Ponatinib induces a sustained deep molecular response in a chronic myeloid leukaemia patient with an early relapse with a T315I mutation following allogeneic hematopoietic stem cell transplantation: a case report.

BACKGROUND: Atypical BCR-ABL1 transcripts are detected in less than 5% of patients diagnosed with chronic myeloid leukaemia (CML), of which e19a2 is the most frequently observed, with breakpoints in the micro breakpoint cluster region (µ-BCR) and coding for the p230 BCR-ABL1 protein. p230 CML is associated with various clinical presentations and courses with variable responses to first-line imatinib. CASE PRESENTATION: Here we report a case of imatinib resistance due to an E255V mutation, followed by early post-transplant relapse with a T315I mutation that achieved a persistent negative deep molecular response (MR5.0) after treatment with single-agent ponatinib. Using CastPCR, we could trace back the presence of the T315I mutation to all the RNA samples up to the detection of T315 mutation by Sanger sequencing shortly after allogeneic hematopoietic stem cell transplantation (HSCT). CONCLUSION: This case illustrates the major interest of ponatinib as a valid treatment option for e19a2 CML patients who present a T315I mutation following relapse after HSCT.

Discontinuation of tyrosine kinase inhibitors in CML patients in real-world clinical practice at a single institution.

BACKGROUND: Most patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs) will relapse if treatment is withdrawn, but various trials have recently demonstrated that a significant proportion of patients who achieved a stable and deep molecular response (DMR) can stop therapy without relapsing. However, most information on treatment cessation was obtained from clinical trials with strict recruiting criteria. METHODS: We evaluated the outcome of 25 patients with CML that discontinued TKI therapy in our institute in real-world clinical practice. RESULTS: Of the 25 patients, 76% discontinued therapy in sustained deep molecular response (SDMR) and 24% were in unsustained DMR (UDMR). Discontinuation of therapy due to adverse effects was observed in 5 and 50% of the patients in the SDMR and UDMR groups, respectively. After TKI discontinuation, patients were followed for a median of 24 months. At the time of this analysis, 56% patients had a molecular relapse after a median of 4 months. SDMR and longer treatment duration were associated with lower probability of molecular relapse: 25% in SDMR patients with TKI treatment > 96 months and 85% in UDMR patients with TKI treatment ≤96 months. All relapsed patients promptly resumed TKI therapy and regained at least major molecular response (MMR). CONCLUSIONS: Our results suggest that TKI discontinuation is safe outside clinical trials and particularly effective in CML patients who are in SDMR with longer TKI treatment duration.

Classical fragile-X phenotype in a female infant disclosed by comprehensive genomic studies.

BACKGROUND: We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities. CASE PRESENTATION: We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation. CONCLUSIONS: Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.

NCOA2 is a candidate target gene of 8q gain associated with clinically aggressive prostate cancer.

Prostate carcinomas harboring 8q gains are associated with poor clinical outcome, but the target genes of this genomic alteration remain to be unveiled. In this study, we aimed to identify potential 8q target genes associated with clinically aggressive prostate cancer (PCa) using fluorescence in situ hybridization (FISH), genome-wide mRNA expression, and protein expression analyses. Using FISH, we first characterized the relative copy number of 8q (assessed with MYC flanking probes) of a series of 50 radical prostatectomy specimens, with available global gene expression data and typed for E26 transformation specific (ETS) rearrangements, and then compared the gene expression profile of PCa subsets with and without 8q24 gain using Significance Analysis of Microarrays. In the subset of tumors with ERG fusion genes (ERG+), five genes were identified as significantly overexpressed (false discovery rate [FDR], ≤ 5%) in tumors with relative 8q24 gain, namely VN1R1, ZNF417, CDON, IKZF2, and NCOA2. Of these, only NCOA2 is located in 8q (8q13.3), showing a statistically higher mRNA expression in the subgroup with relative 8q gain, both in the ERG+ subgroup and in the whole series (P = 0.000152 and P = 0.008, respectively). Combining all the cases with NCOA2 overexpression, either at the mRNA or at the protein level, we identified a group of tumors with NCOA2 copy-number increase, independently of ETS status and relative 8q24 gain. Furthermore, for the first time, we detected a structural rearrangement involving NCOA2 in PCa. These findings warrant further studies with larger series to evaluate if NCOA2 relative copy-number gain presents prognostic value independently of the well-established poor prognosis associated with MYC relative copy-number gain.

POU1F1 is a novel fusion partner of NUP98 in acute myeloid leukemia with t(3;11)(p11;p15).

