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Patients with unfavourable subset cancer of unknown primary (CUP) have a poor prognosis when treated with standard platinum-based chemotherapy. Whether first-line treatment guided by comprehensive genomic profiling (CGP) can improve outcomes is unknown. The CUPISCO trial was designed to inform a molecularly guided treatment strategy to improve outcomes over standard platinum-based chemotherapy in patients with newly diagnosed, unfavourable, non-squamous CUP. The aim of the trial was to compare the efficacy and safety of molecularly guided therapy (MGT) versus standard platinum-based chemotherapy in these patients. This was to determine whether the inclusion of CGP in the initial diagnostic work-up leads to improved outcomes over the current standard of care. We herein report the primary analysis.

Hintergrund: Die akute myeloische Leukämie (AML) ist eine klonale Erkrankung hämatopoetischer Stamm- und Progenitorzellen des Knochenmarkes mit einem Altersgipfel im fortgeschrittenen Erwachsenenalter. Akute myeloische Leukämien können sowohl spontan und de novo als auch hereditär infolge von Keimbahnmutationen, sekundär aus anderen hämatologischen Erkrankungen oder therapie-induziert nach vorausgegangener Chemo- oder Strahlentherapie entstehen. Unabhängig vom Entstehungsmechanismus sind alle Formen der AML auf die klonale Expansion hämatopoetischer Vorläuferzellen mit rekurrenten Treibermutationen in Onkogenen und Tumorsuppressoren oder chromosomalen Aberrationen zurückzuführen. Schlussfolgerung: Insbesondere moderne Sequenzierungsmethoden haben in den letzten Jahren zu der Erkenntnis geführt, dass die Expansion hämatopoetischer Klone mit bestimmten Treibermutationen bei gesunden Personen mit zunehmendem Lebensalter häufig nachzuweisen und mit einem erhöhten Risiko des Auftretens einer AML verbunden ist. Der Nachweis molekularer Aberrationen gehört mittlerweile zur Primärdiagnostik der AML und ist sowohl von prognostischer als auch therapeutischer Bedeutung. Im weiteren Erkrankungsverlauf und nach Abschluss der Therapie werden initial nachgewiesene Mutationen zur Bestimmung der messbaren residuellen Erkrankung (MRD) verwendet, die ebenfalls prognostisch bedeutsam ist und die Möglichkeit präemptiver Therapien bereits vor dem Auftreten eines hämatologischen Rezidivs eröffnet.

Plasma cell disorders are clonal outgrowths of pre-malignant or malignant plasma cells, characterized by extensive chromosomal aberrations. Centrosome abnormalities are a major driver of chromosomal instability in cancer but their origin, incidence, and composition in primary tumor cells is poorly understood. Using cutting-edge, semi-automated high-throughput electron tomography, we characterized at nanoscale 1386 centrioles in CD138posplasma cells from eight healthy donors and 21 patients with plasma cell disorders, and 722 centrioles from different control populations. In plasma cells from healthy individuals, over-elongated centrioles accumulated with age. In plasma cell disorders, centriole over-elongation was notably frequent in early, pre-malignant disease stages, became less pronounced in overt multiple myeloma, and almost entirely disappeared in aggressive plasma cell leukemia. Centrioles in other types of patient-derived B cell neoplasms showed no over-elongation. In contrast to current belief, centriole length appears to be highly variable in long-lived, healthy plasma cells, and over-elongation and structural aberrations are common in this cell type. Our data suggest that structural centrosome aberrations accumulate with age in healthy CD138posplasma cells and may thus play an important role in early aneuploidization as an oncogenic driver in plasma cell disorders.

•Germline RUNX1, GATA2, and DDX41HHMs are associated with driver somatic variants during leukemogenesis which are unique for each syndrome.•Ongoing molecular monitoring of germline carriers without HM is needed to assess the risk profile and clinical actionability of somatic markers.

