Vijay Varma

Molecular Mapping of Tumor Heterogeneity on Clinical Tissue Specimens with Multiplexed Quantum Dots

Tumor heterogeneity is one of the most important and challenging problems not only in studying the mechanisms of cancer development but also in developing therapeutics to eradicate cancer cells. Here we report the use of multiplexed quantum dots (QDs) and wavelength-resolved spectral imaging for molecular mapping of tumor heterogeneity on human prostate cancer tissue specimens. By using a panel of just four protein biomarkers (E-cadherin, high-molecular-weight cytokeratin, p63, and alpha-methylacyl CoA racemase), we show that structurally distinct prostate glands and single cancer cells can be detected and characterized within the complex microenvironments of radical prostatectomy and needle biopsy tissue specimens. The results reveal extensive tumor heterogeneity at the molecular, cellular, and architectural levels, allowing direct visualization of human prostate glands undergoing structural transitions from a double layer of basal and luminal cells to a single layer of malignant cells. For clinical diagnostic applications, multiplexed QD mapping provides correlated molecular and morphological information that is not available from traditional tissue staining and molecular profiling methods.

Multiplexed Detection and Characterization of Rare Tumor Cells in Hodgkin’s Lymphoma with Multicolor Quantum Dots

The multicolor and multiplexing capabilities of semiconductor quantum dots (QDs) are most promising for improving the sensitivity and specificity of in vitro molecular and cellular diagnostics. Here, we report the use of multiplexed QDs and wavelength-resolved imaging to detect and characterize a class of low-abundant tumor cells in Hodgkins lymphoma. Known as the Hodgkins and Reed-Sternberg (HRS) cells, this class of malignant cells is a pathological hallmark in clinical diagnosis, but it comprises only about 1% of the heterogeneous infiltrating cells in lymph node tissues. To overcome this cellular heterogeneity and rarity problem, we have developed multicolor QD-antibody conjugates to simultaneously detect a panel of four protein biomarkers (CD15, CD30, CD45, and Pax5) directly on human tissue biopsies. This multiplexing approach allows rapid detection and differentiation of rare HRS cells from infiltrating immune cells such as T and B lymphocytes. We have also carried out clinical translation studies involving six confirmed Hodgkins lymphoma patients, two suspicious lymphoma cases, and two patients with reactive lymph nodes (but not lymphoma). The results indicate that a distinct QD staining pattern (CD15 positive, CD30 positive, CD45 negative, and Pax5 positive) can be used to not only detect Hodgkins lymphoma but also differentiate it from benign lymphoid hyperplasia.

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.

Reliability of Differential PCR for the Detection of EGFR and MDM2 Gene Amplification in DNA Extracted from FFPE Glioma Tissue

A series of 43 human gliomas, consisting of 30 glioblastomas, 7 anaplastic astrocytomas, 3 low grade astrocytomas, 2 epcndymomas, and I oligodendroglioma, was studied for amplification of the epidermal growth factor receptor (EGFR) and mouse double minute 2 (MDM2) genes. DNA extracted from formalin-fixed, paraffin-embedded tissue sections was analyzed by differential PCR and the results were compared with slot blot examination of DNA extracted from frozen tissue from the same neoplasms. Twelve glioblastomas (40%) showed amplification of the EGFR gene, and overexpression of EGFR was evident in each of these tumors as indicated by the immunoperoxidase technique. Two of the tumors with EGFR gene amplification also revealed amplification of the MDM2 gene, while one additional glioblastoma revealed MDM2 amplification only. A 100% concordance in the detection of amplification was observed between differential PCR and slot blot analysis; consequently, these results indicate that differential PCR using DNA extracted from archival tissue sections is a reliable method of demonstrating gene amplifications in glial tumors.

Sox7 is an independent checkpoint for β-catenin function in prostate and colon epithelial cells

The presence of somatic β-catenin mutations in some prostate cancers implies that aberrant WNT signaling is involved in the cancer development. Although β-catenin stability is regulated by a multicomponent destruction complex, mutational alterations of β-catenin or other components of the destruction complexes are rare in prostate tumors. Therefore, β-catenin may be regulated by another protein in the prostate. In fact, recent linkage and somatic deletion analyses in prostate cancers reveal a 1.4-Mb candidate tumor suppressor locus on 8p23.1, which includes the Sox7 gene. Here we show that Sox7 protein expression was indeed down-regulated in 47% (15 of 32) of prostate adenocarcinomas. In addition, Sox7 mRNA was down-regulated in 60% of snap-frozen tumors. This down-regulation was found to be due to tumor-specific promoter hypermethylation, which was present in 48% (10 of 21) of primary prostate tumors and 44% (11 of 25) of prostate cancer cell lines/xenografts. We discovered that Sox7 protein physically interacts with β-catenin and suppresses β-catenin–mediated transcription by depleting active β-catenin. Furthermore, in HCT116 colorectal cancer cell lines with Sox7 inactivation, ectopic Sox7 expression suppressed cell proliferation and inhibited transcription that was activated by an endogenous mutant β-catenin. Although nearly all colorectal cancers contain mutations in β-catenin or adenomatous polyposis coli/axin, epigenetic silencing of Sox7 was still observed. These data suggest that Sox7 is a tumor suppressor that functions as an independent checkpoint for β-catenin transcriptional activity. Inactivation of Sox7 could promote the development of a majority of colorectal tumors and approximately half of prostate tumors. (Mol Cancer Res 2008;6(9):1421–10)

