Impact of the Cell Cycle Progression Test on Physician and Patient Treatment Selection for Localized Prostate Cancer.

PURPOSE: The cell cycle progression test is a validated molecular assay that assesses prostate cancer specific disease progression and mortality risk when combined with clinicopathological parameters. We present the results from PROCEDE-1000, a large, prospective registry designed to evaluate the impact of the cell cycle progression test on shared treatment decision making for patients newly diagnosed with prostate cancer. MATERIALS AND METHODS: Untreated patients with newly diagnosed prostate adenocarcinoma were enrolled in the study and the cell cycle progression test was performed on the initial prostate biopsy tissue. A set of 4 sequential surveys tracked changes relative to initial therapy recommendations (before cell cycle progression) based on clinicopathological parameters following physician review of the cell cycle progression test result, physician/patient review of the cell cycle progression test results and a minimum of 3 months of clinical followup (actual treatment). RESULTS: Of the 1,596 patients enrolled in this registry 1,206 were eligible for analysis. There was a significant reduction in the treatment burden recorded at each successive evaluation (p <0.0001), with the mean number of treatments per patient decreasing from 1.72 before the cell cycle progression test to 1.16 in actual followup. The cell cycle progression test caused a change in actual treatment in 47.8% of patients. Of these changes 72.1% were reductions and 26.9% were increases in treatment. For each clinical risk category there was a significant change in treatment modality (intervention vs nonintervention) before vs after cell cycle progression testing (p=0.0002). CONCLUSIONS: The cell cycle progression test has a significant impact in assisting physicians and patients reach personalized treatment decisions.

Patients Tested at a Laboratory for Hereditary Cancer Syndromes Show an Overlap for Multiple Syndromes in Their Personal and Familial Cancer Histories.

OBJECTIVE: Hereditary cancer testing guidelines are based on the premise that the common hereditary cancer syndromes have distinct, recognizable phenotypes. However, many syndromes present with overlapping cancers. The aim of this analysis was to identify the proportion of patients tested for Lynch syndrome (LS) or hereditary breast and ovarian cancer (HBOC) who met testing criteria for the other syndrome. METHOD: We analyzed a commercial laboratory database of patients tested for LS and HBOC in a clinical setting from 2006 to 2013. Patient cancer histories were analyzed using the 2012 NCCN criteria for LS and the 2013 NCCN criteria for HBOC. RESULTS: In all, 7% of the patients tested for HBOC met criteria for LS testing. The majority of these patients had a family history of colorectal (30.9%) and/or endometrial cancer (22.7%). Conversely, 29.5% of the patients tested for LS met criteria for HBOC testing. In this group, 30.5% of the patients had a personal history of breast cancer, and 12.6% had a personal history of ovarian cancer. CONCLUSIONS: Our data demonstrate a substantial phenotypic overlap among patients for multiple common inherited cancer syndromes, which likely complicates diagnosis and test selection. This supports the value of multigene panels to identify pathogenic mutations in the absence of a clinically specific phenotype.

Clinical validation of a gene expression signature that differentiates benign nevi from malignant melanoma.

BACKGROUND: Histopathologic examination is sometimes inadequate for accurate and reproducible diagnosis of certain melanocytic neoplasms. As a result, more sophisticated and objective methods have been sought. The goal of this study was to identify a gene expression signature that reliably differentiated benign and malignant melanocytic lesions and evaluate its potential clinical applicability. Herein, we describe the development of a gene expression signature and its clinical validation using multiple independent cohorts of melanocytic lesions representing a broad spectrum of histopathologic subtypes. METHODS: Using quantitative reverse-transcription polymerase chain reaction (PCR) on a selected set of 23 differentially expressed genes, and by applying a threshold value and weighting algorithm, we developed a gene expression signature that produced a score that differentiated benign nevi from malignant melanomas. RESULTS: The gene expression signature classified melanocytic lesions as benign or malignant with a sensitivity of 89% and a specificity of 93% in a training cohort of 464 samples. The signature was validated in an independent clinical cohort of 437 samples, with a sensitivity of 90% and specificity of 91%. CONCLUSIONS: The performance, objectivity, reliability and minimal tissue requirements of this test suggest that it could have clinical application as an adjunct to histopathology in the diagnosis of melanocytic neoplasms.

Bone marrow-induced Mef2c deficiency delays B-cell development and alters the expression of key B-cell regulatory proteins.

The Mef2 family transcriptional regulator Mef2c (myocyte enhancer factor 2c) is highly expressed in maturing bone marrow and peripheral mature B-cells. To evaluate the role of this transcription factor in B-cell development, we generated a B-cell-specific conditional deletion of Mef2c using the Mb-1-Cre transgene that is expressed during the early stages of immunoglobulin rearrangement. Young mice possessing this defect demonstrated a significant impairment in B-cell numbers in bone marrow and spleen. This phenotype was evident in all B-cell subsets; however, as the animals mature, the deficit in the peripheral mature B-cell compartments was overcome. The absence of Mef2c in mature B-cells led to unique CD23+ and CD23- subsets that were evident in Mef2c knockout primary samples as well as Mef2c-deficient cultured, differentiated B-cells. Genome-wide expression analysis of immature and mature B-cells lacking Mef2c indicated altered expression for a number of key regulatory proteins for B-cell function including Ciita, CD23, Cr1/Cr2 and Tnfsf4. Chromatin immunoprecipitation analysis confirmed Mef2c binding to the promoters of these genes indicating a direct link between the presence (or absence) of Mef2c and altered transcriptional control in mature B-cells.

Interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-ATPase localization and function.

The innate response interferon-inducible transmembrane (Ifitm) proteins have been characterized as influencing proliferation, signaling complexes and restricting virus infections. Treatment of cells lacking these proteins (IfitmDel) with IFN-ß resulted in the loss of clathrin from membrane compartments and the inhibition of clathrin-mediated phagocytosis, suggesting a molecular interaction between clathrin and Ifitm proteins. The pH of endosomes of IfitmDel cells, with or without IFN activation, was neutralized, suggesting the function of the vacular ATPase proton pumps in such cells was compromised. Co-immunoprecipitation of Ifitm3 with Atp6v0b demonstrated a direct interaction between the Ifitm proteins and the v-ATPase. These data suggest that the Ifitm proteins help stabilize v-ATPase complexes in cellular membranes which, in turn, facilitates the appropriate subcellular localization of clathrin.

The in vitro derivation of phenotypically mature and diverse B cells from immature spleen and bone marrow precursors.

The capacity of immature B cells of the spleen and bone marrow to differentiate in vitro into cells representing mature end stage cells was investigated using B-cell activating factor belonging to the TNF family (BAFF) and Notch pathway activators. Immature splenic and bone marrow B cells were found, in the presence of both of these activators, to mature into cells with follicular mature (FM) and marginal zone (MZ) cell phenotypes. Such cells were functionally responsive to B-cell-specific activation. The derivation in vitro of cells with an MZ phenotype was more robust from CD23(-) populations than CD23(+) immature/transitional B cells, suggesting a direct immature/T1 B cell to MZ cell differentiation pathway. Transcript analysis of the in vitro-derived B-cell populations demonstrated expression profiles similar to maturing B cells in vivo. FACS-purified populations of B220(+)CD19(+)CD21(-)CD23(-) cells from bone marrow of 2-wk-old mice gave rise to populations of CD21(+)CD23(-) cells with MZ cell phenotypes as well as CD21(+)CD23(+) cells with FM cell phenotypes in percentages similar to those found in vivo. These data suggest that the commitment to an MZ and FM B cell phenotype is set prior to immature B-cell release from the marrow.

Deletion of putative intronic control sequences does not alter cell or stage specific expression of Cr2.

The expression of the mouse Cr2 gene has been shown to be restricted to mature B cells, follicular dendritic cells and, in some reports, to a minor population of activated T cells. In this report, we demonstrate that the expression of antigen(s) recognized by the anti-CR2 antibody on the surface of T cells is co-incident with T cell apoptotic death. Two distinct regions of the Cr2 gene have been implicated as critical for specific expression, the promoter region at the transcription start site and a control region within the first intron of the gene, approximately 1500 bp from the transcription start site. We have created a mouse that is lacking this intronic control sequence which, in the wild type (WT) mouse, contains multiple known binding sites for RBP-jkappa, Oct, NFAT and YY1 proteins. The analysis of this mouse named Cr2iDelta (Cr2 intron deletion) demonstrated normal tissue specific expression of the Cr2 gene including a lack of expression in mouse T cells. B cell expression of the Cr2 gene products, CR1 and CR2, is normal compared to WT, and the FDC of these mice continue to express Cr2 gene products. Therefore the intronic control region of the Cr2 gene, defined in transfection-based reporter gene assays as instrumental in controlling the cell specific expression profile of Cr2, does not influence the expression of the Cr2 gene in vivo nor alter the relative production of the CR1 and CR2 proteins via alternative slicing of Cr2 gene products.

Regulation of murine splenic B cell CR3 expression by complement component 3.

Complement component C3 has established roles in both innate and adaptive immune responses. C3 cleavage products function in B cell activation through the complement receptors CD21/35. Phenotypes of Ab production between CD21/35(-/-) and C3(-/-) mice are not always congruent, implicating additional roles for C3 in B cell responses. To further characterize complement and complement receptors, we have identified a role for C3 in the regulation of CR3 on splenic B cells. Splenic B2 cells are not defined as expressing CR3, yet the analysis of splenic B cells from C3(-/-) animals demonstrate cell surface expression of CR3. B cells from both wild-type (WT) and C3(-/-) animals express CR3/CD11b/Itgam (integrin alpha M) gene transcripts although the level of such transcripts is 2- to 3-fold higher in B cells from the C3(-/-) animal vs WT cells. C3(-/-) and WT animals have similar B cell subpopulations with identical CR3 expression on B220(-) cells from the spleen, marrow, and lymph nodes. The C3-deficient environment is responsible for altered CR3 expression as WT splenic B cells transferred into C3(-/-) animals expressed cell surface CR3 within 48 h while transfer of C3(-/-) splenic B cells into WT animals depressed surface expression of CR3. Furthermore, transfer of C3-producing splenic macrophages into C3(-/-) mice depressed CR3 expression by resident B cells. These data suggest a role for C3 in influencing the level of expression of CR3 by modulating the transcript levels encoding the CD11b alpha integrin protein.

Defining in vivo transcription factor complexes of the murine CD21 and CD23 genes.

The expression of the CD21 and CD23 genes is coincident with differentiation from transition 1 B cells (T1) to transition 2 B cells (T2). To define constituents controlling CD21 and CD23 expression, we conducted chromatin immunoprecipitation analyses for candidate transcription factors. We found constitutive binding of Oct-1, NFAT species, YY1, NF-kappaB-p52, Pax5, E2A, and RBP-Jkappa to CD21 sequences and NF-kappaB-p52, Pax5, NFAT species, E2A, and RBP-Jkappa to CD23 promoter sequences. Splenic T and B cell subsets displayed constitutive binding of YY1, NF-kappaB-p52, Pax5, and Oct-1 proteins to CD21 sequences in B cells but no specific binding of NFATc3 or Pax5 in T cells. Similarly, CD23 sequences demonstrated constitutive binding of NF-kappaB-p52 in splenic T and B cells but only Pax5 in B cells. Of the various NFAT species, only a subset were found forming constitutive DNA/protein complexes with the CD21, CD23, and IL-2 gene sequences. Maturing B cells in the marrow possess stable Pax5 complexes on CD19, CD21, and CD23 gene promoters in the nuclei of such cells, even though only CD19 is expressed. The similarity of genetic controlling elements between the CD21 and CD23 genes does not suggest a mechanism for alternative regulation of these genes; however, separation of splenic B cell subsets into T1, T2, marginal zone (MZ), and mature follicular B cells, followed by quantitative RT-PCR, demonstrated the lack of appreciable CD23 transcripts in CD21(+) MZ cells. We propose an alternative derivation of MZ cells as maturing directly from T1 cells, leaving CD23 transcriptionally inactive in that lineage of cells.

Analysis of the regulatory role of BAFF in controlling the expression of CD21 and CD23.

The TNF family member BAFF serves to promote the survival and differentiation of maturing splenic B cells. The major receptor for BAFF (BAFF-R) is expressed by the transition 2, marginal zone and follicular, mature conventional B-2 cell populations; functional BAFF/BAFF-R signaling is required for T1 to T2 cell B cell maturation. Induced expression of CD23 and CD21 is also coincident with the T1 to T2 maturation stage. A key question we address in this report is if BAFF signaling directly induces CD21 and CD23 gene transcription and expression at this B cell transition point, or if their expression is simply coincident with B cell maturation and differentiation. We present data that supports the contention that BAFF does not preferentially induce the expression of CD23 or CD21 at the T1 to T2 transition, nor does exogenous BAFF lead to preferential increased expression of these proteins/genes in mature B cell populations. The analysis of LPS-induced splenic B cells from BAFF-R defective (A/WySnJ) mice did not show the preferential induction of expression of CD21 or CD23 that might have been expected if NF-kappaB-p52 protein was lacking due to insufficient BAFF-R signaling in cells bearing this mutation. Indeed, chromatin immunoprecipitation analysis demonstrated stable NF-kappaB-p52 complexes on CD21 and CD23 genes obtained from both wild type and A/WySnJ B cells. FACS analysis of splenic B cells from 1-, 2-, 3- and 6-week-old A/WySnJ mice demonstrated a block in differentiation (thus reducing overall B cell numbers) resulting in a failure of such cells to express CD21 but allowing for the expression level of CD23 per cell to reach levels approaching wild type. We have dubbed this CD23(HI)CD21(LO) subset as the T1b transition B cell. These data support the recognized role of BAFF as promoting the survival and differentiation of splenic B cells but do not support a model of BAFF signaling directly inducing the expression of the CD21 and CD23 proteins via translocation of NF-kappaB-p52 species.

Partial rescue of B cells in microphthalmic osteopetrotic marrow by loss of response to type I IFNs.

The microphthalmic (mi) mouse exhibits deficiencies in the development of osteoclasts, melanocytes, mast cells and marrow B cells. Previously, we demonstrated that the marrow of such mice over-express receptor activator of nuclear factor kappaB (RANK) ligand (RANKL). RANKL has been shown to induce the production of IFN-beta, a type I IFN. Additionally, maturing B cells have been shown to undergo apoptosis in response to type I IFNs including IFN-beta during differentiation. We hypothesized that the loss of B cells in the marrow of mi mice was due to the over-expression of IFN-beta as a result of heightened RANK-RANKL signaling. Creating a mouse with the mi genotype that was non-responsive to IFN-beta (lacking the type I IFNR) allowed us to test this hypothesis. These mice demonstrated an elevated number of marrow B cells and marrow precursor cells compared with mi animals possessing the type I IFNR. Intriguingly, type I IFNR-deficient wild-type animals also demonstrated an increased number of precursor cells in the marrow, but not an expansion of B220-positive pre-B cells, compared with wild type, suggesting that modulation of type I IFN responses directly controls the development of marrow constituents.