Hintergrund: Als CUP-Syndrom („cancer of unknown primary“) bezeichnet man eine Krebserkrankung, die histologisch aus Metastasengewebe gesichert ist, bei der aber trotz ausführlicher Diagnostik kein Primärtumor nachgewiesen werden kann. Damit handelt es sich beim CUP-Syndrom um eine Ausschlussdiagnose. Relevante Aktualisierungen: Aktuell hat die European Society of Medical Oncology (ESMO) stark überarbeitete Leitlinien zum CUP-Syndrom herausgegeben. Folgende relevante Aktualisierungen sind vorgenommen worden: Bei der Diagnose des CUP-Syndroms zielen die neuen Leitlinien darauf ab, die Erkrankung präziser zu definieren und die Diagnose anhand von Algorithmen besser zu standardisieren. Auch wird die molekulare Diagnostik am Tumorgewebe in den neuen Empfehlungen verankert. Die Klassifikation des CUP-Syndroms wurde ebenfalls überarbeitet. Zum einen wurde bei der Neudefinition der günstigen Untergruppen, für die eine spezifische Behandlung indiziert ist, das Karzinom mit nierenzellkarzinomtypischer Immunhistochemie („renal-like CUP“) neu aufgenommen. Zum anderen wurde eine lokal mittels Operation und/oder Strahlentherapie potenziell kurativ behandelbare Subgruppe basierend auf einer neu definierten oligometastasierten Situation in die CUP-Klassifikation eingeführt. Bezüglich der Therapie der CUP-Syndrome zeigen die aktuellen Leitlinien auf, in welchen Indikationen jenseits der empirischen Chemotherapie, die immer noch den therapeutischen Goldstandard darstellt, auch Immuntherapien und zielgerichtete Therapien zum Einsatz kommen können. Schlussfolgerung: Ziel dieser Übersichtsarbeit ist es, den aktuellen Stand der Diagnostik, Klassifikation und Therapie des CUP-Syndroms aufzuzeigen und dabei insbesondere die aktuellen Entwicklungen und die in den revidierten ESMO-Leitlinien vorgenommenen Änderungen darzustellen.

Hintergrund: Die akute myeloische Leukämie (AML) ist eine klonale Erkrankung hämatopoetischer Stamm- und Progenitorzellen des Knochenmarkes mit einem Altersgipfel im fortgeschrittenen Erwachsenenalter. Akute myeloische Leukämien können sowohl spontan und de novo als auch hereditär infolge von Keimbahnmutationen, sekundär aus anderen hämatologischen Erkrankungen oder therapie-induziert nach vorausgegangener Chemo- oder Strahlentherapie entstehen. Unabhängig vom Entstehungsmechanismus sind alle Formen der AML auf die klonale Expansion hämatopoetischer Vorläuferzellen mit rekurrenten Treibermutationen in Onkogenen und Tumorsuppressoren oder chromosomalen Aberrationen zurückzuführen. Schlussfolgerung: Insbesondere moderne Sequenzierungsmethoden haben in den letzten Jahren zu der Erkenntnis geführt, dass die Expansion hämatopoetischer Klone mit bestimmten Treibermutationen bei gesunden Personen mit zunehmendem Lebensalter häufig nachzuweisen und mit einem erhöhten Risiko des Auftretens einer AML verbunden ist. Der Nachweis molekularer Aberrationen gehört mittlerweile zur Primärdiagnostik der AML und ist sowohl von prognostischer als auch therapeutischer Bedeutung. Im weiteren Erkrankungsverlauf und nach Abschluss der Therapie werden initial nachgewiesene Mutationen zur Bestimmung der messbaren residuellen Erkrankung (MRD) verwendet, die ebenfalls prognostisch bedeutsam ist und die Möglichkeit präemptiver Therapien bereits vor dem Auftreten eines hämatologischen Rezidivs eröffnet.

Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are among the most frequent alterations in acute myeloid leukemia (AML) and can be found in ∼20% of patients at diagnosis. Among 4930 patients (median age, 56 years; interquartile range, 45-66) with newly diagnosed, intensively treated AML, we identified IDH1 mutations in 423 (8.6%) and IDH2 mutations in 575 (11.7%). Overall, there were no differences in response rates or survival for patients with mutations in IDH1 or IDH2 compared with patients without mutated IDH1/2. However, distinct clinical and comutational phenotypes of the most common subtypes of IDH1/2 mutations could be associated with differences in outcome. IDH1-R132C was associated with increased age, lower white blood cell (WBC) count, less frequent comutation of NPM1 and FLT3 internal tandem mutation (ITD) as well as with lower rate of complete remission and a trend toward reduced overall survival (OS) compared with other IDH1 mutation variants and wild-type (WT) IDH1/2. In our analysis, IDH2-R172K was associated with significantly lower WBC count, more karyotype abnormalities, and less frequent comutations of NPM1 and/or FLT3-ITD. Among patients within the European LeukemiaNet 2017 intermediate- and adverse-risk groups, relapse-free survival and OS were significantly better for those with IDH2-R172K compared with WT IDH, providing evidence that AML with IDH2-R172K could be a distinct entity with a specific comutation pattern and favorable outcome. In summary, the presented data from a large cohort of patients with IDH1/2 mutated AML indicate novel and clinically relevant findings for the most common IDH mutation subtypes.

