Genetic landscape of homologous recombination repair genes in early-onset/familial prostate cancer patients.

Prostate cancer (PrCa) is one of the three most frequent and deadliest cancers worldwide. The discovery of PARP inhibitors for the treatment of tumors with deleterious variants in homologous recombination repair (HRR) genes has placed PrCa on the roadmap of precision medicine. However, the overall contribution of HRR genes to the 10%-20% of carcinomas arising in men with early-onset/familial PrCa has not been fully clarified. We used targeted next-generation sequencing (T-NGS) covering eight HRR genes (ATM, BRCA1, BRCA2, BRIP1, CHEK2, NBN, PALB2, and RAD51C) and an analysis pipeline querying both small and large genomic variations to clarify their global and relative contribution to hereditary PrCa predisposition in a series of 462 early-onset/familial PrCa cases. Deleterious variants were found in 3.9% of the patients, with CHEK2 and ATM being the most frequently mutated genes (38.9% and 22.2% of the carriers, respectively), followed by PALB2 and NBN (11.1% of the carriers, each), and finally by BRCA2, RAD51C, and BRIP1 (5.6% of the carriers, each). Using the same NGS data, exonic rearrangements were found in two patients, one pathogenic in BRCA2 and one of unknown significance in BRCA1. These results contribute to clarify the genetic heterogeneity that underlies PrCa predisposition in the early-onset and familial disease, respectively.

Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study.

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.

When to Stop TKIs in Patients with Chronic Myeloid Leukemia and How to Follow Them Subsequently.

OPINION STATEMENT: ABL1 tyrosine kinase inhibitors (TKI) have dramatically improved the outcome for CML (chronic myeloid leukemia) patients. When TKI therapy is addressed appropriately, it can lead to an optimal molecular response in the majority of CML patients and a life expectancy that approaches that of the general population. However, lifelong TKI therapy may have consequences, including chronic, mostly low-grade, adverse events that can substantially impact patients' quality of life, adherence to therapy and, consequently, success of treatment. In the last few years, several groups have demonstrated that approximately 50% of chronic phase CML patients (CP-CML) who have achieved a stable deep molecular response (DMR) can stop therapy without suffering molecular relapse. Nowadays, treatment-free remission (TFR) has a significant role in the management of CML and should be considered in selected motivated patients that fulfill well-defined requirements to maximize the probability of successful discontinuation of TKI therapy.

Recommendations from a Portuguese Expert Group for Discontinuation of Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia Patients in Clinical Practice.

Until recently, the main goal of chronic myeloid leukemia therapy was disease control with the best overall survival, which required lifelong treatment. However, currently, the treatment-free remission concept is becoming an important goal in clinical practice, and several tyrosine kinase inhibitors discontinuation studies have shown that round 50% of patients with a durable deep molecular response beyond major molecular response successfully interrupt tyrosine kinase inhibitors for at least three years without loss of molecular response. However, and regardless of the existing evidence, the exact conditions for attempting treatment-free remission remain poorly defined. Different authors tried to guide the clinical decision regarding this topic but there are some points that differ, namely with respect to the recommended duration of tyrosine kinase inhibitors therapy and the appropriate molecular response prior to treatment-free remission. The goal of this article is to propose an algorithm to guide clinical practice in Portugal concerning chronic phase-chronic myeloid leukemia patients who wish to attempt treatment-free remission, since there are no national guidelines.

Até recentemente, o objectivo principal do tratamento da leucemia mielóide crónica era o controlo da doença com o maior tempo possível de sobrevivência, o que requeria tratamento durante toda a vida. No entanto, e actualmente, o conceito de remissão livre de tratamento está a tornar-se um objectivo importante na prática clínica, e vários estudos de descontinuação de inibidores de tirosina cinase mostraram que cerca de 50% dos doentes com uma resposta molecular profunda duradoura, para além da resposta molecular major, interrompem tratamento com inibidores de tirosina cinase com sucesso durante pelo menos três anos sem perda da resposta molecular. No entanto, e apesar da evidência existente, as condições concretas e exactas para tentar remissão livre de tratamento são ainda pouco conhecidas. Diferentes autores tentaram guiar a decisão clínica sobre este tópico mas alguns pontos permanecem não consensuais, nomeadamente no que respeita à duração recomendada de tratamento com inibidores de tirosina cinase e à resposta molecular apropriada antes de tentar remissão livre de tratamento. O objectivo desta publicação é propor um algoritmo que permita guiar a prática clínica em Portugal relativa a doentes com leucemia mielóide crónica ou em fase crónica que pretendam tentar remissão livre de tratamento, dada a ausência de recomendações nacionais.

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.

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.

