Application of a digital PCR method for WT1 to myeloid neoplasms in CR and deep ELN WT1 molecular response (< 10 copies).

Bone marrow WT1 mRNA levels assessed by the ELN method are useful to establish prognostic correlations in myeloid malignancies treated with chemotherapy or hematopoietic stem cell transplantation (HCT). Those patients with WT1 levels below ten copies have a good outcome. However, some of these patients relapse. To further characterize this group of cases, we applied a new and sensitive digital (ddPCR) WT1 method. A consecutive series of 49 patients with treated myeloid malignancies and with an ELN WT1 quantitation of < 10 copies were included in the study. All cases (47 AML and 2 MDS) have received intensive chemotherapy or HCT. One to four micrograms of total RNA were retrotranscribed to obtain ≥ 10,000 ABL1 copies using the ELN protocol. Only those cases with a good quality cDNA were used in the ddPCR WT1 test. The ddPCR Gene Expression WT1 Assay of Bio-Rad© was used to perform the PCR amplification, and the microdroplets were quantified in the Bio-Rad's QX200 droplet reader. Eighteen patients showed a negative WT1 ddPCR assay (0 copies/µl), whereas 31 cases were positive (results ranged from 1 to 15.2 copies/µl). Survival analysis showed statistically significant differences in terms of OS between both groups, 83 ± 8% vs. 46 ± 9% (p = 0.024). A statistically significant correlation was also found between ddPCRWT1 results and CD123+ cell number detected by flow cytometry (p = 0.024). Larger series of patients tested with the current ddPCRWT1 method will solve whether it could be used to stratify patients with myeloid malignancies achieving deep WT1 molecular response (< 10 copies).

Bone marrow WT1 levels at diagnosis, post-induction and post-intensification in adult de novo AML.

We retrospectively assessed whether normalized bone marrow WT1 levels could be used for risk stratification in a consecutive series of 584 acute myeloid leukemia (AML) patients. A cutoff value of 5065 copies at diagnosis identified two prognostic groups (overall survival (OS): 44 ± 3 vs 36 ± 3%, P=0.023; leukemia-free survival (LFS): 47 ± 3 vs 36 ± 4%, P=0.038; and cumulative incidence of relapse (CIR): 37 ± 3 vs 47 ± 4%, P=:0.043). Three groups were identified on the basis of WT1 levels post-induction: Group 0 (WT1 between 0 and 17.5 copies, 134 patients, OS: 59 ± 4%, LFS:59 ± 4% and CIR: 26 ± 4%); Group 1 (WT1 between 17.6 and 170.5 copies, 160 patients, OS: 48 ± 5%, LFS:41 ± 4% and CIR: 45 ± 4%); and Group 2 (WT1 >170.5 copies, 71 patients, OS: 23 ± 6%, LFS: 19 ± 7% and CIR: 68 ± 8%) (P<0.001). Post-intensification samples distinguished three groups: patients with WT1 >100 copies (47 patients, 16%); an intermediate group of patients with WT1 between 10 and 100 copies (148 patients, 52%); and a third group with WT1 <10 copies (92 patients, 32%). Outcomes differed significantly in terms of OS (30 ± 7%, 59 ± 4%, 72 ± 5%), LFS (24 ± 7%, 46 ± 4%, 65 ± 5%) and relapse probability (CIR 72 ± 7%, 45 ± 4%, 25 ± 5%), all P<0.001. WT1 levels in bone marrow assayed using the standardized ELN method provide relevant prognostic information in de novo AML.

Adverse impact of IDH1 and IDH2 mutations in primary AML: experience of the Spanish CETLAM group.

The study of genetic lesions in AML cells is helpful to define the prognosis of patients with this disease. This study analyzed the frequency and clinical impact of recently described gene alterations, isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations, in a series of homogeneously treated patients with primary (de novo) AML. Two-hundred and seventy-five patients enrolled in the CETLAM 2003 protocol were analyzed. IDH1 and IDH2 mutations were investigated by well-established melting curve-analysis and direct sequencing (R140 IDH2 mutations). To establish the percentage of the mutated allele a pyrosequencing method was used. Patients were also studied for NPM, FLT3, MLL, CEBPA, TET2 and WT1 mutations. IDH1 or IDH2 mutations were identified in 23.3% AML cases and in 22.5% of those with a normal karyotype. In this latter group, mutations were associated with short overall survival. This adverse effect was even more evident in patients with the NPM or CEBPA mutated/FLT3 wt genotype. In all the cases analyzed, the normal allele was detected, suggesting that both mutations act as dominant oncogenes. No adverse clinical impact was observed in cases with TET2 mutations. IDH1 and IDH2 mutations are common genetic alterations in normal karyotype AML. Favourable genotype NPM or CEBPA mutated/FLT3 wt can be further categorized according to the IDH1 and IDH2 mutational status.

Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size.

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Bilateral and symmetrical palmoplantar punctate keratoses in childhood: a possible clinical clue for an early diagnosis of PTEN hamartoma-tumour syndrome.