Biallelic mutations of the CEBPA gene (CEBPAbi) define a distinct entity associated with favorable prognosis; however, the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adults with acute myeloid leukemia (AML) who had been recruited into the Study Alliance Leukemia trials, to investigate the prognostic impact of CEBPAsm. CEBPA mutations were identified in 240 patients (5.1%): 131 CEBPAbi and 109 CEBPAsm (60 affecting the N-terminal transactivation domains [CEBPAsmTAD] and 49 the C-terminal DNA-binding or basic leucine zipper region [CEBPAsmbZIP]). Interestingly, patients carrying CEBPAbi or CEBPAsmbZIP shared several clinical factors: they were significantly younger (median, 46 and 50 years, respectively) and had higher white blood cell (WBC) counts at diagnosis (median, 23.7 × 109/L and 35.7 × 109/L) than patients with CEBPAsmTAD (median age, 63 years, median WBC 13.1 × 109/L; P < .001). Co-mutations were similar in both groups: GATA2 mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIP vs 6.7% CEBPAsmTAD; P < .001) or NPM1 mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIP vs 38.3% CEBPAsmTAD; P < .001). CEBPAbi and CEBPAsmbZIP, but not CEBPAsmTAD were associated with significantly improved overall (OS; median 103 and 63 vs 13 months) and event-free survival (EFS; median, 20.7 and 17.1 months vs 5.7 months), in univariate and multivariable analyses. Additional analyses revealed that the clinical and molecular features as well as the favorable survival were confined to patients with in-frame mutations in bZIP (CEBPAbZIP-inf). When patients were classified according to CEBPAbZIP-inf and CEBPAother (including CEBPAsmTAD and non-CEBPAbZIP-inf), only patients bearing CEBPAbZIP-inf showed superior complete remission rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutation.

Biallelic mutations of the CEBPAgene (CEBPAbi) define a distinct entity associated with favorable prognosis; however, the role of monoallelic mutations (CEBPAsm) is poorly understood. We retrospectively analyzed 4708 adults with acute myeloid leukemia (AML) who had been recruited into the Study Alliance Leukemia trials, to investigate the prognostic impact of CEBPAsm. CEBPAmutations were identified in 240 patients (5.1%): 131 CEBPAbiand 109 CEBPAsm(60 affecting the N-terminal transactivation domains [CEBPAsmTAD] and 49 the C-terminal DNA-binding or basic leucine zipper region [CEBPAsmbZIP]). Interestingly, patients carrying CEBPAbior CEBPAsmbZIPshared several clinical factors: they were significantly younger (median, 46 and 50 years, respectively) and had higher white blood cell (WBC) counts at diagnosis (median, 23.7 × 109/L and 35.7 × 109/L) than patients with CEBPAsmTAD(median age, 63 years, median WBC 13.1 × 109/L; P< .001). Co-mutations were similar in both groups: GATA2mutations (35.1% CEBPAbi; 36.7% CEBPAsmbZIPvs 6.7% CEBPAsmTAD; P< .001) or NPM1mutations (3.1% CEBPAbi; 8.2% CEBPAsmbZIPvs 38.3% CEBPAsmTAD; P< .001). CEBPAbiand CEBPAsmbZIP, but not CEBPAsmTADwere associated with significantly improved overall (OS; median 103 and 63 vs 13 months) and event-free survival (EFS; median, 20.7 and 17.1 months vs 5.7 months), in univariate and multivariable analyses. Additional analyses revealed that the clinical and molecular features as well as the favorable survival were confined to patients with in-frame mutations in bZIP (CEBPAbZIP-inf). When patients were classified according to CEBPAbZIP-infand CEBPAother(including CEBPAsmTADand non-CEBPAbZIP-inf), only patients bearing CEBPAbZIP-infshowed superior complete remission rates and the longest median OS and EFS, arguing for a previously undefined prognostic role of this type of mutation.

