Guiding the global evolution of cytogenetic testing for hematologic malignancies.

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

Second Neoplasms in Children Following a Treatment for Acute Leukemia and/or Lymphoma: 29 Years of Experience in a Single Institution in Argentina.

INTRODUCTION: Childhood acute leukemias (AL) and lymphomas achieve good survival rates. However, second neoplasms (SN) are a devastating event. METHODS: From August 1987 to December 2016, 34 of 3321 (1%) patients with diagnosis of AL or lymphoma developed SN. SN were AL (n=16), CNS tumors (n=5), endocrinal tumors (n=3), lymphomas (n=2), schwannoma (n=2) assorted sarcomas (n=4), retinal melanoma (n=1), and Vanek tumor (n=1). Median latency was 51 (range, 10 to 110) months for hematological malignancies and 119 (range, 25 to 236) months for solid tumors (P=0.001). RESULTS: A total of 33 patients with SN were treated taking into account cumulative doses of anthracyclines and radiotherapy. Twenty-three (67.6%) patients achieved complete remission (CR), 5 died early during therapy and 5 were refractory or partial responders. Six patients presented relapses of the SN and 1 died in CR. Seventeen patients remain alive in CR, with a median follow-up of 110 (range, 4 to 276) months. CONCLUSIONS: (1) The latency period was significantly longer for patients developing solid tumors than for those developing AL. (2) AL was the most frequent SN. (3) Our results strongly encourage giving standard therapy to SN, considering cumulative doses of previous treatment, since similar probabilities of surviving as "de novo" counterparts can be achieved.

Identification of ZCCHC8 as fusion partner of ROS1 in a case of congenital glioblastoma multiforme with a t(6;12)(q21;q24.3).

Congenital gliobastoma multiforme (GBM) is rare and little is known about the molecular defects underlying the initiation and progression of this tumor type. We present a case of congenital GBM analyzed by conventional cytogenetics, fluorescence in situ hybridization, array comparative genomic hybridization and next generation sequencing. On cytogenetic analysis we detected a reciprocal translocation t(6;12)(q21;q24.3). By sequencing, the translocation was shown to form a fusion between the 5' region of ZCCHC8 and the 3' region of ROS1. RT-PCR analyses confirmed the existence of an in-frame fusion transcript with ZCCHC8 exons 1-3 joined to ROS1 exons 36-43. In addition to the ZCCHC8-ROS1 fusion, we detected a deletion in the short arm of chromosome 9, including homozygous loss of the CDKN2A/2B locus in 9p21.3 and heterozygous deletion of the HAUS6 gene in 9p22.1. The latter encodes a protein involved in faithful chromosome segregation by regulating microtubule nucleation and its deletion might be associated with the marked subclonal changes of ploidy observed in the tumor. This report adds the ZCCHC8-ROS1 fusion as oncogenic driver in GBM and supports the role of ROS1 activation in the pathogenesis of a subset of GBM. © 2016 Wiley Periodicals, Inc.

B-cell acute lymphoblastic leukemia with mature phenotype and MLL rearrangement: report of five new cases and review of the literature.

The association between mature-B phenotype and MLL abnormalities in acute lymphoblastic leukemia (ALL) is a very unusual finding; only 14 pediatric cases have been reported so far. We describe the clinical and biological characteristics and outcome of five pediatric cases of newly diagnosed B lineage ALL with MLL abnormalities and mature immunophenotype based on light chain restriction and surface Ig expression. Blasts showed variable expression of CD10/CD34/TdT. MLL abnormalities with no MYC involvement were detected in all patients by G-banding, FISH, and/or RT-PCR. Three patients were treated according to Interfant protocol, one to ALLIC-09, and one received B-NHL-BFM-2004. All patients achieved complete remission and three of them relapsed. Despite the small cohort size, it could be postulated that B lineage ALL with MLL abnormalities and mature phenotype is a distinct entity that differs both from the typical Pro B ALL observed in infants and mature B-ALL with high MYC expression.

Experience with four consecutive BFM-based protocols for treatment of childhood with non-promyelocytic acute myeloblastic leukemia in Argentina.

Childhood acute myeloid leukemia (AML) achieves event-free-survival (EFS) rates of ∼50%. Double induction phase has been introduced for improving these results. Four consecutive protocols for AML treatment were evaluated to assess the impact of the addition of a second induction course in our setting. From January 1990 to January 2014, 307 evaluable AML patients were accrued. They were classified into low-risk (LR) and high-risk (HR) according to cytogenetic/molecular findings and response on day 15. The first two studies administered one induction cycle while the latter two protocols administered double induction. Relapse was the most frequent event and early-deaths were reduced by 50% in the last protocol. Statistically significant differences were observed when comparing EFS in LR and HR groups. Patients from both risk-groups who received double induction achieved significantly better outcome. EFS improved in protocols with double induction and early-deaths rate was decreased. Cytogenetic/molecular features and early-response were confirmed as prognostic factors.

