The mammary gland and the homeobox gene Otx1.

The mammary gland, the unique organ that primarily form at puberty, is an ideal model to study the functions of homeobox (HB) genes in both development and tumorigenesis. HB genes comprise a large family of developmental regulators that have a critical role in cell growth and differentiation. In the normal mammary gland, homeobox genes are involved in ductal formation, epithelial branching, and lobulo-alveolar development by regulating epithelial proliferation and differentiation. The HB genes are controlled in a spatial and temporal manner in both stromal and epithelial cells. They are coordinately regulated by hormones and extracellular matrix, suggesting that many signaling pathways are involved in homeobox gene functions. When homeobox genes are misexpressed in animal models, different defects are displayed in mammary gland development. Aberrant expression of homeobox genes, overexpressed or downregulated, is found in primary carcinomas and in breast cancer. The Otx1 HB gene is a classic regulatory of nervous system development during embryogenesis. Postnatally Otx1 is transcribed in the anterior pituitary gland, where activates transcription of the pituitary hormones, and plays a role in hematopoiesis, enhancing pluripotent cells, and erythroid differentiation. Otx1 can still be detected in mature cells of the erythroid and megacaryocytic lineage. During cyclical development of mammary gland, the Otx1 gene is overexpressed in lactation, confirming a role of this transcription factor in cell differentiation. Recent studies report that Otx1 is overexpressed in breast cancer. Otx1 is expressed during embryogenesis, and it is expressed again during carcinogenesis, implying its possible function in differentiation of neoplastic cells.

The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman-Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies.

An investigation of 22 new patients with Shwachman-Diamond syndrome (SDS) and the follow-up of 14 previously reported cases showed that (i) clonal chromosome changes of chromosomes 7 and 20 were present in the bone marrow (BM) of 16 out of 36 cases, but if non-clonal changes were taken into account, the frequency of anomalies affecting these chromosomes was 20/36: a specific SDS karyotype instability was thus confirmed; (ii) the recurrent isochromosome i(7)(q10) did not include short arm material, whereas it retained two arrays of D7Z1 alphoid sequences; (iii) the deletion del(20)(q11) involved the minimal region of deletion typical of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML); (iv) only one patient developed MDS, during the rapid expansion of a BM clone with a chromosome 7 carrying additional material on the short arms; (v) the acquisition of BM clonal chromosome anomalies was age-related. We conclude that karyotype instability is part of the natural history of SDS through a specific mutator effect, linked to lacking SBDS protein, with consequent clonal anomalies of chromosomes 7 and 20 in BM, which may eventually promote MDS/AML with the patients' ageing.

Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia.

Reciprocal translocation t(9;22) is central to the pathogenesis of chronic myeloid leukemia. Some authors have suggested that Alu repeats facilitate this process, but supporting analyses have been sparse and often anecdotal. The purpose of this study was to analyze the local structure of t(9;22) translocations and assess the relevance of interspersed repeat elements at breakpoints. Collected data have been further compared with the current models of DNA recombination, in particular the single-strand annealing (SSA) and the nonhomologous end joining (NHEJ) processes. We developed a protocol for the rapid characterization of patient-specific genomic junctions and analyzed 27 patients diagnosed with chronic myeloid leukemia. Sequence analysis revealed microhomologies at the junctions of 21 patients of 27, while interspersed repeats were of relevance (P < 0.05) in at least 16 patients. These findings are more frequent than expected and give an indication that the main mechanisms involved in the t(9;22) translocation are the SSA and NHEJ pathways, both playing a role. Furthermore, our report is consistent with microhomologies facilitating the joining of DNA ends in the translocation process, and with both Alu and a variety of other repeat sequences pairing nonhomologous chromosomes during the SSA pathway.

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

OTX1 and OTX2 as possible molecular markers of sinonasal carcinomas and olfactory neuroblastomas.

