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 Hodgkin's lymphoma. Known as the Hodgkin's 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 Hodgkin's 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 Hodgkin's lymphoma but also differentiate it from benign lymphoid hyperplasia.

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 alpha/beta T-cell receptor (TCR). The TCR repertoire was limited and varied with cell line, but was not the V alpha 24V beta 11 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).

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.

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.