Cytogenetic and Molecular Findings in Children with Acute Lymphoblastic Leukemia: Experience of a Single Institution in Argentina.

The purpose of the current study was to evaluate the cytogenetic findings in 1,057 children with acute lymphoblastic leukemia (ALL) referred to the cytogenetics laboratory at the Hospital de Pediatría Dr. Juan P. Garrahan, between 1991 and 2014. Chromosomal abnormalities were evaluated by G-banding and FISH. Since December 2002, RT-PCR determinations were systematically carried out for BCR-ABL1, KMT2A-AFF1, ETV6-RUNX1, and TCF3-PBX1 rearrangements in children, adding KMT2A-MLLT3 and KMT2A-MLLT1 in infants. The percentage of abnormalities detected by cytogenetics was 70.1%. Four novel abnormalities, t(2;8)(p11.2;p22), inv(4)(p16q25), t(1;7)(q25;q32), and t(5;6)(q21;q21), were found in this cohort. We compared cytogenetic and RT-PCR results for BCR-ABL1, KMT2A-AFF1 and TCF3-PBX1 rearrangements in 497 children evaluated by both methods. The results were highly concordant (p < 0.7), and interestingly, FISH was relevant to confirm G-banding findings that were discordant with RT-PCR studies. This study showed the importance of performing G-banding, FISH and RT-PCR simultaneously to improve the detection of chromosomal abnormalities considering their important value in the diagnosis and prognosis of childhood ALL patients. Finally, to the best of our knowledge, this is the first series of cytogenetic findings in children with ALL reported in Argentina.

Gonadoblastoma in patients with Ullrich-Turner syndrome.

Ullrich-Turner syndrome (UTS) is a common chromosomal abnormality caused by partial or complete X chromosome monosomy. One half of the patients have a 45,X karyotype, whereas the remaining patients display other X chromosome anomalies. In 6% to 11% of UTS, a normal or partly deleted Y chromosome has been found. A 10% to 30% risk of developing gonadoblastoma was found in the latter patients. The aim of this study was to evaluate the prevalence of Y chromosome-derived material, the occurrence of gonadoblastoma, and the incidence of possible neoplasms in patients with UTS. Of 217 patients studied with UTS and chromosome analysis of peripheral-blood lymphocytes, Y chromosome material was found in 20 patients. Fluorescence in situ hybridization (FISH) testing was performed to characterize the structurally abnormal Y chromosome in 13 cases. Molecular analysis of the SRY gene could only be performed in 20 patients with 45,X karyotype. Two patients had the SRY genomes. Of the 20 patients with Y chromosome-derived material, 17 underwent gonadectomy. The incidence of gonadoblastoma development in our series was 35.5%. Furthermore, 1 patient also showed a pure dysgerminoma, and another showed a mixed dysgerminoma and embryonal carcinoma. We emphasize the importance of complete processing of the gonadectomy specimen, including step sections, molecular studies, and FISH, in addition to the classic cytogenetic searching for Y chromosome sequences, in patients who present with a nonmosaic 45,X karyotype. Finally, we propose to routinely collect a sample for storage in the tumor bank for future studies.

Caracterización de mutaciones en el gen GATA-1 en pacientes con Síndrome de Down y diagnóstico de mielopoyesis anormal transitoria o leucemia megacarioblástica aguda / Mutation characterization in the GATA-1 gene in patients with Down's Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia

Los pacientes con síndrome de Down tienen un riesgo más elevado de presentar leucemia megacarioblástica aguda (LMCA). Un 10% de los recién nacidos con ese síndrome presentan un cuadro de mielopoyesis anormal transitoria (MAT), indistinguible de la LMCA, que en general remite espontáneamente. En ambos grupos de pacientes se describió una alta incidencia de mutaciones en el gen GATA-1. Se analizaron 14 muestras de ADN de médula ósea (10 MAT/4 LMCA) correspondientes a 13 pacientes con Síndrome de Down mediante PCR y secuenciación, para describir la frecuencia y las características de las mutaciones en el gen GATA-1 en la población estudiada y sus consecuencias a nivel proteico. Se detectaron mutaciones en 10 de 10 MAT y en 3 de 4 LMCA, que a nivel proteico originarían un codón de terminación prematuro (n= 5), alteraciones en el sitio de corte y empalme (splicing) (n= 6) o cambio de secuencia (n= 3). Se confrmó la alta frecuencia de mutaciones en el gen GATA-1 en recién nacidos con Síndrome de Down y MAT o LMCA.

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

Mutation characterization in the GATA-1 gene in patients with Down's Syndrome diagnosed with transient abnormal myelopoiesis or acute megakaryoblastic leukemia.

