True Donor Cell Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation: Diagnostic and Therapeutic Considerations-Brief Report.

Donor cell leukemia (DCL) is a rare complication after allogeneic hematopoietic stem cell transplantation (HSCT) accounting for 0.1% of relapses and presenting as secondary leukemia of donor origin. Distinct in phenotype and cytogenetics from the original leukemia, DCL's clinical challenge lies in its late onset. Its origin is affected by donor cell anomalies, transplant environment, and additional mutations. A 43-year-old woman, treated for early stage triple-negative breast cancer, developed mixed-phenotype acute leukemia (MPAL), 12 years later. Following induction chemotherapy, myeloablative conditioning, and allo-HSCT from her fully HLA-matched brother, she exhibited multiple cutaneous relapses of the original leukemia, subsequently evolving into DCL of the bone marrow. Cytogenetic analysis revealed a complex male karyotype in 20 out of 21 metaphases, however, still showing the MPAL phenotype. DCL diagnosis was confirmed by 90.5% XY in FISH analysis and the male karyotype. Declining further intensive chemotherapy including a second allo-HSCT, she was subsequently treated with repeated radiotherapy, palliative systemic therapies, and finally venetoclax and navitoclax but died seven months post-DCL diagnosis. This case underlines DCL's complexity, characterized by unique genetics, further complicating diagnosis. It highlights the need for advanced diagnostic techniques for DCL identification and underscores the urgency for early detection and better prevention and treatment strategies.

Mutational patterns in therapy-related acute lymphoblastic leukemia subgroups: one step closer to unveiling the genetic odyssey.

There is increasing evidence that therapy-related acute lymphoblastic leukemia (trALL) resulting from chemo- and/or radiotherapy represents a distinct entity. However, apart from KMT2A rearrangements, which have been repeatedly reported in this subgroup, the relevance of other aberrations remains controversial due to divergent study results and sparse molecular analyses. Within our ALL patient cohort, 15% (n = 19/131) met the criteria for trALL with a high proportion of Ph + and KMT2A rearrangements. On the molecular level, the most frequently observed mutation was KMT2D, followed by CDKN2A, KRAS and DNMT3A. No TP53 mutation was detected. Outcome was particularly poor in Ph + trALL compared to Ph+ de novo ALL, which seemed to be mitigated by allogeneic stem cell transplantation. Our findings further define trALL as a distinct entity but highlight the need for further molecular genome sequencing of somatic and germline variants to advance our understanding of trALL.

Ex vivo drug response heterogeneity reveals personalized therapeutic strategies for patients with multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy defined by complex genetics and extensive patient heterogeneity. Despite a growing arsenal of approved therapies, MM remains incurable and in need of guidelines to identify effective personalized treatments. Here, we survey the ex vivo drug and immunotherapy sensitivities across 101 bone marrow samples from 70 patients with MM using multiplexed immunofluorescence, automated microscopy and deep-learning-based single-cell phenotyping. Combined with sample-matched genetics, proteotyping and cytokine profiling, we map the molecular regulatory network of drug sensitivity, implicating the DNA repair pathway and EYA3 expression in proteasome inhibitor sensitivity and major histocompatibility complex class II expression in the response to elotuzumab. Globally, ex vivo drug sensitivity associated with bone marrow microenvironmental signatures reflecting treatment stage, clonality and inflammation. Furthermore, ex vivo drug sensitivity significantly stratified clinical treatment responses, including to immunotherapy. Taken together, our study provides molecular and actionable insights into diverse treatment strategies for patients with MM.

Near-tetraploid T-cell acute lymphoblastic leukaemia in childhood: Results of the AIEOP-BFM ALL studies.

BACKGROUND: Near-tetraploidy-defined by DNA index 1.79-2.28 or 81-103 chromosomes-is a rare cytogenetic abnormality observed both in children and adults with T-cell acute lymphoblastic leukaemia (T-ALL) and its prognostic value is not yet determined. PATIENTS AND METHODS: We report a retrospective study conducted in paediatric patients with newly diagnosed T-ALL treated in AIEOP-BFM ALL 2000 and 2009 studies. 31 near-tetraploid T-ALL patients (1.4%) are compared to T-ALL patients without near-tetraploidy. RESULTS: Near-tetraploid karyotype was associated with lower frequency of high-risk features: white blood cells count at diagnosis ≥100,000/µL (19.3% versus 41.0%, p-value < 0.001), PPR (13.3% versus 35.8%, p-value = 0.01) and minimal residual disease high-risk at the end of consolidation phase Induction B (4.03% versus 14.6%, p-value = 0.001). Complete remission was achieved at the end of induction phase (day 33) in 100% near-tetraploid T-ALL patients, compared to 93.2% T-ALL without near-tetraploidy. CONCLUSION: Overall, we found that near-tetraploid T-ALL in newly diagnosed paediatric patients is associated with low-risk presenting features, with favourable treatment response and outcome.