Palmoplantar punctate keratoses may be the main cutaneous sign of various diseases (porokeratosis punctata palmaris et plantaris, keratosis punctata of the palmar creases, familial punctate palmoplantar keratoderma) or represent a secondary feature [Cowden's syndrome (CS) and Darier's disease]. In CS, such keratoses usually appear during the second and third decades of life, together with other mucocutaneous features. We present the case of a 3-year-old girl with palmoplantar punctate keratoses in whom diagnosis of new-onset CS was suspected only after the development of other cutaneous lesions. Genetic analysis confirmed the diagnosis. This case highlights the necessity to consider CS in the differential diagnosis when palmoplantar punctate keratoses are found, even in paediatric patients. A prompt diagnosis is important in order to monitor the development of possible underlying associated neoplasms.

Molecular pathology of endometrial hyperplasia and carcinoma.

Four different genetic abnormalities may occur in endometrioid adenocarcinomas of the endometrium (mircosatellite instability and mutations in the PTEN, k-RAS and beta-catenin genes), whereas nonendometrioid carcinomas of the endometrium often have p53 mutations and loss of heterozygosity on several chromosomes. Occasionally, a nonendometrioid carcinoma may develop as a result of dedifferentiation of a preexisting endometrioid carcinoma; in such a case, the tumor exhibits overlapping clinical, morphologic, immunohistochemical, and molecular features of the 2 types. The insaturation of microsatellite instability in endometrial carcinogenesis seems to occur late in the transition from complex hyperplasia to carcinoma, and it is preceded by progressive inactivation of MLH-1 by promoter hypermethylation. Moreover, the endometrioid adenocarcinomas that exhibit microsatellite instability show a stepwise progressive accumulation of secondary mutations in oncogenes and tumor suppressor genes that contain short-tandem repeats in their coding sequences. Mutations in the PTEN and k-RAS genes are also frequent in endometrioid adenocarcinomas of the endometrium, particularly in the tumors that exhibit microsatellite instability, whereas beta-catenin mutations do not seem to be associated with such a phenomenon.

Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases.

Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6- centromeric exon 7- telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6- centromeric exon 7- telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.

Loss of heterozygosity on chromosome 17q in epithelial ovarian tumors: association with carcinomas with serous differentiation.

Loss of heterozygosity (LOH) on chromosome 17q is frequent in epithelial ovarian tumors, but its clinicopathologic significance remains to be elucidated. DNA of 50 patients with epithelial ovarian tumors was extracted from blood and from fresh-frozen and paraffin-embedded tissue (14 benign, 7 borderline, and 29 malignant). Six microsatellite loci were amplified by PCR (D17S250, TRHA1, D17S800, D17S855, D17S579, D17S513). LOH was scored by the absence or reduction of the signal to less than 50% of one of the alleles in tumor DNA compared with normal DNA. LOH was identified on chromosome 17q in at least one locus in 12 tumors (24%), all of them carcinomas (12 of 29 tumors, 41.3%). It occurred more frequently among high-grade serous carcinomas (8 of 14 tumors, 57%) and mixed endometrioid-serous carcinomas (2 of 5, 40%). LOH was detected in all informative markers of 10 tumors, suggesting the complete loss of an entire chromosome 17 homologue. Patients with LOH-positive carcinomas were older than those with LOH-negative malignant tumors (mean ages 67 and 49). The results support the hypothesis that LOH on chromosome 17q may be associated with the development of ovarian cancers in elderly patients, particularly with high-grade serous or mixed endometrioid-serous carcinomas.

Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.

BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.

PTEN mutations in endometrial carcinomas: a molecular and clinicopathologic analysis of 38 cases.

PTEN mutations have been reported to be frequent in endometrioid carinomas of the endometrium (EEC). Some correlation has been found between PTEN mutations and the presence of microsatellite instability (MI) in EEC, but no convincing cause-effect relationship for such association has been offered. DNA of 38 patients with endometrial carcinoma (EC) was extracted from blood and from fresh-frozen and paraffin-embedded tumor tissue. PTEN mutations were detected by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Results were correlated with MI status and clinicopathologic data. PTEN mutations were detected in 17 tumors (44.7%), and they were more frequent in endometrioid (EEC) (17 of 33, 51.5%) than in nonendometrioid carcinomas (NEEC) (0 of 5, 0%). PTEN mutational spectrum differed between MI+ and MItumors. PTEN mutations were detected in 9 of 15 MI+ tumors (60%), but in only 8 of 23 MI- neoplasms (34.8%). In EC with MI, PTEN mutations were detected in short coding mononucleotide repeats (A)s and (A)6 in 4 of 9 carcinomas (44.4%). These results confirm that PTEN is an important target gene in endometrial carcinogenesis. The occurrence of PTEN mutations in short coding mononucleotide repeats in MI-positive tumors suggests that these mutations may be secondary to deficiencies in mismatch repair and gives some explanation for the frequent presence of PTEN mutations in these tumors.

A new autosomal recessive retinitis pigmentosa locus maps on chromosome 2q31-q33.

