Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biological events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs (52%) at the initial time point analyzed, including 12 (25%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution has prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

Refined cytogenetic-risk categorization for overall and leukemia-free survival in primary myelofibrosis: a single center study of 433 patients.

We have previously identified sole +9, 13q- or 20q-, as 'favorable' and sole +8 or complex karyotype as 'unfavorable' cytogenetic abnormalities in primary myelofibrosis (PMF). In this study of 433 PMF patients, we describe additional sole abnormalities with favorable (chromosome 1 translocations/duplications) or unfavorable (-7/7q-) prognosis and also show that other sole or two abnormalities that do not include i(17q), -5/5q-, 12p-, inv(3) or 11q23 rearrangement are prognostically aligned with normal karyotype, which is prognostically favorable. These findings were incorporated into a refined two-tired cytogenetic-risk stratification: unfavorable and favorable karyotype. The respective 5-year survival rates were 8 and 51% (hazard ratio (HR): 3.1, 95% confidence interval (CI): 2.2-4.3; P<0.0001). Multivariable analysis confirmed the International Prognostic Scoring System (IPSS)-independent prognostic value of cytogenetic-risk categorization and also identified thrombocytopenia (platelets <100 × 10(9)/l) as another independent predictor of inferior survival (P<0.0001). A similar multivariable analysis showed that karyotype (P=0.001) and platelet count (P=0.04), but not IPSS (P=0.27), predicted leukemia-free survival; the 5-year leukemic transformation rates for unfavorable versus favorable karyotype were 46 and 7% (HR: 5.5, 95% CI: 2.5-12.0; P<0.0001). This study provides the rationale and necessary details for incorporating cytogenetic findings and platelet count in future prognostic models for PMF.

Monosomal karyotype in myelodysplastic syndromes, with or without monosomy 7 or 5, is prognostically worse than an otherwise complex karyotype.

Monosomal karyotype (MK) refers to the presence of two or more distinct autosomal monosomies or a single monosomy associated with a structural abnormality. In acute myeloid leukemia, MK has been shown to be prognostically worse than an otherwise complex karyotype. The current study examines whether the same holds true for myelodysplastic syndromes (MDS). A total of 127 MDS patients (median age 70 years) with a complex karyotype were considered; 106 (83%) met the above-stipulated criteria for MK and 21 (17%) had a complex karyotype without monosomies. Survival was significantly inferior in patients with MK compared with those with a complex karyotype without monosomies (P=0.01; HR 1.9, 95% confidence interval (95% CI), 1.1-3.3). Multivariable analysis identified MK (P=0.002), advanced age (P=0.0004) and bone marrow blast percentage (0.04) as independent risk factors for survival. There was no difference in survival among MK patients further substratified by the presence or absence of monosomy 7 and/or monosomy 5. Although not statistically significant, leukemia-free survival was also worse with MK compared with complex karyotype without monosomies (P=0.09; HR 2.7, 95% CI 0.8-9.0). MK in MDS identifies a prognostically worse subgroup of patients with a complex karyotype, regardless of whether monosomy 7 or 5 is part of the MK component.

Redefining the risks of prenatally ascertained supernumerary marker chromosomes: a collaborative study.

BACKGROUND: A marker chromosome is defined as a structurally abnormal chromosome that cannot be identified by routine cytogenetics. The risk for phenotypic abnormalities associated with a marker chromosome depends on several factors, including inheritance, mode of ascertainment, chromosomal origin, and the morphology, content, and structure of the marker. METHODS: to understand the karyotype-phenotype relationship of prenatally ascertained supernumerary de novo marker chromosomes, we combined data from prenatal cases obtained from 12 laboratories with those from studies in the literature. We were able to obtain cytogenetic and phenotypic data from 108 prenatally ascertained supernumerary de novo marker chromosomes to refine the phenotypic risk associated with these markers. Because of the growing number of cases and because more techniques are available to delineate marker morphology, we have been able to group risk estimates into subcategories, such as by marker type and whether there are ultrasound abnormalities. RESULTS: If a de novo supernumerary marker chromosome is found prenatally, our data suggest there is a 26% risk for phenotypic abnormality when there is no other information defining the marker (such as chromosomal origin or information about the existing phenotype). However, if high resolution ultrasound studies are normal, this risk reduces to 18%. CONCLUSIONS: Our findings strongly support the value of additional genetic studies for more precisely defining the risk in individual cases involving marker chromosomes.

Deletion of 2q37 and duplication of 10q24: two cases in the same family and review of the literature.