Patients with Down's Syndrome have a higher risk of developing acute megakaryoblastic leukemia (AML). Ten per cent of newborn infants with this syndrome have transient abnormal myelopoiesis (TAM), indistinguishable from AML, which generally remits spontaneously. A high incidence of GATA-1 gene mutations was described in both groups of patients. Fourteen bone marrow DNA samples (10 ATM/4 AML) were analyzed by PCR and sequencing; these samples were obtained from 13 patients with Down's Syndrome to describe the rate and mutation characteristics of the GATA-1 gene in the studied population and its consequences at a protein level. Mutations were detected in 10 out of 10 TAM and in 3 out of 4 AML, which at a protein level would result in an early termination codon (n= 5), alterations in the splicing site (n= 6) or sequence change (n= 3). The high rate of GATA-1 gene mutations was confirmed in newborn infants with Down's Syndrome and MAT or AML.

Lineage switch in childhood acute leukemia: an unusual event with poor outcome.

Although rarely, switches between lymphoid and myeloid lineages may occur during treatment of acute leukemias (AL). Correct diagnosis relies upon confirmation by immunophenotyping of the lineage conversion and certification that the same cytogenetic/molecular alterations remain despite the phenotypic changes. From a total of 1,482 AL pediatric patients, we report nine cases of lineage conversion (0.6%), seven from lymphoid (four Pro-B, two Pre-B, one Common) to myelo-monocytic, and two from myeloid (bilineal, with myeloid predominance) to Pro-B. Eight patients were infants. Switches were suggested by morphology and confirmed with a median of 15 days (range: 8 days-6 months) from initiation of therapy. Of note, in five cases switches occurred before day 15. Stability of the clonal abnormalities was assessed by cytogenetic, RT-PCR/Ig-TCR rearrangement studies in all patients. Abnormalities in 11q23/MLL gene were detected in seven cases. Treatment schedules were ALL (two pts), Interfant-99 (five pts) and AML (two pts) protocols. Despite changing chemotherapy according to the new lineage, all patients died. Our findings support the association of lineage switches with MLL gene alterations and the involvement of a common lymphoid B-myeloid precursor. New therapies should be designed to address these rare cases. Possible mechanisms implicated are discussed.

RT-PCR diagnosis of recurrent rearrangements in pediatric acute lymphoblastic leukemia in Argentina.

The present study was performed to establish the prevalence of the recurrent fusion transcripts in Argentinean pediatric patients with acute lymphoblastic leukemia (ALL). A total of 380 newly diagnosed children (including 50 infants and 44 T-ALL) were screened by RT-PCR; the incidence of recurrent rearrangements was: ETV6-RUNX1, 12.9%; TCF3-PBX1, 5.0%; BCR-ABL1, 1.6%; and MLL rearrangements, 10.5%. STIL-TAL1 was detected in 22.7% of T-ALL cases. In B-ALL cases, the pEFS was significantly influenced by the presence of genetic alterations. RT-PCR studies improved patients' stratification and also the overall outcome of children treated in a pediatric hospital from a developing country.

[Cytogenetics role in pediatrics]. / Rol de la citogenética en pediatría.

Cytogenetics has led to the discovery of multiple chromosomal anomalies associated with mental retardation and hemato-oncologic diseases. From the beginning till now, and with the contribution of the molecular biology, there has been an improvement of the classic cytogenetics, that allow cytogenetists to perform a more precise diagnosis. The present study briefly describe classic and molecular cytogenetic techniques, their advantages and disadvantages, and the application to clinic pediatrics diagnosis.

Prognostic impact of t(1;19)/ TCF3-PBX1 in childhood acute lymphoblastic leukemia in the context of Berlin-Frankfurt-Münster-based protocols.

Historically, t(1;19)(q23;p13.3) has been related to pre-B acute lymphoblastic leukemia (ALL) and associated with a poor prognosis. Current treatments have overcome this dismal outcome, but advantages in survival for the unbalanced group have been reported. We compared the outcome of balanced and unbalanced der(19)t(1;19) cases and also patients with t(1;19)/TCF3-PBX1 versus patients without this translocation, to assess its prognostic value. From January 1990 to December 2010, t(1;19)(q23;p13)/TCF3-PBX1 was detected in 48 cases. Patients were treated with Berlin-Frankfurt-Münster (BFM)-based protocols and classified into balanced (n = 17) and unbalanced (n = 23) groups. The probability of event-free survival (pEFS) (standard error) of patients with t(1;19)/TCF3-PBX1 was 85% (6%), for the unbalanced group 78% (10%), and 88% (8%) for the balanced. The pEFS of patients with t(1;19)/TCF3-PBX1 was significantly superior to that of patients without t(1;19)/TCF3-PBX1 (p-value <0.0001). Patients with t(1;19)/TCF3-PBX1 presented a good outcome with no differences between balanced and unbalanced subgroups. Thus, risk-adjustment therapy would not be necessary for cases with t(1;19)/TCF3-PBX1.