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous disease. To date, mutations in four members of the phototransduction cascade have been implicated in ARRP. Additionally, linkage of the disease to three loci on 1p, 1q, and 6p has been described. However, the majority of cases are still uncharacterised. We have performed linkage analysis in a large nuclear ARRP family with five affected sibs. After exclusion of several regions of the genome known to contain loci for retinal dystrophies, a genomic search for linkage to ARRP was undertaken. Positive lod scores were obtained with markers on 2q31-q33 (Zmax at theta = 0.00 of 4.03, 4.12, and 4.12 at D2S364, D2S118, and D2S389, respectively) defining an interval of about 7 cM for this new ARRP locus, between D2S148 and D2S161. Forty-four out of 47 additional ARRP families, tested with markers on 2q32, failed to show linkage, providing evidence of further genetic heterogeneity.

Cramps and minimal EMG abnormalities as preclinical manifestations of spinal muscular atrophy patients with homozygous deletions of the SMN gene.

The characterization of deletions in the SMN gene provides a helpful tool to confirm the diagnosis of spinal muscular atrophy (SMA). However, there may be homozygous deletions of the SMN gene in some unaffected siblings of SMA type II and III patients. We present two SMA families with affected siblings demonstrating a homozygous deletion of the SMN gene with extremely different phenotypes. We propose a preclinical category of an SMA patient with homozygous deletion of the SMN gene: those with minimal expression of the disease including cramps and EMG abnormalities that may develop the complete SMA phenotype in the future.

A new locus for autosomal recessive retinitis pigmentosa (RP19) maps to 1p13-1p21.

Autosomal recessive retinitis pigmentosa (arRP) is characterized by considerable allelic and nonallelic heterogeneity. Mutations have been described in the rhodopsin gene (RHO), the genes encoding the alpha and beta subunits of rod phosphodiesterase (PDEA and PDEB), and the gene encoding the alpha subunit of the cGMP-gated channel (CNCG). In addition, linkage studies in single extended pedigrees have defined two new arRP loci, at 1q and 6p. To identify the disease gene in a Spanish consanguineous arRP family, a linkage analysis was undertaken. After testing 102 polymorphic markers, a significant positive lod score (Zmax = 3.64 at theta = 0) was obtained with marker D1S188 at 1p13-p21, the same region where the Stargardt and fundus flavimaculatus (FFM) loci were previously defined. Exhaustive ophthalmologic examination of the patients clearly distinguished the disease from the Stargardt and FFM phenotypes and revealed an atypical form of arRP with choroidal atrophy as a distinctive feature.

[The contribution of molecular genetics to the study of spinal muscular atrophy]. / Contribución de la genética molecular al estudio de la atrofia muscular espinal.

Spinal muscular atrophies constitute a group of hereditary diseases characterized by degeneration of the anterior horn of the spinal cord. Molecular studies began in 1990 with the location of the genome region responsible for the disease in chromosome 5q13. New directions for research were opened in 1995 with the identification of the affected region in the survival motor neuron (SMN) genes and the neuronal apoptosis inhibitory protein genes. A main feature of these genes is that they are duplicate, forming part of two elements (centromeric and telomeric) that include mini-satellites that are also repeated, making this zone particularly unstable. The molecular abnormalities found in patients are a consequence of that instability: gene deletions and conversions in the SMN gene have been described independently of whether symptoms were severe or not. Molecular data make it possible to confirm the clinical diagnosis of most patients and to provide certain prenatal diagnosis for couples that are at high risk of passing on the disorder. Determining both the function of these genes and their pathogenesic role will help to ground new therapeutic strategies that will prevent or detain motor neuron degeneration.

Deletion analysis of the simple tandem repeat loci physically linked to the spinal muscular atrophy locus.

Multicopy dinucleotide repeats have been characterized in the spinal muscular atrophy (SMA) region on chromosome 5q13, which reveal deletions in some SMA patients. 119 Italian and Spanish SMA families have been analysed using the C272 and C212 markers. Seventy percent of these families were informative. We found 9.4% de novo deletions in SMA I and 1.5% in SMA II families. A single inherited deletion segregating in a Spanish pedigree was detected in three affected brothers. A SMA II patient showed deletion only of C272. The data presented in this study are relevant to the molecular diagnosis of SMA families in Italy and Spain and provide additional insights toward the understanding of the molecular pathology of SMA.

A frame-shift deletion in the survival motor neuron gene in Spanish spinal muscular atrophy patients.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disease characterized by degeneration of the motor neurons of the spinal cord causing proximal paralysis with muscle atrophy. The region on chromosome 5q13 encompassing the disease gene is particularly unstable and prone to large-scale deletions whose characterization recently led to the identification of the survival motor neuron (SMN) gene. We now present a genetic analysis of 54 unrelated Spanish SMA families that has revealed a 4-basepair (bp) deletion (AGAG) in exon 3 of SMN in four unrelated patients. This deletion, which results in a frameshift and a premature stop codon, occurs on the same haplotype background, suggesting that a single mutational event is involved in the four families. The other patients showed either deletions of the SMN gene (49/54) or a gene conversion event changing SMN exon 7 into its highly homologous copy (cBCD541, 1/54). This observation gives strong support to the view that mutations of the SMN gene are responsible for the SMA phenotype as it is the first frameshift mutation reported in SMA.