OTX Homeobox genes are involved in embryonic morphogenesis and in the development of olfactory epithelium in adult. Mutations occurring in the OTX genes are reported to be associated to tumorigenisis in human. No reports correlate the expression of OTX genes and neoplasms of the nasal cavity. Thus, through immunohistochemical and Real-time PCR analysis we investigated OTX1 and OTX2 expression in the more frequent types of nasal and sinonasal tumours. Variable expression of both genes were found in normal sinonasal mucosa and in tumours. Interestingly, no expression of both OTX genes were detected in sinonasal intestinal-type adenocarcinomas; only OTX1 was found in non-intestinal-type adenocarcinomas and OTX2 was selectively expressed in olfactory neuroblastomas. In conclusion, OTX1 and OTX2 genes might have a role in the pathogenesis of different types of sinonasal neoplasms.

Establishment and study of different real-time polymerase chain reaction assays for the quantification of cells with deletions of chromosome 7.

The evaluation of residual disease, which has prognostic value in the treatment of hematological malignancies, is currently assessed by scoring a limited number of cells by karyotyping and molecular cytogenetics. Real-time polymerase chain reaction (PCR) is an easier and more sensitive technique, enables analysis of a larger number of cells, and decreases sampling error. However, real-time PCR has been applied only to target transcripts of fusion genes. Here, we considered two real-time PCR strategies to quantify a number of cells carrying a partial deletion of chromosome 7 mixed with normal disomic cells. The first strategy was based on the amplification of two sequences, one on chromosome 7 and the other on chromosome 14. In the second strategy residual disease was assessed by the ratio between the two alleles of a bi-allelic marker, mapped on chromosome 7, measured with allele-specific assays. Precision and accuracy of the two approaches were tested by reference samples with nominal values of residual disease ranging from 2 to 95%. As expected the second strategy resulted in more precise and accurate monitoring within the range from 5 to 95%. Furthermore, this method may be applied to assess the number of dysplastic or neoplastic clones carrying any unbalanced chromosome changes.

Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: assays development, characterization and pre-validation.

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5 % to 95 % and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1 % for both the approaches, allowing to extend the range of quantifications from 1 % to 99 %. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

Familial myelodysplastic syndromes, monosomy 7/trisomy 8, and mutator effects.

A family is reported, in which two sisters presented with myelodysplastic syndrome (MDS), namely refractory anemia with excess of blasts in transformation (RAEB-t), and refractory anemia (RA). Bone marrow chromosome changes were present in both: trisomy and tetrasomy 8 (with a pericentric inversion of one chromosome 8) in the older sister, and monosomy 7 (with clones with additional trisomies 19 and 21) in the younger one. Molecular data were obtained on the parental chromosome involved in these numerical anomalies, which proved to be of paternal origin in these cases. The observations of this family, and a review of familial cases of MDS/acute myeloid leukemia (AML), led us to consider that they may be divided into two groups: those which arise on the basis of a Mendelian predisposing disorder exerting a mutator effect, often with the acquisition of monosomy 7, and those in which no specific Mendelian predisposing disease is recognized, as the familial monosomy 7 cases and the one reported here. We postulate that in these families an inherited mutator effect is present and that it causes a karyotype instability, which leads to MDS/AML, often through the acquisition of monosomy 7 and trisomy 8.

Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia.

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Chromosome anomalies in bone marrow as primary cause of aplastic or hypoplastic conditions and peripheral cytopenia: disorders due to secondary impairment of RUNX1 and MPL genes.

BACKGROUND: Chromosome changes in the bone marrow (BM) of patients with persistent cytopenia are often considered diagnostic for a myelodysplastic syndrome (MDS). Comprehensive cytogenetic evaluations may give evidence of the real pathogenetic role of these changes in cases with cytopenia without morphological signs of MDS. RESULTS: Chromosome anomalies were found in the BM of three patients, without any morphological evidence of MDS: 1) an acquired complex rearrangement of chromosome 21 in a boy with severe aplastic anaemia (SAA); the rearrangement caused the loss of exons 2-8 of the RUNX1 gene with subsequent hypoexpression. 2) a constitutional complex rearrangement of chromosome 21 in a girl with congenital thrombocytopenia; the rearrangement led to RUNX1 disruption and hypoexpression. 3) an acquired paracentric inversion of chromosome 1, in which two regions at the breakpoints were shown to be lost, in a boy with aplastic anaemia; the MPL gene, localized in chromosome 1 short arms was not mutated neither disrupted, but its expression was severely reduced: we postulate that the aplastic anaemia was due to position effects acting both in cis and in trans, and causing Congenital Amegakaryocytic Thrombocytopenia (CAMT). CONCLUSIONS: A clonal anomaly in BM does not imply per se a diagnosis of MDS: a subgroup of BM hypoplastic disorders is directly due to chromosome structural anomalies with effects on specific genes, as was the case of RUNX1 and MPL in the patients here reported with diagnosis of SAA, thrombocytopenia, and CAMT. The anomaly may be either acquired or constitutional, and it may act by deletion/disruption of the gene, or by position effects. Full cytogenetic investigations, including a-CGH, should always be part of the diagnostic evaluation of patients with BM aplasia/hypoplasia and peripheral cytopenias.