BACKGROUND: NUP98 gene rearrangements have been reported in acute myeloid leukemia, giving rise to fusion proteins that seem to function as aberrant transcription factors, and are thought to be associated with poor prognosis. FINDINGS: A patient with treatment-related acute myeloid leukemia presented a t(3;11)(p11;p15) as the only cytogenetic abnormality. FISH and molecular genetic analyses identified a class 1 homeobox gene, POU1F1, located on chromosome 3p11, as the fusion partner of NUP98. In addition, we have found that the patient harbored an FLT3-ITD mutation, which most likely collaborated with the NUP98-POU1F1 fusion gene in malignant transformation. CONCLUSIONS: We have identified POU1F1 as the NUP98 fusion partner in therapy-related AML with a t(3;11)(p11;p15). This is the first POU family member identified as a fusion partner in human cancer.

Genetic and clinical characterization of 45 acute leukemia patients with MLL gene rearrangements from a single institution.

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant.

Acute megakaryoblastic leukemia with a four-way variant translocation originating the RBM15-MKL1 fusion gene.

Acute megakaryoblastic leukemia (AMKL) with t(1;22)(p13;q13) is a subset of acute myeloid leukemia (AML) representing <1% of all cases and about 70% of pediatric AMKL in the first year of life. We present a case of a 7-month-old female in whom the bone marrow karyotype showed the derivative chromosome der(22)t(1;22)(p13;q13). The RBM15-MKL1 fusion transcript was detected by RT-PCR and confirmed by sequencing analyses. FISH analyses revealed the presence of the four-way translocation t(1;22;17;18)(p13;q13;q22;q12).

A novel spliced fusion of MLL with CT45A2 in a pediatric biphenotypic acute leukemia.

BACKGROUND: Abnormalities of 11q23 involving the MLL gene are found in approximately 10% of human leukemias. To date, nearly 100 different chromosome bands have been described in rearrangements involving 11q23 and 64 fusion genes have been cloned and characterized at the molecular level. In this work we present the identification of a novel MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia. METHODS: Cytogenetics, fluorescence in situ hybridization (FISH), molecular studies (RT-PCR and LDI-PCR), and bioinformatic sequence analysis were used to characterize the CT45A2 gene as novel MLL fusion partner in pediatric acute leukemia. RESULTS: Fluorescence in situ hybridization of bone marrow G-banded metaphases demonstrated a cryptic insertion of 11q23 in Xq26.3 involving the MLL gene. Breakpoint fusion analysis revealed that a DNA fragment of 653 kb from 11q23, containing MLL exons 1-9 in addition to 16 other 11q23 genes, was inserted into the upstream region of the CT45A2 gene located at Xq26.3. In addition, a deletion at Xq26.3 encompassing the 3' region of the DDX26B gene (exons 9-16) and the entire CT45A1 gene was identified. RNA analysis revealed the presence of a novel MLL-CT45A2 fusion transcript in which the first 9 exons of the MLL gene were fused in-frame to exon 2 of the CT45A2 gene, resulting in a spliced MLL fusion transcript with an intact open reading frame. The resulting chimeric transcript predicts a fusion protein where the N-terminus of MLL is fused to the entire open reading frame of CT45A2. Finally, we demonstrate that all breakpoint regions are rich in long repetitive motifs, namely LINE/L1 and SINE/Alu sequences, but all breakpoints were exclusively identified outside these repetitive DNA sequences. CONCLUSION: We have identified CT45A2 as a novel spliced MLL fusion partner in a pediatric patient with de novo biphenotypic acute leukemia, as a result of a cryptic insertion of 11q23 in Xq26.3. Since CT45A2 is the first Cancer/Testis antigen family gene found fused with MLL in acute leukemia, future studies addressing its biologic relevance for leukemogenesis are warranted.

Coexistence of alternative MLL-SEPT9 fusion transcripts in an acute myeloid leukemia with t(11;17)(q23;q25).

We present the characterization at the RNA level of an acute myeloid leukemia with a t(11;17)(q23;q25) and a MLL rearrangement demonstrated by FISH. Molecular analysis led to the identification of two coexistent in-frame MLL-SEPT9 fusion transcripts (variants 1 and 2), presumably resulting from alternative splicing. Real-time quantitative RT-PCR analysis showed that the relative expression of the MLL-SEPT9 fusion variant 2 was 1.88 fold higher than the relative expression of MLL-SEPT9 fusion variant 1. This is the first description of a MLL-SEPT9 fusion resulting in coexistence of two alternative splicing variants, each of which previously found isolated in myeloid leukemias.

Expression pattern of the septin gene family in acute myeloid leukemias with and without MLL-SEPT fusion genes.

