lavage cell count adequacy. In known BRCA1/2 mutation carriers, only breast-feeding (OR, 2.5; P = 0.01) and the presence of NAF (OR, 3.0; P = 0.01) were independent correlates of ductal lavage cell count adequacy.
Ductal lavage is unlikely to be useful in breast cancer screening among BRCA1/2 mutation carriers because the procedure fails to yield adequate specimens sufficient for reliable cytologic diagnosis or to support translational research activities.
[Show abstract][Hide abstract] ABSTRACT: A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.