Evaluating chromosomal mosaicism by array comparative genomic hybridization in hematological malignancies: the proposal of a formula.

Array-based comparative genomic hybridization (aCGH) has proven indispensable to the study of unbalanced constitutional and acquired chromosomal anomalies, but its sensitivity for detecting mosaicism is still not well established. On the basis of the ADM2 algorithm used for microarray image analysis with one of the most widely used oligomer-based aCGH platforms [the whole genome 244K system by Agilent Technologies (Santa Clara, CA)] we suggest a formula to infer the percentage of cells bearing a chromosome imbalance in cases with constitutional or acquired mosaicism. Three examples of acquired mosaicism in which this formula was applied are reported together with parallel fluorescence in situ hybridization (FISH) to interphase nuclei with informative probes. Although some approximation affects both the results inferred from aCGH and FISH data, the proposed formula was successful in the three patients studied.

Comparative genomic hybridization on microarray (a-CGH) in constitutional and acquired mosaicism may detect as low as 8% abnormal cells.

BACKGROUND: The results of cytogenetic investigations on unbalanced chromosome anomalies, both constitutional and acquired, were largely improved by comparative genomic hybridization on microarray (a-CGH), but in mosaicism the ability of a-CGH to reliably detect imbalances is not yet well established. This problem of sensitivity is even more relevant in acquired mosaicism in neoplastic diseases, where cells carrying acquired imbalances coexist with normal cells, in particular when the proportion of abnormal cells may be low.We constructed a synthetic mosaicism by mixing the DNA of three patients carrying altogether seven chromosome imbalances with normal sex-matched DNA. Dilutions were prepared mimicking 5%, 6%, 7%, 8%, 10% and 15% levels of mosaicism. Oligomer-based a-CGH (244 K whole-genome system) was applied on the patients' DNA and customized slides designed around the regions of imbalance were used for the synthetic mosaics. RESULTS AND CONCLUSIONS: The a-CGH on the synthetic mosaics proved to be able to detect as low as 8% abnormal cells in the tissue examined. Although in our experiment some regions of imbalances escaped to be revealed at this level, and were detected only at 10-15% level, it should be remarked that these ones were the smallest analyzed, and that the imbalances recurrent as clonal anomalies in cancer and leukaemia are similar in size to those revealed at 8% level.

Molecular monitoring of residual disease in chronic myeloid leukemia by genomic DNA compared with conventional mRNA analysis.

Translocation t(9;22), which produces the BCR-ABL gene, is pathognomonic of chronic myeloid leukemia. For clinical purposes, the amount of chimeric transcript is considered proportional to the leukemic clone; thus, mRNA is commonly used for molecular monitoring of patients. However, there is no consensus regarding the degree of increase in mRNA that should cause concern or whether the absence of transcript indicates a "cure." In this study, we analyzed 57 samples from 10 chronic myeloid leukemia patients undergoing imatinib treatment. For each sample, we compared BCR-ABL mRNA levels with the actual proportion of leukemic cells, which were measured through a novel genomic approach based on the quantitative amplification of DNA breakpoints. The two approaches gave similar patterns of residual disease, and the majority of patients were still positive after an average treatment period of 2 years. Nevertheless, in one of two patients with confirmed undetectable levels of chimeric transcript, DNA still revealed the persistence of leukemic cells at 42 months. These findings appear to justify the clinical practice of maintaining imatinib treatment indefinitely. However, the absence of leukemic DNA (observed in 1 of 10 patients) could be used to identify possible candidates for drug discontinuation. In conclusion, DNA analysis proved to be a reliable index of residual disease with potential applications in the field of clinical diagnostics and research.

