Characterization of resident B cells of vascular walls in human atherosclerotic patients.

Animal models of atherosclerosis suggest that B cells have contradictory protective or proatherogenic effects that are also subset and context dependent. To further understand the pathophysiology of human atheroma, we characterized local Ig production and functional properties of resident B cells in human arterial lesions. Ig repertoires were analyzed by RT-PCR in carotid endarterectomy samples. Cytokine, differentiation marker and transcription factor mRNA expression was studied on arterial wall lymphocytes isolated by laser capture microdissection. Ig sequence analysis revealed that individual samples each contained a limited number of B cell clones. Functional α and γ mRNAs made up the majority of H chain mRNAs in the adventitia. Clonal evolution of Ig V regions, expression of activation-induced cytidine deaminase, clonal H chain switch, and an inverted λ/κ ratio of Ig L chain usage indicated that a local differentiation process was taking place in arterial walls. Clonotypic markers revealed different plaque and adventitia Ig repertoires and a B cell recirculation between adventitia and draining lymph nodes. Microdissected mononuclear cells had an activated phenotype expressing IL-6, GM-CSF, and TNF-α, whereas IL-2, IL-4, IL-10, M-CSF, and IFN-γ were not detected. Adventitial oligoclonal resident B cells of atherosclerotic patients are mainly mature B2 (conventional) CD20⁻ plasmablasts lacking markers of terminal differentiation to plasma cell (CD138 and Blimp-1). They present hallmarks of Ag-driven maturation and could act on inflammation and disease progression directly or by promoting polarization of other immune cells.

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment.

KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with anti-epidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allele-specific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

Prognostic significance of TRIM24/TIF-1α gene expression in breast cancer.

In this study, we have analyzed the expression of TRIM24/TIF-1α, a negative regulator of various transcription factors (including nuclear receptors and p53) at the genomic, mRNA, and protein levels in human breast tumors. In breast cancer biopsy specimens, TRIM24/TIF-1α mRNA levels (assessed by Real-Time Quantitative PCR or microarray expression profiling) were increased as compared to normal breast tissues. At the genomic level, array comparative genomic hybridization analysis showed that the TRIM24/TIF-1α locus (7q34) exhibited both gains and losses that correlated with mRNA levels. By re-analyzing a series of 238 tumors, high levels of TRIM24/TIF-1α mRNA significantly correlated with various markers of poor prognosis (such as the molecular subtype) and were associated with worse overall survival. By using a rabbit polyclonal antibody for immunochemistry, the TRIM24/TIF-1α protein was detected in nuclei of normal luminal epithelial breast cells, but not in myoepithelial cells. Tissue microarray analysis confirmed that its expression was increased in epithelial cells from normal to breast infiltrating duct carcinoma and correlated with worse overall survival. Altogether, this work is the first study that shows that overexpression of the TRIM24/TIF-1α gene in breast cancer is associated with poor prognosis and worse survival, and it suggests that this transcription coregulator may play a role in mammary carcinogenesis and represent a novel prognostic marker.

Is the mannose-6-phosphate/insulin-like growth factor 2 receptor coded by a breast cancer suppressor gene?

The multifunctional growth factor mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2-R) binds proteins sharing M6P signals, including cathepsins and IGF2. It is involved in targeting newly synthesized mannose-6-phosphorylated lysosomal enzymes, activating transforming growth factor beta (TGFbeta), and neutralising the mitogen IGF2 by transporting it to lysosomes. The M6P/IGF2-R was proposed as being coded by a tumor suppressor gene. We measured gene expression at the protein level by quantitative immunohistochemistry, using chicken high affinity IgY antibodies directed against human M6P/IGF2-R. Chicken immunization was performed with human purified M6P/IGF2-R, and IgY antibodies were extracted from egg yolk by polyethylene glycol precipitation method. The biosensor analysis showed that IgY antibodies bind M6P/IGF2-R with high affinity (Kd = 7.5 nM). Quantitative immunohistochemical studies in sections from invasive breast carcinoma and ductal carcinoma in situ (DCIS) indicated various levels (from 5 to 400 units) of the M6P/IGF2-R protein, which did not correlate with tumor size, histological grade, estrogen and progesterone receptors. Moreover, the M6P/IGF2-R level was increased in DCIS relative to adjacent normal tissue (p < 0.005) and then decreased in invasive carcinoma compared with DCIS (p < 0.02). The hypothesis of tumor suppressor gene is not supported by these studies. However, it is not excluded for a small proportion of the tumors. Its assay might help to complement the cathepsin D assay to predict breast cancer prognosis and physiopathology.

Peroxisome proliferator-activated receptor gamma regulates E-cadherin expression and inhibits growth and invasion of prostate cancer.

Peroxisome proliferator-activated receptor gamma (PPARgamma) might not be permissive to ligand activation in prostate cancer cells. Association of PPARgamma with repressing factors or posttranslational modifications in PPARgamma protein could explain the lack of effect of PPARgamma ligands in a recent randomized clinical trial. Using cells and prostate cancer xenograft mouse models, we demonstrate in this study that a combination treatment using the PPARgamma agonist pioglitazone and the histone deacetylase inhibitor valproic acid is more efficient at inhibiting prostate tumor growth than each individual therapy. We show that the combination treatment impairs the bone-invasive potential of prostate cancer cells in mice. In addition, we demonstrate that expression of E-cadherin, a protein involved in the control of cell migration and invasion, is highly up-regulated in the presence of valproic acid and pioglitazone. We show that E-cadherin expression responds only to the combination treatment and not to single PPARgamma agonists, defining a new class of PPARgamma target genes. These results open up new therapeutic perspectives in the treatment of prostate cancer.

RCL2, a new fixative, preserves morphology and nucleic acid integrity in paraffin-embedded breast carcinoma and microdissected breast tumor cells.

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions.