Alfred Au

Leukocyte composition of human breast cancer

Retrospective clinical studies have used immune-based biomarkers, alone or in combination, to predict survival outcomes for women with breast cancer (BC); however, the limitations inherent to immunohistochemical analyses prevent comprehensive descriptions of leukocytic infiltrates, as well as evaluation of the functional state of leukocytes in BC stroma. To more fully evaluate this complexity, and to gain insight into immune responses after chemotherapy (CTX), we prospectively evaluated tumor and nonadjacent normal breast tissue from women with BC, who either had or had not received neoadjuvant CTX before surgery. Tissues were evaluated by polychromatic flow cytometry in combination with confocal immunofluorescence and immunohistochemical analysis of tissue sections. These studies revealed that activated T lymphocytes predominate in tumor tissue, whereas myeloid lineage cells are more prominant in “normal” breast tissue. Notably, residual tumors from an unselected group of BC patients treated with neoadjuvant CTX contained increased percentages of infiltrating myeloid cells, accompanied by an increased CD8/CD4 T-cell ratio and higher numbers of granzyme B-expressing cells, compared with tumors removed from patients treated primarily by surgery alone. These data provide an initial evaluation of differences in the immune microenvironment of BC compared with nonadjacent normal tissue and reveal the degree to which CTX may alter the complexity and presence of selective subsets of immune cells in tumors previously treated in the neoadjuvant setting.

Determination of HER2 Gene Amplification by Chromogenic In Situ Hybridization (CISH) in Archival Breast Carcinoma

Purpose: To compare the efficacy of chromogenic in situ hybridization (CISH™) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer. Materials and Methods: HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT™), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase–streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40 × objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest. Results: HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively. Conclusion: By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.

Magnetic Resonance Imaging Captures the Biology of Ductal Carcinoma In Situ

PURPOSE Magnetic resonance imaging (MRI) is an important tool for characterizing invasive breast cancer but has proven to be more challenging in the setting of ductal carcinoma in situ (DCIS). We investigated whether MRI features of DCIS reflect differences in biology and pathology. PATIENTS AND METHODS Forty five of 100 patients with biopsy-proven DCIS who underwent MRI and had sufficient tissue to be characterized by pathologic (nuclear grade, presence of comedo necrosis, size, and density of disease) and immunohistochemical (IHC) findings (proliferation, Ki67; angiogenesis, CD34; and inflammation, CD68). Pathology and MRI features (enhancement patterns, distribution, size, and density) were analyzed using pairwise and canonical correlations. RESULTS Histopathologic and IHC variables correlated with MRI features (r = 0.73). The correlation was largely due to size, density (by either MRI or pathology), and inflammation (P < .05). Most small focal masses were estrogen receptor-positive. MRI enhancement patterns that were clumped were more likely than heterogeneous patterns to be high-grade lesions. Homogenous lesions were large, high grade, and rich in macrophages. Presence of comedo necrosis and size could be distinguished on MRI (P < .05). MRI was most likely to over-represent the size of less dense, diffuse DCIS lesions. CONCLUSION The heterogeneous presentation of DCIS on MRI reflects underlying histopathologic differences.

Ethnicity and spirituality in breast cancer survivors.

IntroductionMany women are incorporating spirituality as a way of coping with cancer. However, few studies have examined the role of spirituality in mood and quality of life among breast cancer survivors from different ethnic groups.MethodsOne hundred and seventy-five women who had completed treatment for breast cancer participated in in-depth interviews about their experiences. Transcripts were available for 161 women.ResultsThe majority (83%) of the women talked about their spirituality. The main themes were: (1) God as a Comforting Presence; (2) Questioning Faith; (3) Anger at God; (4) Spiritual Transformation of Self and Attitude Towards Others/Recognition of Own Mortality; (5) Deepening of Faith; (6) Acceptance; and (7) Prayer by Self. A higher percentage of African-Americans, Latinas, and Christians felt comforted by God than the other groups.ConclusionsThese results are consistent with the common assumption that more African-American and Latinas engage in spiritual activities and that African-Americans are more fatalistic than the other groups. Implications for Cancer Survivors: The present findings suggest that there are several dimensions of spirituality experienced among cancer survivors. For many the trauma of a cancer diagnosis might deepen their faith and appreciation of life as well as changing the way they view at themselves, their lives, and how they relate to those around them, including God.

The benefits of prayer on mood and well-being of breast cancer survivors

ObjectivesPrayer is becoming more widely acknowledged as a way to cope with cancer. The goal of this study was to compare differences in use of prayer between breast cancer survivors from different ethnic groups and examine how use of prayer is related to mood and quality of life.MethodsThis study used a mixed methods design. One hundred and seventy-five breast cancer survivors participated in a longitudinal study of survivorship. Women completed in-depth qualitative interviews and a battery of measures including quality of life, spirituality, social support, and mood.ResultsEighty-one percent of the women prayed. There were no significant differences between the groups for any of the psychological, social support, or quality of life variables with the exception of higher benefit finding and spiritual well-being among those who prayed. The data did show that women who prayed were able to find more positive contributions from their cancer experience than women who did not pray. The interviews showed that those who prayed tended to be African American or Asian, Catholic or Protestant. The prayers were for petitioning, comfort, or praise. Some of the women stated that they had difficulty praying for themselves.ConclusionsWhile there seems to be few differences in terms of standardized measures of quality of life, social support, and mood between those who prayed and those who did not, the interviews showed that certain ethnic minority groups seem to find more comfort in prayer, felt closer to God, and felt more compassion and forgiveness than Caucasian women.

Molecular profiling of human mammary gland links breast cancer risk to a p27(+) cell population with progenitor characteristics.