We describe two patients (first cousins, once removed) with an unusual head shape, high arched palate, flat nasal bridge, abnormal ears, hand and feet abnormalities and other anomalies. The patients were ascertained independently and it was initially unknown that they were related to each other. Cytogenetic and fluorescent in situ hybridization (FISH) analysis identified a der(2)t(2;10)(q37.3;q24.1) unbalanced translocation resulting in loss of 2q37.3-qter and duplication 10q24.1-qter. The clinical features of these two patients are compared with previously described cases of 2q deletion and 10q duplication. These patients also emphasize the difficulty in some families of understanding and sharing genetic information and in the difficulties in obtaining an accurate pedigree in a genetics clinic.

Cytogenetic analysis and construction of a BAC contig across a common neocentromeric region from 9p.

Over 40 cases of neocentric marker chromosomes, without detectable alpha-satellite DNA, have been reported. Although these have originated from many different chromosomes, a few of these chromosomes have been involved in multiple cases of marker formation. In this study, two different markers originating from the short arm of chromosome 9 were analyzed, identifying a common neocentromeric region. A bacterial artificial chromosome (BAC) contig extending over more than 900 kb has been assembled across this neocentromeric region. Fluorescent in situ hybridization and immunofluorescence assays (CENP-C and CENP-E) have localized the neocentromere to a 500 kb region. Preliminary analysis of DNA sequences in this neocentromere revealed a highly AT-rich region, which also has an increase in the level of retroviral elements compared with the average levels in the genome.

Prenatal diagnosis of 22q11.2 deletion when ultrasound examination reveals a heart defect.

PURPOSE: The incidence of 22q11.2 deletion syndrome is approximately 1 in 5,000 births, and accounts for 5-30% of all heart defects, making it one of the more common genetic conditions in the population. METHODS: We employed fluorescence in situ hybridization (FISH) to study the incidence of 22q11.2 deletions in fetuses with cardiac anomalies detected on ultrasound examination. RESULTS: Of 64 cases, 18 had visible chromosome anomalies. FISH testing for 22q11.2 deletion was performed on the remaining 46 cases, and five exhibited a 22q11.2 deletion. Three of the five had de novo deletions, one was maternally inherited, and one family declined testing. CONCLUSION: FISH analysis for 22q11.2 deletion should be performed on all fetuses with cardiac defects (excluding hypoplastic left heart and echogenic focus) and a normal G-banded karyotype.

Significance of p53 mutations in patients with chronic lymphocytic leukemia: a sequential study of 30 patients.

BACKGROUND: In patients with B-cell chronic lymphocytic leukemia (CLL), considerable disease heterogeneity within clinical stages necessitates the search for relevant prognostic indicators, particularly those that may help to determine the need for early therapeutic intervention. In the current study, the authors investigated the role of p53 mutations and chromosomal abnormalities in 30 patients with CLL. METHODS: Thirty patients were screened for p53 mutations. Half of the group had aggressive disease characterized by leucocytosis, lymph node enlargement, organomegaly, and shortened tumor doubling time. Because 95% of p53 mutations reside in "hot-spot" regions of exons 5-9 of the p53 gene, the authors sequenced these exons completely for mutation detection. RESULTS: Sequence analysis identified p53 mutations in 14 of 30 patients that were distributed equally among patients with aggressive disease and nonaggressive disease. There were six mutations in exon 7, five mutations in exon 5, and one mutation each in exons 6 and 8. Five of 15 patients with clinically aggressive disease had mutations in exon 7. Only one patient with nonaggressive disease had an exon 7 mutation. Abnormal cytogenetics were present in 22 of 30 patients (73%). Most patients with the p53 mutation (13 of 14 patients; 93%) displayed abnormal cytogenetics. Twelve of 15 patients with aggressive disease and 9 of 15 patients with average disease exhibited abnormal karyotypes. CONCLUSIONS: The presence of p53 mutations did not predict clinical behavior or disease outcome, although the frequency of mutations appears to be higher than reported previously. In this study, mutations of exon 7 (5 of 6 patients) occurred in patients with clinically aggressive disease. The significance of this observation warrants further examination.

Developmental delay and multiple congenital anomalies in a child with a unique combination of partial monosomy 18 and partial trisomy 16.

A male child with multiple congenital anomalies and developmental delay is described. Cytogenetic evaluation showed that the patient was partially monosomic for the short arm of chromosome 18 and partially trisomic for the short arm of chromosome 16: a combination of chromosomal syndromes not previously described.

Analphoid 3qter markers.