Monitoring the isochromosome i(7)(q10) in the bone marrow of patients with Shwachman syndrome by real-time quantitative PCR.

Clonal chromosome anomalies may be found in the bone marrow (BM) of patients with Shwachman syndrome, who are at risk to develop myelodysplastic syndromes and/or acute myeloid leukemias. In particular, an isochromosome i(7)(q10) is frequent, and is usually monitored by chromosome analyses. We tested an approach by real-time quantitative polymerase chain reaction (RQ-PCR) on a chromosome 7 polymorphism. Five DNA samples of 2 Shwachman syndrome patients with clonal i(7)(q10) in the BM were used. Both were heterozygous for the diallelic indel polymorphism MID1064, which maps in 7q35. The percentage of i(7)(q10)-positive cells was extrapolated from the ratio of the 2 alleles measured by means of an allele-specific RQ-PCR assay. The results were compared with cytogenetic analyses on the same material used for RQ-PCR. In 1 patient, the RQ-PCR results matched well with those of chromosome analyses, whereas in the other one RQ-PCR showed that around 40% of the BM cells were abnormal, while they resulted to be nearly 80% with conventional monitoring assays. As the results obtained by RQ-PCR refer to the DNA of around 128,000 BM cells, our method proved to be feasible and more efficient in the quantitative evaluation of the i(7)(q10)-positive clone than conventional ones.

Trisomy 8 in myelodysplasia and acute leukemia is constitutional in 15-20% of cases.

The trisomy 8 found in malignancies may derive from a constitutional trisomy 8 mosaicism (CT8M), and in these cases the trisomy itself may be regarded as the first mutation in a multistep carcinogenetic process. To assess the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8, an informative sample of 14 patients was collected. The data ascertained included chromosome analyses of fibroblast cultures and of PHA-stimulated blood cultures in patients with normal blood differential count, as well as possible CT8M clinical signs. One patient showed trisomy 8 in all cell types analyzed and undoubtedly has a CT8M; a second patient consistently showed trisomy 8 in PHA-stimulated blood cultures when no immature myeloid cells were present in blood and should be considered as having CT8M; a third patient, with Philadelphia-positive chronic myelocytic leukemia, was more difficult to interpret, but the possibility that she had CT8M is likely. A few clinical signs of CT8M were also present in these three patients. Our data indicate that the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8 is approximately 15-20%.

Familial platelet disorder with propensity to acute myelogenous leukemia: genetic heterogeneity and progression to leukemia via acquisition of clonal chromosome anomalies.

Familial platelet disorder with propensity to acute myelogenous leukemia, or FPD/AML (OMIM #601399), is a rare autosomal dominant condition, with only 12 families reported. It is characterized by qualitative and quantitative platelet defects and predisposition to the development of myeloid malignancies. Causal mutations have been identified in the RUNX1 gene (also known as AML1, CBFA2) in the 11 families so far analyzed. RUNX1 is a gene frequently involved in the pathogenesis of sporadic leukemia and myelodysplastic syndromes, through acquired chromosome rearrangements and point mutations. We report an Italian family with three members affected with FPD/AML, two sibs and their father, who developed myelodysplastic syndromes (which in one subsequently evolved into AML). Direct sequencing and polymorphisms haplotype analysis of the region of chromosome 21 where RUNX1 is mapped demonstrated that FPD/AML in this family was not caused by any mutation of the RUNX1 gene, thus providing evidence for the genetic heterogeneity of this disorder. Cytogenetic studies showed monosomy 7 in the marrow of all the three affected subjects, as well as an independent clone with trisomy 8 in the father. The importance of mutator effects in the pathogenesis of familial myeloid malignancies characterized by relevant chromosome changes, in the presence or absence of an underlying Mendelian disorder, has already been suggested. Our results and a review of the cytogenetic literature led us to postulate that mutations also causing FPD/AML may have a mutator effect that could give origin to myelodysplastic syndromes and acute myeloid leukemias through acquired chromosome changes.