Immunohistochemical and molecular markers in the diagnosis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has distinct morphologic features and can be identified in the majority of cases by routine hematoxylin and eosin (H&E)-stained formalin-fixed paraffin-embedded sections. However, distinguishing a well-differentiated HCC from normal or regenerative tissue may be very difficult in some cases, particularly in small needle aspiration or core biopsies. Furthermore, some of the unusual morphologic variants, including clear-cell, pleomorphic, and sarcomatoid variants, may be mistaken for metastases. Similarly, metastases from various primary tumors to the liver may be mistaken for primary hepatic tumors. In this overview, we summarize the immunohistochemical and molecular markers that have been developed to address these diagnostic challenges. Among the numerous diagnostic markers studied, pCEA, HepPar 1, CD34, CK 7, CK 19, CK 20, and albumin in situ (ISH) have been found to be valuable in distinguishing HCC from metastatic neoplasms of extrahepatic sites.

Ex Vivo Expansion of CD8+CD56+ and CD8+CD56− Natural Killer T Cells Specific for MUC1 Mucin

Prostate cancers express MUC1, but nearly all metastatic cells lack HLA class I molecules. Thus, a lymphocyte population that can sense its antigenic environment, while also able to react to stimuli of natural killer (NK) cells, may be a more versatile effector cell population for antitumor immune responses. Herein, we report that tumor-specific MUC1 peptide, interleukin 2, and interleukin 12 act synergistically to stimulate the ex vivo expansion of CD8+CD56− T cells and CD8+CD56+ natural killer T (NKT) cells from the peripheral blood mononuclear cells of prostate cancer patients, as well as healthy male and female donors. Both the CD56+ NKT cells and CD56− T cells lysed allogeneic mucin-bearing target cells, as well as NK target cells, but not lymphokine-activated killer target cells. However, the CD56+ NKT cells displayed a 2-fold greater cytolytic activity than the CD56− T cells. The mucin-specific cytolytic activity and NK cytolytic activities for both lymphocyte populations were independent of HLA class I and CD1 molecules. The CD56− T cells up-regulated CD56 with continued antigenic stimulation in the presence of interleukin 12, suggesting that CD8+CD56− T cells are NKT cells. However, CD56+ NKT cells expand poorly to continued stimulation. All mucin-stimulated NKT cells exhibited the activated/memory CD45RO phenotype. The NKT cell lines express the α/β T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the Vα24Vβ11 TCR typically associated with NKT cells. Whereas CD161 is generally considered a marker of NKT cells, the mucin-stimulated NKT cells did not express this marker. Thus, we have described two phenotypically distinct NKT types that do not display a biased TCR repertoire, but do display specificity for a tumor-specific peptide antigen (CTL-like activity), as well as HLA class I-deficient target cells (NK-like activity).

Non-isotopic in situ hybridization method for mitochondria in oncocytes

We used in situ hybridization to specifically identify mitochondria in a series of formalin-fixed, paraffin-embedded oncocytic lesions. Digoxigenin-labeled DNA probes were generated by the polymerase chain reaction (PCR), with primers designed to amplify a mitochondrion-specific 154 BP sequence within the ND4 coding region. Probes were hybridized with mitochondrial DNA under stringent conditions. Oncocytes were strongly and consistently stained, reflecting the high copy number of mitochondrial DNA within these cells. Because of the presence of endogenous biotin within mitochondria, digoxigenin is preferable to biotin as a label for detection of mitochondria.

CHFR protein expression predicts outcomes to taxane-based first line therapy in metastatic NSCLC

Purpose: Currently, there is no clinically validated test for the prediction of response to tubulin-targeting agents in non–small cell lung cancer (NSCLC). Here, we investigated the significance of nuclear expression of the mitotic checkpoint gene checkpoint with forkhead and ringfinger domains (CHFR) as predictor of response and overall survival with taxane-based first-line chemotherapy in advanced stage NSCLC. Methods: We studied a cohort of 41 patients (median age 63 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. CHFR expression by immunohistochemistry (score 0–4) was correlated with clinical outcome using chi-square test and Cox proportional models. A cutoff score of “3” was determined by receiver operator characteristics analysis for “low” CHFR expression. Results were validated in an additional 20 patients who received taxane-based chemotherapy at Emory University Hospital and the Atlanta VAMC. Results: High expression (score = 4) of CHFR is strongly associated with adverse outcomes: the risk for progressive disease after first-line chemotherapy with carboplatin–paclitaxel was 52% in patients with CHFR-high versus only 19% in those with CHFR-low tumors (P = 0.033). Median overall survival was strongly correlated with CHFR expression status (CHFR low: 9.9 months; CHFR high: 6.2 months; P = 0.002). After multivariate adjustment, reduced CHFR expression remained a powerful predictor of improved overall survival (HR = 0.24; 95% CI, 0.1–0.58%; P = 0.002). In the validation set, low CHFR expression was associated with higher likelihood of clinical benefit (P = 0.03) and improved overall survival (P = 0.038). Conclusions: CHFR expression is a novel predictive marker of response and overall survival in NSCLC patients treated with taxane-containing chemotherapy. Clin Cancer Res; 19(6); 1603–11. ©2013 AACR.

Disseminated pyoderma gangrenosum: Role for vascular endothelial growth factor and hypoxia inducible factor-2

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