21q22 amplification detection in three patients with acute myeloid leukemia: cytogenomic profiling and literature review.

BACKGROUND: 21q22 amplification is a rare cytogenetic aberration in acute myeloid leukemia (AML). So far, the cytogenomic and molecular features and clinical correlation of 21q22 amplification in AML have not been well-characterized. CASE PRESENTATION: Here, we describe a case series of three AML patients with amplified 21q22 identified by fluorescence in situ hybridization using a RUNX1 probe. Two of these patients presented with therapy-related AML (t-AML) secondary to chemotherapy, while the third had de novo AML. There was one case each of FAB M0, M1 and M4. Morphologic evidence of dysplasia was identified in both t-AML cases. Phenotypic abnormalities of the myeloblasts were frequently observed. Extra copies of 21q22 were present on chromosome 21 and at least one other chromosome in two cases. Two showed a highly complex karyotype. Microarray analysis of 21q22 amplification in one case demonstrated alternating levels of high copy number gain split within the RUNX1 locus at 21q22. The same patient also had mutated TP53. Two patients died at 1.5 and 11 months post-treatment, while the third elected palliative care and died within 2 weeks. CONCLUSIONS: Our results provide further evidence that 21q22 amplification in AML is associated with complex karyotypes, TP53 aberrations, and poor outcomes. Furthermore, we demonstrate that 21q22 amplification is not always intrachromosomally localized to chromosome 21 and could be a result of structural aberrations involving 21q22 and other chromosomes.

Clinical utility and cost-effectiveness analysis of chromosome testing concomitant with chromosomal microarray of patients with constitutional disorders in a U.S. academic medical center.

Chromosomal microarray (CMA) is now widely used as first-tier testing for the detection of copy number variants (CNVs) and absence of heterozygosity (AOH) in patients with multiple congenital anomalies (MCA), autism spectrum disorder (ASD), developmental delay (DD), and/or intellectual disability (ID). Chromosome analysis is commonly used to complement CMA in the detection of balanced genomic aberrations. However, the cost-effectiveness and the impact on clinical management of chromosome analysis concomitant with CMA were not well studied, and there is no consensus on how to best utilize these two tests. To assess the clinical utility and cost-effectiveness of chromosome analysis concomitant with CMA in patients with MCA, ASD, DD, and/or ID, we retrospectively analyzed 3,360 postnatal cases for which CMA and concomitant chromosome analysis were performed in the Colorado Genetic Laboratory (CGL) at the University Of Colorado School Of Medicine. Chromosome analysis alone yielded a genetic diagnosis in two patients (0.06%) and contributed additional information to CMA results in 199 (5.92%) cases. The impact of abnormal chromosome results on patient management was primarily related to counseling for reproductive and recurrence risks assessment (101 cases, 3.01%) while a few (5 cases, 0.15%) led to changes in laboratory testing and specialist referral (25 cases, 0.74%). The incremental cost-effectiveness ratio (ICER) of combined testing demonstrated the cost of each informative chromosome finding was significantly higher for patients with clinically insignificant (CI) CMA findings versus clinically significant (CS) CMA results. Our results suggest that a stepwise approach with CMA testing with reflex to chromosome analysis on cases with CS CMA findings is a more cost-effective testing algorithm for patients with MCA, ASD, and/or DD/ID.

Current concepts in breast cancer genomics: An evidence based review by the CGC breast cancer working group.

BACKGROUND: Genomic abnormalities in breast cancer have been described according to diverse conceptual frameworks, including histologic subtypes, clinical molecular subtypes, intrinsic DNA, RNA, and epigenetic profiles, and activated molecular pathways. METHODS: The Cancer Genomics Consortium (CGC) Breast Cancer Workgroup performed an evidence based literature review to summarize current knowledge of clinically significant genomic alterations in breast cancer using CGC levels of evidence. Targetable or disease-defining alterations were prioritized. RESULTS: We summarized genomic alterations in breast cancer within a framework of existing clinical tools for diagnosis, risk stratification, and therapeutic management. Using CGC levels of evidence, we catalog copy number profiles, gene expression profiles, and mutations in clinically significant genes. We also describe emerging molecular markers such as methylation profiling and immunotherapy biomarkers. CONCLUSION: A summary of currently available information on breast cancer genomics will enhance precision medicine by serving as an interpretive resource for clinical laboratory geneticists, providing a foundation for future practice guidelines, and identifying knowledge gaps to address in future research.

Clinicopathologic and genetic spectrum of infantile B-lymphoblastic leukemia: a multi-institutional study.

Acute lymphoblastic leukemia (ALL) in infants <1-year-old is biologically different from ALL in older children. Although KMT2A rearrangement is the predominant genetic signature in infantile B-ALL, disease course is heterogenous, behaving more aggressively in younger infants. We investigated clinicopathological differences throughout the first year to understand the transition to pediatric B-ALL. In a multi-institutional review involving four medical institutions, 54 cases of infantile B-ALL were identified. Patients were divided into congenital and non-congenital groups with multiple age subgroups. Male predominance was seen in congenital cases compared to female in non-congenital cases. There were decreasing trends of hyperleukocytosis, central nervous system involvement, KMT2A rearrangements, lineage switch, and mortality, versus increasing trends of CD10 expression and non-KMT2A abnormalities. Statistically significant differences emerged at 3 and 9 months, the latter was not previously described. Poor-prognostic risk factors decreased with age, the last trimester of infantile B-ALL essentially merging with pediatric B-ALL.

Clonality of neutrophilia associated with plasma cell neoplasms: report of a SETBP1 mutation and analysis of a single institution series.

A rare but well-known association between plasma cell neoplasms and neutrophilia is known to exist. Whether the neutrophilia is secondary to the plasma cell neoplasm or this convergence represents two independent clonal disorders is unclear. We reviewed five consecutive cases from a single institution over a 3-year period, applying molecular, cytogenetic and cytokine-profiling approaches to determine whether neutrophilia associated with plasma cell neoplasms represents a reactive or clonal process. We report, for the first time, the occurrence of a SETBP1 mutation in two cases, as well as changes in G-CSF and IL-6 in SETBP1 wild type vs. mutated patients that are supportive of a hypothesis that neutrophilia associated with plasma cell neoplasms may sometimes be reactive and may sometimes represent a second clonal entity. Finally, using an ex vivo drug screening platform we report the potential efficacy of the multi-kinase inhibitor dasatinib in select patients.

Heterogeneity of Abnormal RUNX1 Leading to Clinicopathologic Variations in Childhood B-Lymphoblastic Leukemia.

OBJECTIVES: Abnormalities of the RUNX1 gene in childhood B-acute lymphoblastic leukemia (B-ALL) are manifested by ETV6-RUNX1 or RUNX1 amplification. A detailed comparison between the two regarding clinicopathologic features with genetic analysis has not been performed previously. This parallel study assessed how different RUNX1 abnormalities affect the clinicopathology of B-ALL. METHODS: We compared clinicopathologic factors, including age, sex, WBC count, cerebrospinal fluid (CSF) involvement, immunophenotype, and blast proliferation rate between B-ALL with RUNX1 amplification (10 cases) and B-ALL with ETV6-RUNX1 translocation (67 cases) in childhood B-ALL. RESULTS: CD7 was often expressed in RUNX1 amplification but not in ETV6-RUNX1 (44% vs 0%, P = .0001) and appeared to correlate with CSF involvement in the former group (3/4 [75%]). CD13 was often detected in ETV6-RUNX1 with additional RUNX1 gain (38%) with an even higher frequency in double ETV6-RUNX1 translocation (77%), but was not detected in RUNX1 amplification (0%, P < .05). Children with RUNX1 amplification were older and more often CSF positive, while those with ETV6-RUNX1 were younger, more frequently had hyperleukocytosis, and had higher blast proliferation rates. CONCLUSIONS: RUNX1 copy numbers seem to be proportional to the age of B-ALL onset and the frequency of CSF involvement, while RUNX1 amplification vs translocation causes aberrant expression of CD7 and CD13, respectively.

Rare double-hit with two translocations involving IGH both, with BCL2 and BCL3, in a monoclonal B-cell lymphoma/leukemia.

BACKGROUND: Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by multiple recurring clonal cytogenetic anomalies and is the most common leukemia in adults. Chromosomal abnormalities associated with CLL include trisomy 12 and IGH;BCL3 rearrangement [t(14;19)(q32;q13)] that juxtaposes a proto-oncogenic gene BCL3 and an immunoglobulin heavy chain, a translocation that may be associated with shorter survival. In addition to the IGH;BCL3 rearrangement, other translocations involving 14q32 locus are involved in various lymphoproliferative pathologies pointing toward the significance of IGH locus in oncogenic progression. Significantly, in the majority of B-cell neoplasms that carry an IGH;BCL3 rearrangement, it is a sole translocation involving an IGH locus. CASE PRESENTATION: We report a patient who, in addition to trisomy 12, carried a rare double-hit translocation characterized by the IGH;BCL3 translocation and an additional clonal IGH;BCL2 translocation involving IGH and another proto-oncogene BCL2, t(14;18)(q32;q21), commonly found in follicular lymphoma. Further single nucleotide polymorphism (SNP) array-based analysis detected a duplication of the 58.8 kb region at 19q13.32 adjacent to the BCL3 translocation junction on chromosome 19q13. Interestingly, the duplicated region contained ERCC2 gene, which encodes a DNA excision repair protein involved in the cancer-prone syndrome, xeroderma pigmentosum. CONCLUSIONS: Taken together our findings indicate the existence of double-translocation driven oncogenic events involving both IGH loci and proto-oncogenes BCL2 and BCL3. Importantly, the IGH;BCL3 translocation was characterized by the duplication of the genomic region adjacent to BCL3, containing a major DNA repair factor, ERCC2.

Isochromosome 13 in a patient with childhood-onset schizophrenia, ADHD, and motor tic disorder.

BACKGROUND: A small percentage of all cases of schizophrenia have a childhood onset. The impact on the individual and family can be devastating. We report the results of genetic analyses from a patient with onset of visual hallucinations at 5 years, and a subsequent diagnosis at 9 years of schizophrenia, attention deficit hyperactivity disorder (ADHD) with hyperactivity and impulsivity, and chronic motor tic disorder. RESULTS: Karyotypic analysis found 45,XX,i(13)(q10) in all cells examined. Alpha satellite FISH of isochromosome 13 revealed a large unsplit centromeric region, interpreted as two centromeres separated by minimal or undetectable short-arm material or as a single monocentric centromere, indicating that the isochromosome likely formed post-zygotically by a short arm U-type or centromeric exchange. Characterization of chromosome 13 simple tandem repeats and Affymetrix whole-genome 6.0 SNP array hybridization found homozygosity for all markers, and the presence of only a single paternal allele in informative markers, consistent with an isodisomic isochromosome of paternal origin. Analysis of two chromosome 13 schizophrenia candidate genes, D-amino acid oxidase activator (DAOA) and 5-hydroxytryptamine (serotonin) receptor 2A (5-HTR2A), failed to identify non-synonymous coding mutations but did identify homozygous risk polymorphisms. CONCLUSIONS: We report a female patient with childhood-onset schizophrenia, ADHD, and motor tic disorder associated with an isodisomic isochromosome 13 of paternal origin and a 45,XX,i(13)(q10q10) karyotype. We examined two potential mechanisms to explain chromosome 13 involvement in the patient's pathology, including reduction to homozygosity of a paternal mutation and reduction to homozygosity of a paternal copy number variation, but were unable to identify any overtly pathogenic abnormality. Future studies may consider whether epigenetic mechanisms resulting from uniparental disomy (UPD) and the lack of chromosome 13 maternal alleles lead to the patient's features.

Primary high-grade B-cell lymphoma of the breast with concurrent IGH-BCL2 and MYC-IGL translocations in an adolescent patient.

BCL2 and MYC are oncogenes often deregulated in lymphomas. Concurrent IGH-BCL2 and MYC translocations result in a highly aggressive behavior of these tumors. Both primary breast lymphoma and lymphoma with concurrent BCL2-IGH and MYC translocations are rare and are primarily seen in adult patients. As a result of limited clinician experience and the condition's rarity, it poses a great challenge to pediatric pathologists and oncologists in terms of making an accurate diagnosis and choosing better treatment regimens. In this article, we report a case of an adolescent patient who presented with high-grade breast lymphoma with concurrent BCL2-IGH and MYC-IGL translocations, and we review the clinical, pathological, and genetic features; management strategies; and outcomes associated with this unusual neoplasm.

Male with mosaicism for supernumerary ring X chromosome: analysis of phenotype and characterization of genotype using array comparative genome hybridization.

Supernumerary, derivative, and ring X chromosomes are relatively common in Turner syndrome females but have been reported rarely in males. To date, less than 10 cases have been published, of which only 2 have been partially characterized in defining the breakpoints and genetic content of the derivative X chromosome. We describe a male with mosaicism for a supernumerary X chromosome (46,XY/47,XY, r(X)) who has multiple congenital anomalies, including features of craniofrontonasal dysplasia (Mendelian Inheritance in Man 304110) and the presence of ectopic female reproductive organs. Using comparative genomic hybridization array mapping, we determined that the derivative X is composed of a 24-Mb fragment that contains the regions Xp11.3 through Xq13.1 and lacks the XIST gene. This is the first report to describe a detailed molecular characterization of a ring X chromosome in a male by comparative genomic hybridization array analysis. We compare the clinical and molecular findings in this patient to other 46,XY, r(X) patients reported in the literature and discuss the potential role of disomy for known genes contained on the ring X chromosome.

Werner syndrome gene variants in human sarcomas.

Werner syndrome is an autosomal inherited disease that is characterized by premature aging. The gene mutated in Werner syndrome (WS), WRN, encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Among the WS phenotypes is an exceptionally high incidence of sarcomas. We asked whether spontaneous sarcomas, not known to be associated with WS, also harbor mutations or unreported single nucleotide polymorphisms (SNPs) in WRN. We analyzed RNA or DNA sequences within the helicase and exonuclease domains from 51 and 69 matched sarcoma and adjacent normal tissues, respectively. Among a total of 13 nucleotide variants detected, we identified three novel nonsynonymous substitutions: c.611C>T, c.809_810insT, and c.1882C>G. We further characterized one, c.611C>T, which results in substitution of an evolutionarily conserved proline at amino acid 204 in the exonuclease domain with leucine. We show that P204L WRN exhibits a reduction of WRN exonuclease activity; the specific activity is approximately 10-fold lower than that of wild-type WRN. In contrast, the helicase activity of P204L WRN is reduced less than twofold.

High prevalence of array comparative genomic hybridization abnormalities in adults with unexplained intellectual disability.

PURPOSE: Array comparative genomic hybridization is now a widely used clinical tool for the evaluation of intellectual disability. The current 10% yield of positive findings is based largely on pediatric data. Adults with unexplained intellectual disability have not been systematically studied with array comparative genomic hybridization. Here, we report our initial experience with array comparative genomic hybridization testing on 45 adults with unexplained intellectual disability referred to an adult genetics clinic. METHODS: Beginning in 2006, we applied clinically available array comparative genomic hybridization testing to adults referred with an intellectual disability phenotype. The initial platform used was an early generation targeted or constitutional array, which was replaced by our current platform using more than 5000 bacterial artificial chromosome clones with an average resolution of 500 Kb and targeting 114 disease loci. All patients also underwent high-resolution karyotype analysis and molecular testing for Fragile X syndrome. RESULTS: Our population comprised 45 patients with unexplained intellectual disability (18 men and 27 women) with an average age of 35.1 years. Most patients had not been evaluated by genetics clinics since childhood or had never undergone a genetic evaluation; only two had documentation of prior normal karyotype studies. Three subjects had abnormal high-resolution chromosome studies, which were also confirmed by array comparative genomic hybridization. Seven of the remaining 42 patients (17%) had novel genomic losses identified only by array comparative genomic hybridization. CONCLUSION: Abnormal genomic losses detected by array comparative genomic hybridization are prevalent in adults with unexplained intellectual disability. Our data showing abnormalities in 22% and 17% of overall patients and of cases with normal karyotypes, respectively, suggest that the yield of array comparative genomic hybridization in adults with unexplained intellectual disability may be higher than in pediatric populations.

Induction of p53 renders ATM-deficient mice refractory to hepatocarcinogenesis.

BACKGROUND & AIMS: p53 Mutations are very common in human hepatocellular carcinoma, and induction of hepatic p53 expression causes lysis of implanted hepatoblastoma cells in a chimeric mouse. Ataxia Telangiectasia Mutated (ATM) kinase senses DNA strand breaks and induces p53. Our aims were to establish whether ATM deficiency alters the carcinogenic response of hepatocytes to diethylnitrosamine (DEN). METHODS: Male ATM-deficient (ATM(-/-)), heterozygote (ATM(+/-)), and wild-type (WT) mice were injected with DEN at age 15 days, and animals were killed up to 12 months to assess p53, cell cycle, apoptosis, and liver tumor development. RESULTS: Whereas >80% of WT and ATM(+/-) mice developed hepatocellular carcinoma (HCC), at 9-12 months, ATM(-/-) mice remained refractory to DEN-induced HCC up to 15 months. At 6 and 9 months, and compared with WT mice, p53 and p19(ARF) expression were greatly enhanced in ATM(-/-) liver associated with up-regulation of ATR and Chk1; cleaved caspase-3 immunohistochemistry and caspase-3 activity were also significantly increased. Whereas livers of DEN-treated ATM(-/-) mice showed markers of senescence (beta-galactosidase, Cxcl-1), up-regulation of telomerase occurred concurrently. The possibility that such balanced senescence could result in immortalization was demonstrated in hepatocytes prepared at 9 months from DEN-treated ATM(-/-) liver. CONCLUSIONS: Hepatocarcinogenesis is abrogated in ATM-deficient mice in association with induction of ATR, Chk1, p53, and p19(ARF). Resultant cell cycle arrest and apoptosis of DNA-damaged cells are possible mechanisms that underlie this unique "refractoriness" to malignant transformation in DEN-initiated ATM(-/-) hepatocytes. The findings also show that prolonged up-regulation of p53 associated with some features of senescence does not inevitably cause organ failure.

Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells.

Normal human epithelial cells in culture have generally shown a limited proliferative potential of approximately 10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence-associated beta-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice.

UNLABELLED: Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. CONCLUSION: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC.

Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

PURPOSE: This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts. METHODS: Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses. RESULTS: This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes. CONCLUSIONS: The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

Dickkopf-1 mediated tumor suppression in human breast carcinoma cells.

Dickkopf-1 (DKK-1) is a secreted inhibitor of the Wnt signaling pathway. We previously identified DKK-1 as a candidate tumor suppressor and demonstrated that ectopic expression of the DKK-1 suppressed the tumorigenicity of HeLa cells in vitro and in vivo. Since suppression of tumorigenicity of HeLa cells by DKK-1 overexpression was not mediated by effects on beta-catenin dependent transcription, we hypothesized that DKK-1 might also inhibit tumorigenicity of breast carcinoma cell lines lacking an activated canonical Wnt pathway. In the present study we show that ectopic expression of DKK-1 in various breast cancer cell lines resulted in a change in the cell phenotype, increased sensitivity to apoptosis, inhibition of anchorage independent growth in vitro, and suppression of tumorigenicity in vivo. Consistent with known effects of DKK-1 on the canonical Wnt signaling pathway, ectopic expression of DKK-1 in breast carcinoma cells was associated with increased phosphorylation and degradation of beta-catenin. However, none of the breast tumor cells used in this study showed detectable levels of beta-catenin dependent activation of TCF/Lef promoter activity measured by reporter constructs. Consistent with the results of these transient transfection assays, we were unable to demonstrate the expected beta-catenin dependent, TCF/Lef mediated inhibition of cyclin D1 and c-myc gene transcription in breast cells overexpressing DKK-1. However, we found that cells with DKK-1 overexpression have increased activity of CamKII pathway. Overexpression of the constitutively active form of CamKII (T286D) resulted in inhibition of breast cancer cell tumorigenicity. Thus, our study supports the hypothesis that DKK-1 mediated tumor suppressor effect is independent of beta-catenin dependent transcription and identified the CamKII pathway that contributes into DKK-1 signaling.

Truncated RAR beta isoform enhances proliferation and retinoid resistance.

We previously reported the existence of a truncated isoform of the retinoic acid receptor beta, termed beta prime. Beta prime lacks the N-terminal domains of beta 2 and beta 4, including the DNA-binding domain. However, beta prime is able to heterodimerize and interact with transcription cofactors. To determine the effects of different retinoic acid receptor isoforms on cell proliferation and apoptosis, we transduced retinoid sensitive (MCF7) and retinoid-resistant (MDA-MB-231) cells with retinoic acid receptor beta 2, beta 4, or beta prime. Expression of the truncated beta prime isoform induces resistance to retinoic acid treatment in retinoid sensitive MCF7 cells. In both retinoid sensitive and resistant cells, expression of full-length beta 2 and beta 4 isoforms results in elevated sensitivity to retinoic acid treatment and caspase-independent cell death. Cell death in beta 4 transduced MDA-MB-231 cells was accompanied by metaphase chromosome decondensation and breakage suggestive of mitotic catastrophe. Our results provide evidence that: (a) the truncated form of the retinoic acid receptor beta induces retinoid resistance rather than sensitivity; and (b) alternative pathways of cell death are mediated by different isoforms in breast cancer cells.

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells.

BACKGROUND: The retinoic acid receptor beta 2 (RARbeta2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARbeta2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. METHODS: RNA from MDA-MB-435 human breast cancer cells transduced with RARbeta2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RESULTS: RARbeta2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 x 10(-8)), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARbeta2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). CONCLUSION: Antimetastatic RARbeta2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARbeta2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN.

So-called "inflammatory leiomyosarcoma'': a series of 3 cases providing additional insights into a rare entity.

Inflammatory leiomyosarcoma, a rare entity first described in 1995, has been characterized by smooth muscle differentiation, a near-haploid karyotype, and a surprisingly good prognosis. The morphology is similar to that of conventional leiomyosarcoma admixed with a chronic inflammatory infiltrate. Thus far, only 15 cases have been reported in the English language literature. We report the clinical and pathological features of 3 additional cases of inflammatory leiomyosarcoma. Two women (ages 64 and 25, respectively) and 1 man (age 32) presented with a thigh, ovary, and lung mass, respectively. Inflammatory symptoms, such as anorexia, fever, night sweats, abdominal pain, and diarrhea, coincided with the thigh and ovarian primaries. Immunohistochemical studies revealed diffuse positivity for desmin and poor expression for other smooth muscle and skeletal muscle markers (muscle-specific actin [0/3], alpha-smooth muscle actin 1/3 [focal], calponin [1/3], caldesmon [0/3], and myogenin [0/3]). CD68 was diffusely positive in both the histiocytes and spindle cell component in all cases. Ultrastructural evaluation of 1 case (lung primary) lacked definitive smooth muscle differentiation. Cytogenetic analysis in 1 of 2 cases that were karyotyped, identified a near-haploid karyotype, which has been reported in other cases of inflammatory leiomyosarcoma. The other case showed 2 clonal populations of cells with interstitial deletions of the short arm of chromosome 8 and the long arm of chromosome 9, respectively. The case without cytogenetic data was intimately associated with an ovarian mature teratoma. These data also suggest that inflammatory leiomyosarcoma may lack smooth muscle differentiation, characterized by diffuse immunoreactivity for desmin but lack of immunoreactivity for alpha-smooth muscle actin, calponin, and caldesmon. In addition, 2 of the 3 cases developed distant metastases to the lungs, which suggests that these lesions may have a worse prognosis than previously believed.