Two cases of marker chromosomes derived from a non-centromeric location were studied to determine the characteristics of these markers with respect to the presence of functional centromeres and whether an associated phenotype could be described. The markers were characterized by fluorescence in situ hybridization and centromeric protein studies. Assessments were done to identify clinical features. Case 1 is a girl referred at age 1.5 years with swirly areas of hyperpigmentation, bilateral preauricular pits, hypotonia, developmental delay, and seizures. Case 2 is a male first evaluated as a newborn and then later during the first year of life. He had streaky hypopigmentation, right preauricular pit, accessory nipples, postaxial polydactyly, asymmetric cerebral ventricles, duplicated right kidney, a right pulmonary artery stenosis, and seizures. Mosaicism for an extra marker from the 3qter region was present in both cases. Both markers had a constriction near one end and were C-band negative. Centromeric protein studies indicated absence of CENP-B, presence of CENP-C (data for case 1 only), and presence of CENP-E. Marker chromosomes were thus identified with a chromosomal origin far from their usual centromeric region and yet appeared to have functional centromeres. These two cases did not permit a specific clinical phenotype to be ascribed to the presence of tetrasomy for 3q26.2 approximately 3q27.2-->3qter.

Pharmacogenetic screening for susceptibility to fetal malformations in women.

OBJECTIVE: To present a review of the literature and research on the pharmacogenetics of congenital defects, with a focus on the need for predictive maternal genotype assays. DATA SOURCE: MEDLINE searches (January 1985-January 1999), past reference reviews, and unpublished research. STUDY SELECTION: Review of relevant human, animal, and basic science studies. DATA EXTRACTION: Data on research on polymorphisms, genotyping, cytochrome P450 enzyme systems, epoxide hydrolase, folate metabolism, metabolism of anticonvulsant medications, molecular genetics of neural tube defects, variations in drug metabolism, and environmental exposures were evaluated. DATA SYNTHESIS: Data synthesis includes not only a review of the literature but suggests ways such data might be used to facilitate the development of maternal genotype assays, with the goal of preventing birth defects. CONCLUSIONS: Individuals vary in how they metabolize drugs and handle toxic environmental exposures. In an ideal pregnancy, there is no or limited exposure to medications and environmental agents. However, in women with chronic medical conditions such as heart disease and seizures, this is often not possible. Unfortunately, no techniques have been available to identify those at risk in this population. Gene polymorphisms for a specific enzyme may result in an absence or reduction in the level of enzyme activity or in no change at all, with little effect on the structure/function of the gene product(s); they are not associated with clinical phenotypes in either the mother or the fetus. Other polymorphisms may be only markers. Thus, developing genotyping assays for women that are predictive of phenotype expression in the fetus is the key to screening for polymorphisms. As more mutations are identified and clinical, pharmacologic, biologic, and pharmacokinetic relationships are established, using these polymorphisms to develop a genotyping assay for women may become a clinical reality, possibly leading to preventive prepregnancy or prenatal treatment that may play an increasingly effective role in maternal care.

Trisomy 15, sex chromosome loss, and hematological malignancy.

We report 6 patients with myelodysplasia who, on routine cytogenetic studies, demonstrated trisomy 15. Four of these also had sex chromosome loss. A review of the literature revealed 6 other cases of trisomy 15 with sex chromosome loss and 22 cases of trisomy 15 as the sole chromosomal abnormality. All cases had hematologic malignancy or myelodysplasia. Trisomy 15 is uncommon but tends to be associated with myelodysplasia in older subjects, and with sex chromosome loss in about one third of cases.

Common trisomy mosaicism diagnosed in amniocytes involving chromosomes 13, 18, 20 and 21: karyotype-phenotype correlations.

Karyotype-phenotype correlations of common trisomy mosaicism prenatally diagnosed via amniocentesis was reviewed in 305 new cases from a collaboration of North American cytogenetic laboratories. Abnormal outcome was noted in 10/25 (40%) cases of 47,+13/46, 17/31 (54%) cases of 47,+18/46, 10/152 (6.5%) cases of 47,+20/46, and in 49/97 (50%) cases of 47,+21/46 mosaicism. Risk of abnormal outcome in pregnancies with less than 50% trisomic cells and greater than 50% trisomic cells were: 26% (4/15) versus 60% (6/10) for 47,+13/46, 52% (11/21) versus 75% (6/8) for 47,+18/46, 4.5% (6/132) versus 20% (4/20) 47,+20/46, and 45% (27/60) versus 59% (22/37) for 47,+21/46. Phenotypically normal liveborns were observed with mean trisomic cell lines of 9.3% for 47,+13/46, 8.6% for 47,+18/46, 27% for 47, +20/46, and 17% for 47,+21/46. Cytogenetic confirmation rates were 46% (6/13 cases) for 47,+13/46 mosaicism, 66% (8/12 cases) for 47, +18/46, 10% (10/97 cases) for 47,+20/46, and 44% (24/54 cases) for 47,+21/46. There were higher confirmation rates in pregnancies with abnormal versus normal outcome: 50% versus 44% for 47,+13/46 mosaicism, 100% versus 33% for 47,+18/46, 66% versus 7% for 47, +20/46, and 55% versus 40% for 47,+21/46. Repeat amniocentesis is not helpful in predicting clinical outcome. It may be considered when there is insufficient number of cells or cultures to establish a diagnosis. Fetal blood sampling may have a role in mosaic trisomy 13, 18, and 21 as the risk for abnormal outcome increases with positive confirmation: 1/5 (20%) normal cases versus 5/8 (62%) abnormal cases. High resolution ultrasound examination(s) is recommended for clinical correlation and to facilitate genetic counselling.