Septins are proteins associated with crucial steps in cell division and cellular integrity. In humans, 14 septin genes have been identified, of which five (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) are known to participate in reciprocal translocations with the MLL gene in myeloid neoplasias. We have recently shown a significant down-regulation of both SEPT2 and MLL in myeloid neoplasias with the MLL-SEPT2 fusion gene. In this study, we examined the expression pattern of the other 13 known septin genes in altogether 67 cases of myeloid neoplasia, including three patients with the MLL-SEPT2 fusion gene, four with MLL-SEPT6 fusion, and three patients with the MLL-SEPT9 fusion gene. When compared with normal controls, a statistically significant down-regulation was observed for the expression of both MLL (6.4-fold; p=0.008) and SEPT6 (1.7-fold; p=0.002) in MLL-SEPT6 leukemia. Significant down-regulation of MLL was also found in MLL-MLLT3 leukemias. In addition, there was a trend for SEPT9 down-regulation in MLL-SEPT9 leukemias (4.6-fold; p=0.077). Using hierarchical clustering analysis to compare acute myeloid leukemia genetic subgroups based on their similarity of septin expression changes, we found that MLL-SEPT2 and MLL-SEPT6 neoplasias cluster together apart from the remaining subgroups and that PML-RARA leukemia presents under-expression of most septin family genes.

Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia.

BACKGROUND: A relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature. METHODS: Cytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient. RESULTS: When compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023) CONCLUSION: We found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.

Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions.

One of the MLL fusion partners in leukemia is the SEPT6 gene, which belongs to the evolutionarily conserved family of genes of septins. In this work we aimed to characterize at both the RNA and DNA levels three acute myeloid leukemias with cytogenetic evidence of a rearrangement between 11q23 and Xq24. Molecular analysis led to the identification of several MLL-SEPT6 fusion transcripts in all cases, including a novel MLL-SEPT6 rearrangement (MLL exon 6 fused with SEPT6 exon 2). Genomic DNA breakpoints were found inside or near Alu or LINE repeats in the MLL breakpoint cluster region, whereas the breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II consensus cleavage site. These data suggest that a non-homologous end-joining repair mechanism may be involved in the generation of MLL-SEPT6 rearrangements in acute myeloid leukemia.

Molecular characterization of a rare MLL-AF4 (MLL-AFF1) fusion rearrangement in infant leukemia.

The t(4;11)(q21;q23) involving the genes MLL and AF4 (alias for AFF1) is detected in 50-70% of infant leukemia. We characterize at both the DNA and RNA level a rare MLL-AF4 fusion transcript identified in a 15-month-old girl with acute lymphoblastic leukemia. Direct sequence analysis of the reverse transcriptase-polymerase chain reaction product showed an in-frame fusion between MLL exon 9 and AF4 exon 6. We further demonstrated that the genomic breakpoints were located 1,553 bp downstream of MLL exon 9 and 1,239 bp upstream of AF4 exon 6. Four Alu repeats were detected in MLL intron 9 and two Alu repeats and one LINE1 repetitive element were identified downstream of AF4 exon 5. Finally, a 9-bp polypurine (A) tract and an 8-bp polypyrimidine (T) tract were found flanking the translocation breakpoint. In summary, we have characterized at both the RNA and the DNA level a rare MLL-AF4 fusion variant that was presumably mediated by Alu repeats or polypurine and polypyrimidine tracts located in the vicinity of genomic breakpoints.

[Worster-Drought syndrome: case report and distinction in relation to Foix-Chavany-Marie syndrome]. / Síndrome de Worster-Drought: relato de caso e distinção em relação à síndrome de Foix-Chavany-Marie.

Worster-Drought syndrome is a clinical entity resulting from cortico-nuclear tract or cerebral cortex injury, without defined etiology, characterized by alteration of the muscles that control the lips, jaw, tongue, soft palate and pharynx. The main clinical manifestations are alterations in the fonation and deglutition. We describe the case of a 15 year old female patient, displaying the cited syndrome. The clinical, radiological and neurophysiological findings are compared with the described data in the literature. We question the connection between Worster-Drought and Foix-Chavany-Marie syndromes, as well as the possible relationship with use of abortive drugs and similar oromotor problems as found in these two syndromes.

Síndrome de Worster-Drought: relato de caso e distinção em relação à síndrome de Foix-Chavany-Marie / Worster-Drought syndrome: case report and distinction in relation to Foix-Chavany-Marie syndrome

A síndrome de Worster-Drought é entidade clínica decorrente de lesão do trato cortico-nuclear. Não existe etiologia definida e caracteriza-se por alteração do controle voluntário e involuntário dos músculos que movimentam lábios, mandíbula, língua, pálato mole e faringe. As principais manifestações clínicas são alterações na fonação e deglutição. Descrevemos o caso de paciente do sexo feminino, 15 anos, que apresenta a referida síndrome. Os achados clínicos e radiológicos são comparados com os dados descritos na literatura. Questiona-se a ligação entre síndrome de Worster-Drought e a de Foix-Chavany-Marie, bem como a possível relação entre o uso de drogas abortivas e transtorno oromotor semelhante ao identificado nessas enfermidades.