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.

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.

Multiple amyloidomas in an HIV patient presented as inguinal and mediastinal masses: a case report diagnosed by fine-needle aspiration.

Systemic amyloidosis can occur rarely as multiple masses with replacement of lymph nodes, causing lymphadenopathy and potentially mimicking neoplasms. We report a case of multiple amyloidomas in the inguinal lymph nodes and mediastinum in a 30-yr-old HIV positive woman with remote history of high grade dysplasia. Abundant amorphous material was obtained on fine-needle aspiration, making metastatic squamous cell carcinoma with abundant keratin a potential differential diagnosis. The prudent use of special stains such as Congo red, crystal violet, and immunohistochemical stains for keratin and kappa and beta light chains can be invaluable in helping to clarify the diagnosis. In patients with chronic inflammation, amyloidomas must be considered in the differential diagnosis of mass lesions so the appropriate work-up can be undertaken to minimize this potential pitfall.

Lower expression of CXCR4 in lymph node metastases than in primary breast cancers: potential regulation by ligand-dependent degradation and HIF-1alpha.

Stromal-derived factor-1 (SDF-1) is a unique ligand of the CXC chemokine receptor 4 (CXCR4), which is critically involved in the metastasis of breast cancer. High levels of SDF-1 in the common destination organs of metastasis, such as the lymph nodes, lungs, liver, and bones, attract CXCR4-positive tumor cells. The interaction between SDF-1 and CXCR4 leads to the activation of specific signaling pathways, allowing for homing and metastatic progression. However, regulation of CXCR4 expression at the metastatic organ site is not well-documented. We detected the expression of CXCR4 and hypoxia inducible factor (HIF)-1alpha in breast tumor tissues by immunohistochemical staining and analyzed SDF-1 in primary tumors and lymph nodes using real-time RT-PCR. Compared to the corresponding metastasized tumors in the lymph nodes, primary invasive carcinomas showed more intense staining for CXCR4, particularly on the cellular membrane. Both primary tumors and lymph node metastases exhibited higher levels of CXCR4 expression compared to non-neoplastic breast tissues. Therefore, we hypothesized that the tumor environment in the lymph nodes may cause the reduction of CXCR4 levels in the metastatic tumor cells because of: (1) high SDF-1 levels and (2) lower levels of HIF-1alpha. Our in vitro data demonstrated that high levels of SDF-1 can induce the internalization and degradation of CXCR4 through the lysosome pathway. In addition, lower levels of HIF-1alpha in the lymph node metastases, probably induced by the less hypoxic environment, further lowered CXCR4 levels. These results indicate that ligand-dependent degradation and lower HIF-1alpha levels may be potential causes of lowered levels of CXCR4 in the lymph nodes compared to the primary tumors. Our study suggests that CXCR4 levels in tumor cells are regulated by its microenvironment. These findings may enhance our ability to understand the biological behavior of breast cancers.

Expression of e-cadherin in high-risk breast cancer.

PURPOSE: E-cadherin expression is diverse, and differences in patient characteristics may produce variability in expression. Whereas some studies have indicated that downregulation of e-cadherin, associated with loss of cellular adhesiveness, was correlative with poor prognosis and metastasis, other studies have failed to confirm this. The present study uses a highly homogenous population of patients at high-risk for breast cancer, on the basis of ethnic and socio-economic status, to examine the relationship between e-cadherin and other prognostic markers in breast cancer. METHODS: Immunohistochemical staining was undertaken for estrogen (ER) and progesterone (PR) receptors, epidermal growth factor receptor 2 (Her-2), p53, vascular endothelial factor (VEGF), and hypoxia inducible factor 1alpha (HIF-1alpha) and the levels of these markers was compared to e-cadherin expression in a high-risk African-American patient population. RESULTS: E-cadherin expression persisted into the later stagers of the disease, and was strongly associated with Her-2 and HIF-1alpha expression, but not p53, ER/PR or VEGF. CONCLUSIONS: In contrast to other studies on heterogeneous populations, e-cadherin is preserved in aggressive tumors in this high-risk population. The ethnic and socio-economic risk stratification needs to be accounted for in studies correlating markers and prognosis.

The negative predicative value of breast fine-needle aspiration biopsy: the Massachusetts General Hospital experience.

Breast fine-needle aspiration biopsy (FNAB) has been increasingly accepted as an important triage tool for the evaluation of breast lumps. We examined the clinical utility and diagnostic accuracy of a negative breast FNAB result by studying 450 breast aspirates in 413 patients (average age 45 years) with a "negative" or benign cytologic interpretation performed at Massachusetts General Hospital over a 4-year period. Of these patients, 121 (29%) underwent subsequent biopsy and 17 (4%) were found to have malignancy (3% of total negative FNABs; 14% with histology). None of these 17 patients had a triple negative test. A cohort of 115 patients had documentation of negative physical, radiologic, and cytologic examinations (the triple negative), none of whom were found to have malignancy on histologic or at least 2-year clinical follow-up (negative predictive value [NPV] = 100% with a triple-negative test). Outside of the triple-negative test, the NPV of a negative breast FNAB is reduced with a false-negative rate of 7%. However, in the setting of a triple-negative test, the NPV in our patient population was 100%, reassuring the patient and clinician that clinical follow-up and not surgical intervention was sufficient for proper patient care.

