Long-term hematopoietic dysfunction in patients with large-scale mitochondrial DNA deletion syndromes.

BACKGROUND: Pearson syndrome (PS) and Kearns-Sayre syndrome (KSS) are single large-scale mitochondrial DNA deletion (SLSMD) syndromes. PS is characterized by severe, transient childhood cytopenia, whereas KSS typically manifests later in life without hematologic abnormalities. Despite distinct clinical presentations, both share a common mitochondrial DNA deletion. Recent observations suggest a potential link between PS progression and myeloid malignancy development, indicating that bone marrow failure (BMF) may be a key aspect of PS pathology and potentially universal across SLSMDs. METHODS: This study explores longitudinal hematological manifestations of SLSMD syndromes, focusing on bone marrow (BM) dysfunction. RESULTS: Sixteen patients with SLSMDs (13 PS and 3 KSS) were followed, of whom 75% experienced cytopenia, necessitating blood transfusions in 56%. Despite achieving transfusion independence at a median age of 24 months, persistent hematological abnormalities were noted. Comprehensive longitudinal BM studies were conducted in 62% of subjects and consistently revealed signs of marrow dysfunction, even without concurrent cytopenia. Median BM cellularity at a median age of four years and eight months was 50%, with histological signs of dyserythropoiesis, abnormal megakaryocytes, and signs suggesting myelodysplasia. Reduced CD34+ counts and BM colony-forming unit capacity were noted, alongside chromosome 7 aberrations in 16% of patients on cytogenetic studies. CONCLUSIONS: Our findings establish BM dysfunction as a persistent hallmark of SLSMD syndromes, posing a risk of clonal evolution and acquisition of chromosome 7 aberrations. This aligns with recent literature, emphasizing enduring BMF in SLSMD syndromes and advocating for tailored hematological monitoring guidelines for this unique patient cohort.

Celiac disease resolution after allogeneic bone marrow transplantation is associated with absence of gliadin-specific memory response by donor-derived intestinal T-cells.

PURPOSE: To elucidate the relative role of the immune system and intestinal epithelium in the ethiopatogenesis of Celiac disease (CD). METHODS: A patient with childhood CD who underwent allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia was followed for 5 years after resumption of gluten containing diet. Immunological memory to gliadin epitopes was assessed in the index patient and in 5 newly diagnosed CD patients by standard serology testing and by CFSE-based proliferation assays of peripheral blood CD4+ cells and of intestinal LPL towards gliadin-TTG antigens. Intestinal lymphocytes' origin was determined by combined immuno-histochemistry and fluorescent in-situ hybridiazation (FISH). RESULTS: Over 5 years of follow-up after receiving BMT from a HLA-matched woman and cessation of gluten-free diet, the patient has remained well, with negative periodic antibodies assays and unremarkable serial duodenal biopsies. In vitro proliferation assays showed lack of a memory response of the patient's peripheral blood and lamina propria CD4+ T-cells towards TTG, gliadin or TTG-treated gliadin, whereas memory responses were evident in the newly diagnosed CD patients. Immuno-FISH of post-BMT duodenal mucosa showed that the chromosomal phenotype of all the epithelial cells was XY. In contrast, CD45+ lymphocytic lineage cells were all donor-derived XX cells, presumably originating in the transplanted bone marrow and re-populating the intestinal wall. CONCLUSIONS: CD resolution following allogeneic BMT is associated with absent gliadin-specific memory response, and with a dichotomous lymphocyte-epithelial chimeric intestine. These observations suggest that the pathogenesis of CD is critically dependent upon the immune system rather than the epithelial compartment.

Breast cancer HER2 equivocal cases: is there an alternative to FISH testing? A pilot study using two different antibodies sequentially.

BACKGROUND: HER2 is an important prognostic and predictive marker in invasive breast cancer. It is currently assessed by immunohistochemistry for protein over-expression and by fluorescence in situ hybridization for gene amplification. The immunohistochemistry-equivocal cases (2+) are currently retested by FISH to determine eligibility for trastuzumab treatment. Retesting by FISH significantly raises the cost of patient management and sometimes delays treatment. The 4B5 is a new, FDA-approved, rabbit monoclonal antibody for HER2 testing. OBJECTIVES: To examine the reliability of 4B5 IHC HER2 testing in cases found by CB11 IHC to be HER2 status equivocal. METHODS: Twenty-eight invasive breast cancer cases, with an equivocal HER2 status by CB11 IHC, were retested by the 4B5 antibody as well as by FISH analysis. The scoring was performed using the same guidelines as HercepTest and was correlated with the FISH ratio. RESULTS: Of the original 28 CB11 clone designated equivocal cases, 14 (50%) showed negative HER2 staining using the 4B5 clone (HercepTest score 0 and 1+). Five cases (18%) proved to be positive (HercepTest score 3+) and 9 cases (32%) remained equivocal (HercepTest score 2+). The corresponding FISH ratio results showed that all 4B5 negative cases were negative by FISH testing, with a negative predictive value of 100%; 4 of 5 of the 4B5-positive cases were positive by FISH testing, with a positive predictive value of 80%. One 4B5-positive case was borderline-high (2.2 ratio) by FISH. The correlation between 4B5 IHC and FISH was statistically significant (P = 0.0013) by chi-square test. CONCLUSIONS: Sequential testing by 4B5 IHC could greatly reduce the need for FISH testing in cases considered HER2 equivocal by CB11 IHC.

Genomic analysis of metastatic rhabdomyosarcoma masquerading as acute leukemia.

Blast appearing cells in the peripheral blood and bone marrow may occasionally arise from non-hematopoietic tissues. We present a 58 year old female who presented at our emergency room with symptomatic pancytopenia. Several months earlier she was diagnosed and treated for rhabdomyosracoma of the nasopharynx and entered remission. When we examined the bone-marrow aspirate we estimated the number of blasts at 25 %. Based on this evaluation, a provisional diagnosis of acute leukemia was made. However, immunohistochemistry and flow cytometry analysis revealed that the cells presumed to be blasts were in fact rhabdomyosarcoma cells masquerading as leukemia. The mutational landscapes of the primary tumor and the bone marrow metastasis had similar yet distinct profiles. Annotation analysis suggested that the primary and metastatic tumors use alternate mutations to activate the RAS/AKT signaling pathways. In this case, looking beyond the mutational profiling revealed an additional layer of similarity between both the original and metastatic samples, exposing a common and possibly targetable pathway. Application of annotation tools in clinical practice could enable extraction of valuable information from somatic mutational gene panels.

Orbital marginal zone lymphomas: an immunohistochemical, polymerase chain reaction, and fluorescence in situ hybridization study.

Many studies have been performed on chromosomal aberrations of extranodal marginal zone lymphomas. However, only a few have been published so far on ocular adnexal marginal zone lymphomas. We studied 18 cases of orbital lymphoid cell infiltrates. Using fluorescence in situ hybridization (FISH), we studied some of the most common chromosomal aberrations found in extranodal marginal zone lymphomas as: trisomies 3, and rearrangements of the 18q21 MALTI gene to detect the translocations t(11;18)(q21;q21) and t(14;18)(q32;q21)MALT1. Our goals were as follows: (1) study those aberrations in our material and compare them with the literature, (2) check their prognostic significance, and (3) check whether studying those aberrations with FISH can be used as a diagnostic tool to differentiate reactive from neoplastic infiltrates, in addition to immunohistochemistry and polymerase chain reaction. We found a high frequency of trisomies 3 (68%) and 18 (56.6%), the highest published so far in orbital lymphomas. On the other hand, no rearrangement was seen in any of our cases. The histologic picture and the clinical course were the same when there was one or more aberrations. As for the diagnostic significance, the presence of a prior, concurrent, or subsequent lymphoma in almost all the positive for aberrations cases suggests that either the orbital infiltrates in these cases are lymphomas, or they have, at least, a malignant potential or a genetic instability. Therefore, the demonstration of these numerical aberrations by FISH may be an additional sensitive, reliable, and relatively simple tool to differentiate reactive from neoplastic orbital lymphoid cell infiltrates when the immunohistochemistry and polymerase chain reaction, performed in a busy and routine-based histopathology laboratory, are unsatisfactory.

MiRNA expression in psoriatic skin: reciprocal regulation of hsa-miR-99a and IGF-1R.

BACKGROUND: Psoriasis is a complex disease at the cellular, genomic and genetic levels. The role of microRNAs in skin development was shown in a keratinocyte-specific Dicer knockout mouse model. Considering that two main characteristics of psoriasis are keratinocytes hyperproliferation and abnormal skin differentiation, we hypothesized that aberrant microRNA expression contributes to the psoriatic phenotype. Here, we describe the differential expression of miRNAs in psoriatic involved and uninvolved skin as compared to normal skin, revealing an additional aspect of this complex disorder. METHODOLOGY/PRINCIPAL FINDINGS: Expression arrays were used to compare microRNA expression in normal skin versus psoriatic involved and uninvolved skin. Fourteen differentially expressed microRNAs were identified, including hsa-miR-99a, hsa-miR-150, hsa-miR-423 and hsa-miR-197. The expression of these microRNAs was reevaluated by qPCR. IGF-1R, which is involved in skin development and the pathogenesis of psoriasis, is a predicted target of hsa-miR-99a. In an in situ hybridization assay, we found that IGF-1R and miR-99a are reciprocally expressed in the epidermis. Using a reporter assay, we found that IGF-1R is targeted by hsa-miR-99a. Moreover, over expression of miR-99a in primary keratinocytes down-regulates the expression of the endogenous IGF-1R protein. Over expression of miR-99a also inhibits keratinocyte proliferation and increases Keratin 10 expression. These findings suggest that overexpression of hsa-miR-99a in keratinocytes drives them towards differentiation. In primary keratinocytes grown in high Ca(++), miR-99a expression increases over time. Finally, we found that IGF1 increases the expression of miR-99a. CONCLUSIONS/SIGNIFICANCE: We identified several microRNAs that are expressed differentially in normal and psoriatic skin. One of these miRNAs is miR-99a that regulates the expression of IGF-1R. Moreover, miR-99a seems to play a role in the differentiation of keratinocytes. We suggest that miR-99a is one of the regulators of the IGF-1R signaling pathway in keratinocytes. Activation of IGF1 signaling results in elevation of miR-99a which represses the expression of IGF-1R.

A novel translocation breakpoint within the BPTF gene is associated with a pre-malignant phenotype.

Partial gain of chromosome arm 17q is an abundant aberrancy in various cancer types such as lung and prostate cancer with a prominent occurrence and prognostic significance in neuroblastoma--one of the most common embryonic tumors. The specific genetic element/s in 17q responsible for the cancer-promoting effect of these aberrancies is yet to be defined although many genes located in 17q have been proposed to play a role in malignancy. We report here the characterization of a naturally-occurring, non-reciprocal translocation der(X)t(X;17) in human lung embryonal-derived cells following continuous culturing. This aberrancy was strongly correlated with an increased proliferative capacity and with an acquired ability to form colonies in vitro. The breakpoint region was mapped by fluorescence in situ hybridization (FISH) to the 17q24.3 locus. Further characterization by a custom-made comparative genome hybridization array (CGH) localized the breakpoint within the Bromodomain PHD finger Transcription Factor gene (BPTF), a gene involved in transcriptional regulation and chromatin remodeling. Interestingly, this translocation led to elevation in the mRNA levels of the endogenous BPTF. Knock-down of BPTF restricted proliferation suggesting a role for BPTF in promoting cellular growth. Furthermore, the BPTF chromosomal region was found to be amplified in various human tumors, especially in neuroblastomas and lung cancers in which 55% and 27% of the samples showed gain of 17q24.3, respectively. Additionally, 42% percent of the cancer cell lines comprising the NCI-60 had an abnormal BPTF locus copy number. We suggest that deregulation of BPTF resulting from the translocation may confer the cells with the observed cancer-promoting phenotype and that our cellular model can serve to establish causality between 17q aberrations and carcinogenesis.