The tyrosine-protein phosphatase nonreceptor type 11 (PTPN11) is an important regulator of RAS signaling and frequently affected by mutations in patients with acute myeloid leukemia (AML). Despite the relevance for leukemogenesis and as a potential therapeutic target, the prognostic role is controversial. To investigate the prognostic impact of PTPN11mutations, we analyzed 1529 adult AML patients using next-generation sequencing. PTPN11mutations were detected in 106 of 1529 (6.93%) patients (median VAF: 24%) in dominant (36%) and subclonal (64%) configuration. Patients with PTPN11mutations were associated with concomitant mutations in NPM1(63%), DNMT3A(37%), and NRAS(21%) and had a higher rate of European LeukemiaNet (ELN) favorable cytogenetics (57.8% vs 39.1%; P< .001) and higher white blood cell counts (P= .007) compared with PTPN11wild-type patients. In a multivariable analysis, PTPN11mutations were independently associated with poor overall survival (hazard ratio [HR]: 1.75; P< .001), relapse-free survival (HR: 1.52; P= .013), and a lower rate of complete remission (odds ratio: 0.46; P= .008). Importantly, the deleterious effect of PTPN11mutations was confined predominantly to the ELN favorable-risk group and patients with subclonal PTPN11mutations (HR: 2.28; P< .001) but not found with dominant PTPN11mutations (HR: 1.07; P= .775), presumably because of significant differences within the rate and spectrum of associated comutations. In conclusion, our data suggest an overall poor prognostic impact of PTPN11mutations in AML, which is significantly modified by the underlying cytogenetics and the clonal context in which they occur.

The tyrosine-protein phosphatase nonreceptor type 11 (PTPN11) is an important regulator of RAS signaling and frequently affected by mutations in patients with acute myeloid leukemia (AML). Despite the relevance for leukemogenesis and as a potential therapeutic target, the prognostic role is controversial. To investigate the prognostic impact of PTPN11 mutations, we analyzed 1529 adult AML patients using next-generation sequencing. PTPN11 mutations were detected in 106 of 1529 (6.93%) patients (median VAF: 24%) in dominant (36%) and subclonal (64%) configuration. Patients with PTPN11 mutations were associated with concomitant mutations in NPM1 (63%), DNMT3A (37%), and NRAS (21%) and had a higher rate of European LeukemiaNet (ELN) favorable cytogenetics (57.8% vs 39.1%; P < .001) and higher white blood cell counts (P = .007) compared with PTPN11 wild-type patients. In a multivariable analysis, PTPN11 mutations were independently associated with poor overall survival (hazard ratio [HR]: 1.75; P < .001), relapse-free survival (HR: 1.52; P = .013), and a lower rate of complete remission (odds ratio: 0.46; P = .008). Importantly, the deleterious effect of PTPN11 mutations was confined predominantly to the ELN favorable-risk group and patients with subclonal PTPN11 mutations (HR: 2.28; P < .001) but not found with dominant PTPN11 mutations (HR: 1.07; P = .775), presumably because of significant differences within the rate and spectrum of associated comutations. In conclusion, our data suggest an overall poor prognostic impact of PTPN11 mutations in AML, which is significantly modified by the underlying cytogenetics and the clonal context in which they occur.

Early results of the randomized placebo-controlled SORAML trial showed that, in patients with newly diagnosed acute myeloid leukaemia (AML), sorafenib led to a significant improvement in event-free (EFS) and relapse-free survival (RFS). In order to describe second-line treatments and their implications on overall survival (OS), we performed a study after a median follow-up time of 78 months. Newly diagnosed fit AML patients aged ≤60 years received sorafenib (n= 134) or placebo (n= 133) in addition to standard chemotherapy and as maintenance treatment. The 5-year EFS was 41 versus 27% (HR 0.68; p= 0.011) and 5-year RFS was 53 versus 36% (HR 0.64; p= 0.035). Allogeneic stem cell transplantation (allo SCT) was performed in 88% of the relapsed patients. Four years after salvage allo SCT, the cumulative incidence of relapse was 54 versus 35%, and OS was 32 versus 50%. The 5-year OS from randomization in all study patients was 61 versus 53% (HR 0.82; p= 0.282). In conclusion, the addition of sorafenib to chemotherapy led to a significant prolongation of EFS and RFS. Although the OS benefit did not reach statistical significance, these results confirm the antileukaemic activity of sorafenib.

•Quizartinib enabled more patients to undergo hematopoietic stem cell transplantation (HSCT) versus chemotherapy.•Continuation of quizartinib after HSCT was tolerable, with no new safety signals.•Quizartinib before and after HSCT may confer clinical benefits in relapsed/refractory FLT3-ITD acute myelogenous leukemia.