Correlation and expression of p53, HER-2, vascular endothelial growth factor (VEGF), and e-cadherin in a high-risk breast-cancer population.

BACKGROUND: Many genetic traits common to aggressive breast carcinoma have been identified; yet little is known about the interrelationships of such traits during tumor development, especially in women prone to aggressive cancer. This study examined the expression of four biological markers associated with poor prognosis at each stage of breast cancer progression in primary tumors from women of lower economic status and assessed the relationship between these markers. METHODS: Archived primary breast tumors from 77 patients were assessed by immunohistochemical analysis for expression of human epidermal growth receptor 2 (HER-2), p53, vascular endothelial growth factor (VEGF), and e-cadherin, and the relationships between the expressions of these molecules were studied. RESULTS: Twenty-two (29%) patients had advanced (stage III or IV) disease. HER-2, VEGF, e-cadherin, and p53 signal were positive for 31 (40%), 58 (75%), 63 (82%), and 37 (48%) of patients, respectively. Among the markers tested, only p53 exhibited a significant association between expression and stage of the disease ( P = 0.012). Expression of e-cadherin was positively associated with HER-2 overexpression ( P = 0.004), and high levels of HER-2 occurred with strongly positive e-cadherin tumors. Marginally significant positive associations were observed between HER-2 and p53 signal ( P = 0.06), and between disease stage and e-cadherin expression ( P = 0.08). CONCLUSION: The significant tendency toward expression of e-cadherin in conjunction with HER-2 overexpression in breast cancer is a novel finding. The association of p53 with more advanced stages of cancer emphasizes it as a key participant in metastatic processes in breast cancer. Many genetic traits common to aggressive breast carcinoma have been identified; yet little is known about the interrelationships of such traits during tumor development, especially in women prone to aggressive cancer. This study examined the expression of four biological markers associated with poor prognosis at each stage of breast cancer progression in primary tumors from women of lower economic status and assessed the relationship between these markers.

Inhibition of breast cancer metastasis by selective synthetic polypeptide against CXCR4.

Metastasis shares many similarities with leukocyte trafficking. Among those chemokine receptors thought to be involved in hemopoietic cell homing, stromal cell-derived factor-1 and its receptor CXC chemokine receptor-4 (CXCR4) have received considerable attention. Like hemopoietic cell homing, levels of stromal cell-derived factor-1 are high at sites of breast cancer metastasis including lymph node, lung, liver, and the marrow. Moreover, CXCR4 expression is low in normal breast tissues and high in malignant tumors, suggesting that a blockade of CXCR4 might limit tumor metastasis. We therefore investigated the role of a synthetic antagonist 14-mer peptide (TN14003) in inhibiting metastasis in an animal model. Not only was TN14003 effective in limiting metastasis of breast cancer by inhibiting migration, but it may also prove useful as a diagnostic tool to identify CXCR4 receptor-positive tumor cells in culture and tumors in paraffin-embedded clinical samples.

Primary small bowel carcinoid tumor with bilateral breast metastases: report of 2 cases with different clinical presentations.

CONTEXT: Carcinoid tumor metastatic to the breast is uncommon and can closely mimic a mammary carcinoma. The differentiation of metastatic carcinoid tumor from primary breast tumor is important, however, owing to different clinical management and prognosis. OBJECTIVE: The purpose of this study was to describe 2 patients with bilateral metastatic carcinoid tumors to the breast with different clinical manifestations. DESIGN: We examined the radiological, clinical, cytologic, histologic, immunohistochemical, and ultrastructural features of these 2 cases. RESULTS: In case 1, the tumor presented initially as a stellate mass on mammogram and was diagnosed as grade II infiltrating ductal carcinoma. It was only after the discovery of small intestinal, liver, ovarian, and contralateral breast masses, as well as careful morphologic and immunohistochemical evaluations, that the true nature of the tumor was realized. In case 2, the tumor initially presented as a small intestinal tumor with liver metastases and bilateral breast masses. The breast masses were diagnosed accurately as metastatic carcinoid tumor by morphologic and immunohistochemical evaluations. CONCLUSIONS: Metastatic carcinoid tumor to the breast is uncommon, but poses a diagnostic challenge in that morphologically it can closely mimic a primary breast tumor. Careful attention to clinical features and the use of auxiliary immunohistochemical studies can help in arriving at the correct diagnosis.