Early full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44(+) progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44(+)p27(+) cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27(+) cells and their proliferation. Our results suggest that pathways controlling p27(+) mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention.

Older women, breast cancer, and social support

IntroductionOne in ten women over the age of 65 will develop breast cancer. Despite this high incidence of breast cancer among older women, social support for them is often inadequate. This paper describes a qualitative study of the impact of a breast cancer diagnosis on older women from racially/ethnically diverse populations and their subsequent need for social support.MethodsForty-seven older African American, Asian American, Caucasian and Latina women between the ages of 65 to 83 participated in a larger study examining the impact of breast cancer on women from racially/ethnically diverse populations and the meaning and nature of social support. The women completed an in-depth qualitative interview on the psychosocial impact of breast cancer and the meaning and nature of social support.Results and ConclusionThe results indicate that there are variations in reactions to a breast cancer diagnosis among older women, and that these reactions impact their experiences with seeking social support at diagnosis and during treatment. Respondents were concerned about their aging bodies, potential dependency on others, and loss of autonomy. At the same time, the severity of cancer treatment and existing co-morbidities often meant they needed to learn to receive support, and to reach out if they had no support. The implications of these findings underscore the older cancer patient’s need to strengthen her supportive networks at the time of diagnosis, during treatment, and post-treatment.

Inhibition of CK2α down-regulates Notch1 signalling in lung cancer cells.

Protein kinase CK2 is frequently elevated in a variety of human cancers. The Notch1 signalling pathway has been implicated in stem cell maintenance and its aberrant activation has been shown in several types of cancer including lung cancer. Here, we show, for the first time, that CK2α is a positive regulator of Notch1 signalling in lung cancer cell lines A549 and H1299. We found that Notch1 protein level was reduced after CK2α silencing. Down‐regulation of Notch1 transcriptional activity was demonstrated after the silencing of CK2α in lung cancer cells. Furthermore, small‐molecule CK2α inhibitor CX‐4945 led to a dose‐dependent inhibition of Notch1 transcriptional activity. Conversely, forced overexpression of CK2α resulted in an increase in Notch1 transcriptional activity. Finally, the inhibition of CK2α led to a reduced proportion of stem‐like CD44 + /CD24− cell population. Thus, we report that the inhibition of CK2α down‐regulates Notch1 signalling and subsequently reduces a cancer stem‐like cell population in human lung cancer cells. Our data suggest that CK2α inhibitors may be beneficial to the lung cancer patients with activated Notch1 signalling.

The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma

The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, are involved in tumour progression, metastasis, and survival. We investigated the expression of CXCR4, CXCL12, and CXCR7 in malignant pleural mesothelioma to determine if they are possible biomarkers and potential therapeutic targets. Forty‐one mesothelioma tumour tissues, ten normal human pleural tissues, and two mesothelioma cell lines were stained with anti‐CXCR4, anti‐CXCL12, anti‐CXCR7, and anti‐p‐Akt antibodies. RT‐PCR was performed to determine the expression of CXCR4, CXCL12, and CXCR7 in six human mesothelioma cell lines (H28, 211H, H2052, ms‐1, H290, and H513) and one human normal mesothelial cell line, LP9. These seven cell lines were also stained with anti‐CXCR7. We found that CXCR4 and CXCL12 were expressed in 97.6% and 78.0% mesothelioma tissue samples, concurrently with strong expression of p‐Akt (R2 = 0.739 and 0.620, respectively). In addition, CXCR7 expression was weaker than CXCR4 expression in mesothelioma tissues. Furthermore, RT‐PCR showed that CXCR4 and CXCL12 were overexpressed in 5/6 mesothelioma cell lines (211H, H2052, ms‐1, H290, and H513), whereas CXCR7 was overexpressed in only 2/6 (H513 and H2052). Moreover, we found that the CXCR4 antagonist AMD3100 inhibited the growth of all five mesothelioma cell lines that overexpress CXCR4 and CXCL12. Our results suggest that the Akt–mTOR pathway is involved during the interruption of the CXCL12/CXCR4 axis in these five mesothelioma cell lines. In conclusion, CXCR4 and CXCL12 are highly expressed in most mesothelioma cell lines and tumour tissues, suggesting that CXCR4 and CXCL12 may be used as biomarkers for patients with mesothelioma. The CXCL12–CXCR4 interaction may be a potential therapeutic target for mesothelioma. Copyright

Molecular Profiling of Tumor Cells in Cerebrospinal Fluid and Matched Primary Tumors from Metastatic Breast Cancer Patients with Leptomeningeal Carcinomatosis

Although leptomeningeal carcinomatosis is a well-established clinical syndrome, virtually nothing is known about the tumor cells responsible for this particularly aggressive metastatic process. To isolate cerebrospinal fluid-derived tumor cells (CSFTC) from 15 patients with metastatic breast cancer diagnosed with leptomeningeal carcinomatosis, CSF samples were subjected to a two-step method involving immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS), a technique previously used for isolating circulating tumor cells (CTC) from blood. CSFTCs were subjected to genome-wide copy number analysis by array comparative genomic hybridization. Genomic profiling was successfully performed for 13 of 15 patients (87%). Copy number analysis in CSFTCs revealed genomic alterations commonly observed in primary breast cancer and CTCs, indicating their malignant origin. Interestingly, 12 (92%) harbored high-level gains on the 8q24 locus, which includes the MYC oncogene. Comparison of CSFTCs against corresponding archival primary tumors in six patients revealed clonal relationships with some divergence. Good concordance among serial samples attested to the reproducibility of the assay. Our approach for isolation and molecular analysis of CSFTCs yielded new insights into the molecular nature of these cells. Further genomic and functional analyses may help elucidate mechanisms by which tumor cells metastasize to the central nervous system.