•Quizartinib enabled more patients to undergo hematopoietic stem cell transplantation (HSCT) versus chemotherapy.•Continuation of quizartinib after HSCT was tolerable, with no new safety signals.•Quizartinib before and after HSCT may confer clinical benefits in relapsed/refractory FLT3-ITD acute myelogenous leukemia.

The mutant IDH1 (mIDH1) inhibitor BAY1436032 demonstrated robust activity in preclinical AML models, supporting clinical evaluation. In the current dose-escalation study, BAY1436032 was orally administered to 27 mIDH1 AML subjects across 4 doses ranging from 300 to 1500 mg twice-daily. BAY1436032 exhibited a relatively short half-life and apparent non-linear pharmacokinetics after continuous dosing. Most subjects experienced only partial target inhibition as indicated by plasma R-2HG levels. BAY1436032 was safe and a maximum tolerated dose was not identified. The median treatment duration for all subjects was 3.0 months (0.49–8.5). The overall response rate was 15% (4/27; 1 CRp, 1 PR, 2 MLFS), with responding subjects experiencing a median treatment duration of 6.0 months (3.9–8.5) and robust R-2HG decreases. Thirty percent (8/27) achieved SD, with a median treatment duration of 5.5 months (3.1–7.0). Degree of R-2HG inhibition and clinical benefit did not correlate with dose. Although BAY1436032 was safe and modestly effective as monotherapy, the low overall response rate and incomplete target inhibition achieved at even the highest dose tested do not support further clinical development of this investigational agent in AML.

In fit patients with newly diagnosed acute myeloid leukemia (AML), immediate treatment start is recommended due to the poor prognosis of untreated acute leukemia. We explored the relationship between time from diagnosis to treatment start (TDT) and prognosis in a large real-world data set from the German Study Alliance Leukemia-Acute Myeloid Leukemia (SAL-AML) registry. All registered non-acute promyelocytic leukemia patients with intensive induction treatment and a minimum 12 months of follow-up were selected (n = 2263). We analyzed influence of TDT on remission, early death, and overall survival (OS) in univariable analyses for each day of treatment delay, in groups of 0 to 5, 6 to 10, 11 to 15, and >15 days of TDT, adjusted for influence of established prognostic variables on outcomes. Median TDT was 3 days (interquartile range, 2-7). Unadjusted 2-year OS rates, stratified by TDT of 0 to 5, 6 to 10, 11 to 15, and >15 days, were 51%, 48%, 44%, and 50% (P = .211). In multivariable Cox regression analysis accounting for established prognostic variables, the TDT hazard ratio as a continuous variable was 1.00 (P = .617). In OS analyses, separately stratified for age ≤60 and >60 years and for high vs lower initial white blood cell count, no significant differences between TDT groups were observed. Our study suggests that TDT is not related to survival. As stratification in intensive first-line AML treatment evolves, TDT data suggest that it may be a feasible approach to wait for genetic and other laboratory test results so that clinically stable patients are assigned the best available treatment option. This trial was registered at www.clinicaltrials.gov as #NCT03188874.

In fit patients with newly diagnosed acute myeloid leukemia (AML), immediate treatment start is recommended due to the poor prognosis of untreated acute leukemia. We explored the relationship between time from diagnosis to treatment start (TDT) and prognosis in a large real-world data set from the German Study Alliance Leukemia–Acute Myeloid Leukemia (SAL-AML) registry. All registered non–acute promyelocytic leukemia patients with intensive induction treatment and a minimum 12 months of follow-up were selected (n = 2263). We analyzed influence of TDT on remission, early death, and overall survival (OS) in univariable analyses for each day of treatment delay, in groups of 0 to 5, 6 to 10, 11 to 15, and >15 days of TDT, adjusted for influence of established prognostic variables on outcomes. Median TDT was 3 days (interquartile range, 2-7). Unadjusted 2-year OS rates, stratified by TDT of 0 to 5, 6 to 10, 11 to 15, and >15 days, were 51%, 48%, 44%, and 50% (P= .211). In multivariable Cox regression analysis accounting for established prognostic variables, the TDT hazard ratio as a continuous variable was 1.00 (P= .617). In OS analyses, separately stratified for age ≤60 and >60 years and for high vs lower initial white blood cell count, no significant differences between TDT groups were observed. Our study suggests that TDT is not related to survival. As stratification in intensive first-line AML treatment evolves, TDT data suggest that it may be a feasible approach to wait for genetic and other laboratory test results so that clinically stable patients are assigned the best available treatment option. This trial was registered at www.clinicaltrials.govas #NCT03188874.