Myoepithelial Carcinoma of Soft Tissue With an EWSR1-KLF15 Gene Fusion in an Infant.

Overall, neonatal cancer is uncommon. Because of its rarity and heterogeneity, diagnosis can be challenging. We report a unique case of a myoepithelial carcinoma in a 7 week old girl. Molecular diagnostic workup revealed a EWSR1-KLF15 gene fusion which was previously described in only six cases of myoepithelial tumors so far. All cases occurred in children and adolescents. To our knowledge, this is the first report of a congenital EWSR1-KLF15 fusion positive myoepithelial tumor in an infant.

Phenotypic profiling with a living biobank of primary rhabdomyosarcoma unravels disease heterogeneity and AKT sensitivity.

Cancer therapy is currently shifting from broadly used cytotoxic drugs to patient-specific precision therapies. Druggable driver oncogenes, identified by molecular analyses, are present in only a subset of patients. Functional profiling of primary tumor cells could circumvent these limitations, but suitable platforms are unavailable for most cancer entities. Here, we describe an in vitro drug profiling platform for rhabdomyosarcoma (RMS), using a living biobank composed of twenty RMS patient-derived xenografts (PDX) for high-throughput drug testing. Optimized in vitro conditions preserve phenotypic and molecular characteristics of primary PDX cells and are compatible with propagation of cells directly isolated from patient tumors. Besides a heterogeneous spectrum of responses of largely patient-specific vulnerabilities, profiling with a large drug library reveals a strong sensitivity towards AKT inhibitors in a subgroup of RMS. Overall, our study highlights the feasibility of in vitro drug profiling of primary RMS for patient-specific treatment selection in a co-clinical setting.

CRISPR-Directed Therapeutic Correction at the NCF1 Locus Is Challenged by Frequent Incidence of Chromosomal Deletions.

Resurrection of non-processed pseudogenes may increase the efficacy of therapeutic gene editing, upon simultaneous targeting of a mutated gene and its highly homologous pseudogenes. To investigate the potency of this approach for clinical gene therapy of human diseases, we corrected a pseudogene-associated disorder, the immunodeficiency p47 phox -deficient chronic granulomatous disease (p47 phox CGD), using clustered regularly interspaced short palindromic repeats-associated nuclease Cas9 (CRISPR-Cas9) to target mutated neutrophil cytosolic factor 1 (NCF1). Being separated by less than two million base pairs, NCF1 and two pseudogenes are closely co-localized on chromosome 7. In healthy people, a two-nucleotide GT deletion (ΔGT) is present in the NCF1B and NCF1C pseudogenes only. In the majority of patients with p47 phox CGD, the NCF1 gene is inactivated due to a ΔGT transfer from one of the two non-processed pseudogenes. Here we demonstrate that concurrent targeting and correction of mutated NCF1 and its pseudogenes results in therapeutic CGD phenotype correction, but also causes potentially harmful chromosomal deletions between the targeted loci in a p47 phox -deficient CGD cell line model. Therefore, development of genome-editing-based treatment of pseudogene-related disorders mandates thorough safety examination, as well as technological advances, limiting concurrent induction of multiple double-strand breaks on a single chromosome.

Detection of rare reciprocal RUNX1 rearrangements by next-generation sequencing in acute myeloid leukemia.

Reciprocal RUNX1 fusions are traditionally found in up to 10% of acute myeloid leukemia (AML) patients, usually associated with a translocation (8;21)(q22;q22) corresponding to the RUNX1-RUNX1T1 fusion gene. So far, alternative RUNX1 rearrangements have been reported only rarely in AML, and the few reports so far have focused on results based on cytogenetics, fluorescence in situ hybridization, and polymerase chain reaction. Acknowledging the inherent limitations of these diagnostic techniques, the true incidence of rare RUNX1 rearrangements may be underestimated. In this report, we present two cases of adult AML, in which we detected rare RUNX1 rearrangements not by conventional cytogenetics but rather by next-generation panel sequencing. These include t(16;21)(q24;q22)/RUNX1-CBFA2T3 and t(7;21)(p22;q22)/RUNX1-USP42, respectively. In both patients the AML was therapy-related and associated with additional structural and numerical alterations thereby conferring bad prognosis. This is in line with previous reports on rare RUNX1 fusions in AML and emphasizes the clinical importance of their detection. In summary, our report not only confirms the clinical utility of NGS for diagnostics of rare reciprocal rearrangements in AML in a real-life scenario but also sheds light on the variety and complexity within AML. It further emphasizes the need for collection of additional cases for deepening insights on their clinical meaning as well as their frequency.

Histological and molecular characterization of TFEB-rearranged renal cell carcinomas.