Clinical significance of Y chromosome loss in hematologic disease.

We have studied 215 male patients (aged 45-97 years) whose sole cytogenetic abnormality was clonal loss of the Y chromosome in metaphase cells from unstimulated cultures. The patients comprised a control group with no evidence of hematologic disease and four disease case groups: 1) myelodysplastic syndrome (MDS), refractory anemia, refractory anemia with excess blasts (RAEB), RAEB in transformation, and chronic myelomonocytic leukemia; 2) acute myelogenous leukemia; 3) myeloproliferative disorder (MPD), chronic granulocytic leukemia, and polycythemia vera; and 4) B-cell lymphoma/leukemia. The frequency of cells with Y loss increased with age and was significantly greater in cases than in controls, but it was not correlated with survival or with prior therapy. The frequency of cases with a -Y clone was 6.3% of male karyotypes and represented 16.4% of all abnormal male cytogenetic reports. Much of the difference between cases and controls appears to be accounted for by a greater frequency of cases with > 75% Y loss. A value of 81% chromosome Y loss maximized the combined sensitivity (28%) and specificity (100%) for predicting disease status, but a 75% cutoff provided the best estimate of disease risk. Even in older males, if > 75% of metaphase cells are 45,X,-Y, they probably represent a disease-associated clonal population, and it is possible that the critical genetic change is not visible through the microscope. This observation is true for MDS, MPD, B-cell disease, and especially acute myelogenous leukemia. The prognostic association of Y chromosome loss for survival appears to be neutral or favorable. Genes Chromosomes Cancer 27:11-16, 2000.

Chromosomal aberrations identified in culture of squamous carcinomas are confirmed by fluorescence in situ hybridisation.

AIMS: Chromosomal aberrations in tumour cells are often not discernable by direct analysis. Although cell culture allows qualitative analysis of the karyotype, potential selection and evolution during growth in vitro may yield misleading data. To determine whether aberrations observed in vitro are representative of the original lesion, chromosomal aberrations found after prolonged growth in vitro of two squamous cell carcinomas of the head and neck (SSCHN) were evaluated with fluorescence in situ hybridisation (FISH) on the original tumour nuclei. METHODS: Specific karyotypic aberrations identified in cultures of two squamous cell carcinomas were targets for FISH analysis on tumour sections. Chromosome painting mixtures were selected based on in vitro karyotypic data. FISH was performed on cultured interphase and metaphase cells, and on histological sections from the original tumours. RESULTS: The 9cen and 17cen probes yielded FISH signals consistent with the aneusomies predicted for the respective chromosomes from the culture karyotypes. Whole chromosome 9 paint confirmed the prior existence in the tumours of i(9p) and i(9q), although only the latter hybridised with the 9cen probe. FISH data also supported in vivo representation of the diploid and tetraploid tumour subclones observed in cultures. In tumour HFH-SCC-8a, FISH results were generally concordant between cultured interphase and metaphase cells and the histological sections, and improved the interpretation of marker chromosomes identified in culture. CONCLUSION: The karyotypes obtained in these cases after prolonged passage in culture were consistent with the genetic alterations in the original tumours.

Premature sexual development in individuals with neurodevelopmental disabilities.

Studies on precocious puberty have primarily focused on children with typical patterns of growth and cognitive development. This study reviewed diagnostic data from the records of 15,719 patients with neurodevelopmental disabilities for diagnoses associated with premature sexual development/precocious puberty. Thirty-two individuals with premature sexual development were identified, with the earliest changes seen in one girl at 1 year 7 months of age. In this group, the mean age at onset was 7 years 2 months in boys and 5 years 11 months in girls. Central precocious puberty, which was the most common cause of onset of early pubertal changes, was present in 15 of the 32 children. The results of this study suggest that children with a neurodevelopmental disability are at increased risk of premature pubertal changes when compared to children without a neurodevelopmental disability. This study indicates the need for health-care providers to be vigilant in screening for early pubertal changes in children with neurodevelopmental disabilities.