No advantage of a rotational continuation phase in acute lymphoblastic leukemia in childhood treated with a BFM back-bone therapy.

BACKGROUND: Our aim was to compare two different schedules of maintenance in pediatric acute lymphoblastic leukemia (ALL) treated with a BFM-based therapy, in a randomized study: an Arm with 6-MP + MTX (with or without vincristine and dexamethasone pulses) versus a more intensive continuation phase. PROCEDURE: From January 1996 to November 2002, 429 eligible children with ALL were enrolled in a protocol with BFM-based back-bone, followed by a randomized continuation phase in standard (SRG) and intermediate (IRG) risk groups. Patients were randomized between Arms A and B for SRG and B or C for IRG. Arms A and C consisted of 6-MP and MTX and in Arm C, six pulses of VCR and dexamethasone were added. Arm B combined four pairs of drugs rotated weekly. All risk-groups received maintenance until completing 2 years of therapy from diagnosis. RESULTS: With a median follow-up of 138 (range: 96-178) months, the overall pEFS (SE) was 72 (6)% for all patients and the different risk groups showed: SRG: 85 (3)%, IRG: 71 (1)%, and HRG: 42 (7)% (P-value ≤ 0.0001). The pDFS (SE) according to the assigned arm of maintenance was, for Arm A: 89 (3)% and for Arm B: 85 (4)% in SRG, and, for Arm B: 77 (4)% and for Arm C: 75 (4)% in IRG, at 10 years follow-up. There were no statistically significant differences in outcome between arms of maintenance for both risk groups. CONCLUSIONS: In protocols with initial BFM-based strategy, a more intensive continuation phase did not benefit any risk group of patients.

A complex karyotype in an atypical teratoid/rhabdoid tumor: case report and review of the literature.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly aggressive and uncommon neoplasm of the central nervous system that usually occurs in children less than 2 years of age. It is characterized by deletions and/or mutations of the INI1 tumor suppressor gene located in chromosome band 22q11.2. We performed cytogenetic and molecular studies of an AT/RT on a 15-month-old boy. The tumor showed a complex karyotype with one cell line showing monosomy 22 and another near-tetraploid one with additional chromosomal abnormalities, involving chromosomes 2, 3, 5, 6, and Y, which had not been previously described. Sequence analysis of the tumor did not identify mutations of the INI1 gene. The karyotypic evolution observed in this tumor suggests that INI1 has an epigenetic role in the maintenance of genome integrity by affecting genes, which produces mitotic defects and polyploidy. Finally, this case is the first to support the theory that loss of INI1 could induce the chromosomal instability that might be responsible for the genesis of this tumor.

Cytogenetic findings in a rare pediatric mixed glioneuronal tumor and review of the literature.

OBJECTIVE: The aim of the present study was to report the chromosomal abnormalities findings in rare pediatric mixed glioneuronal tumor (GNT), which could not be classified according to the WHO classification. METHODS: Cytogenetic studies were performed using G-banding and fluorescence in situ hybridization (FISH) techniques. RESULTS: Cytogenetic analyses showed a deletion of 1p as primary genetic event and gain of chromosome 7 as secondary change. Furthermore, we present a review of available cytogenetic data of 72 pediatric patients with GNT. Taken into account these data and the present case, we found that the most frequent chromosomal anomalies involved gains of chromosomes 7 (15.1%), 5 (8.2%), 1q32-qter (6.8%), 8p21-qter (6.8%), 12 (5.5%), 18 (5.5%), 20q11-qter (5.5%), and X (5.5%). Frequent losses were detected on chromosome regions 1p (8.2%) and 22q (5.5%). CONCLUSION: The findings of our case combined with those of previous reports suggest that chromosomes 1 and 7 may contain candidate genes involved in the tumorigenesis of GNT.

Complex chromosome 8;21 translocation with associated hyperdiploidy in acute myeloid leukemia (FAB-M2).

We present a case of acute myeloblastic leukemia (AML-M2) with a complex t(8;21) translocation and additional acquired chromosomes yielding a hyperdiploid karyotype. AML1/ETO transcript was observed by reverse transcription-polymerase chain reaction. Fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and comparative genomic hybridization (CGH) were performed to further identify the chromosomes observed by G banding. The patient was treated according to our current protocol for AML. He remains in complete remission +11 months from diagnosis. Further follow-up of this patient and the analysis of a larger number of children are needed to define whether the gains of the specific extra chromosomes modify the good prognosis that t(8;21) confers to this subgroup of AML.