The 2016 WHO Classification of Tumors of the Urinary System recognizes microphthalmia transcription factor (MiT) family translocation carcinomas as a separate entity among renal cell carcinomas. TFE3 and transcription factor EB (TFEB) are members of the MiT family for which chromosomal rearrangements have been associated with renal cell carcinoma formation. TFEB translocation renal cell carcinoma is a rare tumor harboring a t(6;11)(p21;q12) translocation. Recently, renal cell carcinomas with TFEB amplification have been identified. TFEB amplified renal cell carcinomas have to be distinguished from TFEB-translocated renal cancer, because they may demonstrate a more aggressive behavior. Herein, we present a TFEB-translocated and a TFEB-amplified carcinoma cases and describe their distinct histological, immunohistochemical, and molecular characteristics. In addition, we review conventional morphology, immunophenotype, genetic background, and clinical outcome of TFEB-rearranged RCCs in the literature, with a special emphasis on important differential diagnoses and the diagnostic approach.

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia.

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2-inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.

Intragenic amplification of PAX5: a novel subgroup in B-cell precursor acute lymphoblastic leukemia?

Intragenic PAX5 amplification defines a novel, relapse-prone subtype of B-cell precursor acute lymphoblastic leukemia with a poor outcome.

Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening.

BACKGROUND: Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge. RESULTS: To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation. CONCLUSIONS: We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes.

Changes in cytogenetics and molecular genetics in acute myeloid leukemia from childhood to adult age groups.

BACKGROUND: To obtain better insight into the biology of acute myeloid leukemia (AML) in various age groups, this study focused on the genetic changes occurring during a lifetime. METHODS: This study analyzed the relation between age and genetics from birth to 100 years in 5564 patients with de novo AML diagnosed from 1998 to 2012 (1192 patients from nationwide pediatric studies [AML Berlin-Frankfurt-Münster studies 98 and 2004] and 4372 adults registered with the Munich Leukemia Laboratory). RESULTS: The frequencies of cytogenetic subgroups were age-dependent. Favorable subtypes (t(8;21), inv(16)/t(16;16), and t(15;17)) decreased in general from the pediatric age group (2 to < 18 years; 33%) to the oldest groups (<5% for > 70 years; P < .0001). Unfavorable cytogenetics (-7/del(7), -5/del(5q) or 5p, inv(3)/t(3;3), t(6;9), complex karyotype, 12p, 17p, and 11q23/mixed-lineage leukemia aberrations, excluding t(9;11)) were frequent (42%) in infants (<2 years), had a low frequency in children and young adults (<22%), and increased in frequency up to 36% in patients older than 85 years (P = .01). This was even more significant for complex karyotypes (P ≤ .0001), which also showed a strong increase in the absolute age-specific incidence with age. Interestingly, the frequency of 11q23 abnormalities decreased from infants to older patients. The proportion of clinically relevant molecular aberrations of CCAAT/enhancer binding protein α, nucleophosmin (NPM1), and NPM1/fms-related tyrosine kinase 3-internal tandem duplication increased with age. CONCLUSIONS: Altogether, with the exclusion of infants, a significant decrease in the proportion of favorable cytogenetic subtypes and an increase in unfavorable cytogenetics were observed with increasing age. These findings indicate different mechanisms for the pathogenesis of AML; these different mechanisms also suggest directions for etiological research and contribute to the more unfavorable prognosis with increasing age. Cancer 2016;122:3821-3830. © 2016 American Cancer Society.

Explant culture of sarcoma patients' tissue.

Human sarcomas comprise a heterogeneous group of rare tumors that affect soft tissues and bone. Due to the scarcity and heterogeneity of these diseases, patient-derived cells that can be used for preclinical research are limited. In this study, we investigated whether the tissue explant technique can be used to obtain sarcoma cell lines from fresh as well as viable frozen tissue obtained from 8 out of 12 soft tissue and 9 out of 13 bone tumor entities as defined by the World Health Organization. The success rate, defined as the percent of samples that yielded sufficient numbers of outgrowing cells to be frozen, and the time to freeze were determined for a total of 734 sarcoma tissue specimens. In 552 cases (75%) enough cells were obtained to be frozen at early passage. Success rates were higher in bone tumors (82%) compared with soft tissue tumors (68%), and the mean time to freezing was lower in bone tumors (65 days) compared with soft tissue tumors (84 days). Overall, from 40% of the tissues cells could be frozen at early passage within <2 month after tissue removal. Comparable results as with fresh tissue were obtained after explant of viable frozen patient-derived material. In a selected number of bone and soft tissue sarcoma entities, conventional karyotyping and/or FISH (fluorescence in situ hybridization) analysis revealed a high amount (>60%) of abnormal cells in 41% of analyzed samples, especially in bone sarcomas (osteosarcoma and Ewing sarcoma). In conclusion, the explant technique is well suited to establish patient-derived cell lines for a large majority of bone and soft tissue sarcoma entities with adequate speed. This procedure thus opens the possibility for molecular analysis and drug testing for therapeutic decision making even during patient treatment.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Successful Combination of Sequential Gene Therapy and Rescue Allo-HSCT in Two Children with X-CGD - Importance of Timing.

We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.