Diagnosis, complications and management of chronic neutrophilic leukaemia: A case report.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm characterized by sustained neutrophilia and the absence of the Philadelphia chromosome or the BCR-ABL1 fusion gene. The present study reports the case of a 59-year-old Caucasian female that was referred to The Francisco Gentil Portuguese Institute of Oncology (Porto, Portugal) with constitutional symptoms (mainly asthenia), marked leukocytosis (51.33×109/l with 90% neutrophils), macrocytic anemia and splenomegaly. Bone marrow aspiration and biopsy revealed hypercellular marrow with clear predominance of segmented neutrophils. The karyotype was normal and the BCR-ABL1 fusion gene was not detected. After excluding a leukemoid reaction, a diagnosis of CNL was established. The clinical follow-up was complicated by hemorrhagic brain lesions and relapsing episodes of erythematous, well-demarcated and painful subcutaneous nodular lesions, consistent with Sweet's syndrome (SS). Multiple treatment strategies were administered, including use of hydroxyurea, imatinib and intensive chemotherapy. Nevertheless, progression was documented and the patient succumbed at 28 months post-diagnosis. The clinical course of CNL varies, and can be complicated by cerebral hemorrhage, blastic transformation or infection. Dermatological manifestations such as SS have seldom been reported in association. No evidence-based treatment currently exists and the majority of our knowledge is based on results from case reports and small series.

Assessment of fusion gene status in sarcomas using a custom made fusion gene microarray.

Sarcomas are relatively rare malignancies and include a large number of histological subgroups. Based on morphology alone, the differential diagnoses of sarcoma subtypes can be challenging, but the identification of specific fusion genes aids correct diagnostication. The presence of individual fusion products are routinely investigated in Pathology labs. However, the methods used are time-consuming and based on prior knowledge about the expected fusion gene and often the most likely break-point. In this study, 16 sarcoma samples, representing seven different sarcoma subtypes with known fusion gene status from a diagnostic setting, were investigated using a fusion gene microarray. The microarray was designed to detect all possible exon-exon breakpoints between all known fusion genes in a single analysis. An automated scoring of the microarray data from the 38 known sarcoma-related fusion genes identified the correct fusion gene among the top-three hits in 11 of the samples. The analytical sensitivity may be further optimised, but we conclude that a sarcoma-fusion gene microarray is suitable as a time-saving screening tool to identify the majority of the correct fusion genes.

Novel 5' fusion partners of ETV1 and ETV4 in prostate cancer.

Gene fusions involving the erythroblast transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, ETV5, and FLI1 are a common feature of prostate carcinomas (PCas). The most common upstream fusion partner described is the androgen-regulated prostate-specific gene TMPRSS2, most frequently with ERG, but additional 5' fusion partners have been described. We performed 5' rapid amplification of cDNA ends in 18 PCas with ETV1, ETV4, or ETV5 outlier expression to identify the 5' fusion partners. We also evaluated the exon-level expression profile of these ETS genes in 14 cases. We identified and confirmed by fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction the two novel chimeric genes OR51E2-ETV1 and UBTF-ETV4 in two PCas. OR51E2 encodes a G-protein-coupled receptor that is overexpressed in PCas, whereas UBTF is a ubiquitously expressed gene encoding an HMG-box DNA-binding protein involved in ribosome biogenesis. We additionally describe two novel gene fusion combinations of previously described genes, namely, SLC45A3-ETV4 and HERVK17-ETV4. Finally, we found one PCa with TMPRSS2-ETV1, one with C15orf21-ETV1, one with EST14-ETV1, and two with 14q133-q21.1-ETV1. In nine PCas (eight ETV1 and one ETV5), exhibiting ETS outlier expression and genomic rearrangement detected by FISH, no 5' fusion partner was found. Our findings contribute significantly to characterize the heterogeneous group of ETS gene fusions and indicate that all genes described as 5' fusion partners with one ETS gene can most likely be rearranged with any of the other ETS genes involved in prostate carcinogenesis.

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.

Potential downstream target genes of aberrant ETS transcription factors are differentially affected in Ewing's sarcoma and prostate carcinoma.

FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing's sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+) and without (PCa ETS-) ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.

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.

MLL-SEPTIN gene fusions in hematological malignancies.

The mixed lineage leukemia (MLL) locus is involved in more than 60 different rearrangements with a remarkably diverse group of fusion partners in approximately 10% of human leukemias. MLL rearrangements include chromosomal translocations, gene internal duplications, chromosome 11q deletions or inversions and MLL gene insertions into other chromosomes, or vice versa. MLL fusion partners can be classified into four distinct categories: nuclear proteins, cytoplasmatic proteins, histone acetyltransferases and septins. Five different septin genes (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) have been identified as MLL fusion partners, giving rise to chimeric fusion proteins in which the N terminus of MLL is fused, in frame, to almost the entire open reading frame of the septin partner gene. The rearranged alleles result from heterogeneous breaks in distinct introns of both MLL and its septin fusion partner, originating distinct gene fusion variants. MLL-SEPTIN rearrangements have been repeatedly identified in de novo and therapy related myeloid neoplasia in both children and adults, and some clinicopathogenetic associations are being uncovered. The fundamental roles of septins in cytokinesis, membrane remodeling and compartmentalization can provide some clues on how abnormalities in the septin cytoskeleton and MLL deregulation could be involved in the pathogenesis of hematological malignancies.

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.