The fusion genes CBFB/MYH11and RUNX1/RUNX1T1block differentiation through disruption of the core binding factor (CBF) complex and are found in 10–15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum (‘mutatome’) in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2,and ZBTB7Abeing strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A,and DHX15and identified novel, recurrent mutations in NFE2(3%), MN1(4%), HERC1(3%), and ZFHX4(5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3Aand the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

Abnormal karyotypes are common in cancer cells and frequently observed in acute myeloid leukemia (AML), in which complex karyotype aberrations are associated with poor prognosis. How exactly abnormal karyotypes arise and are propagated in AML is unclear. TP53mutations and deletions are frequent in complex karyotype AML, suggesting a role of TP53alterations in the development of chromosome abnormalities. Here, we generated isogenic TP53-knockout versions of the euploid AML cell line EEB to investigate the impact of TP53on karyotype stability. We show that chromosome abnormalities spontaneously arise in TP53-deficient cells. Numerical aneuploidy could, to some extent, be propagated in a TP53-proficient setting, indicating that it does not necessarily trigger TP53 activation. In contrast, tolerance to structural chromosome aberrations was almost entirely restricted to TP53-knockout clones, all of which were able to continue proliferation in the presence of damaged DNA. Mechanistically, as a source of chromosome aberrations, limited numerical but not structural chromosomal instability was tolerated by TP53-wildtype cells. In contrast, structural instability was found only in TP53-knockout cells. Together, in myeloid cells TP53loss allows for the development of complex karyotype aberrations and karyotype heterogeneity by perpetuation of chromosome segregation errors.

Analysis of intraclonal heterogeneity has yielded insights into the clonal evolution of hematologic malignancies. We compared the clonal and subclonal compositions of the underlying plasma cell dyscrasia in 544 systemic light chain amyloidosis (PC-AL) patients with 519 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or symptomatic MM; ie, PC–non-AL patients). Using interphase fluorescence in situ hybridization, subclones were stringently defined as clone size below two thirds of the largest clone and an absolute difference of ≥30%. Subclones were found less frequently in the PC-AL group, at 199 (36.6%) of 544 as compared with 267 (51.4%) of 519 in the PC–non-AL group (P< .001), and were not associated with the stage of plasma cell dyscrasia in either entity. In both groups, translocation t(11;14), other immunoglobulin heavy chain translocations, and hyperdiploidy were typically found as main clones, whereas gain of 1q21 and deletions of 8p21, 13q14, and 17p13 were frequently found as subclones. There were no shifts in the subclone/main clone ratio depending on the MGUS, SMM, or MM stage of plasma cell dyscrasia. In multivariate analysis, t(11;14) was associated with lower rates of subclone formation and hyperdiploidy with higher rates. PC-AL itself lost statistical significance, demonstrating that the lower subclone frequency in AL is a reflection of its exceptionally high t(11;14) frequency. In summary, the subclone patterns in PC-AL and PC–non-AL are closely related, implying that subclone formation depends on the main cytogenetic categories and is independent of disease entity and stage.

Analysis of intraclonal heterogeneity has yielded insights into the clonal evolution of hematologic malignancies. We compared the clonal and subclonal compositions of the underlying plasma cell dyscrasia in 544 systemic light chain amyloidosis (PC-AL) patients with 519 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or symptomatic MM; ie, PC–non-AL patients). Using interphase fluorescence in situ hybridization, subclones were stringently defined as clone size below two thirds of the largest clone and an absolute difference of =30%. Subclones were found less frequently in the PC-AL group, at 199 (36.6%) of 544 as compared with 267 (51.4%) of 519 in the PC–non-AL group (P < .001), and were not associated with the stage of plasma cell dyscrasia in either entity. In both groups, translocation t(11;14), other immunoglobulin heavy chain translocations, and hyperdiploidy were typically found as main clones, whereas gain of 1q21 and deletions of 8p21, 13q14, and 17p13 were frequently found as subclones. There were no shifts in the subclone/main clone ratio depending on the MGUS, SMM, or MM stage of plasma cell dyscrasia. In multivariate analysis, t(11;14) was associated with lower rates of subclone formation and hyperdiploidy with higher rates. PC-AL itself lost statistical significance, demonstrating that the lower subclone frequency in AL is a reflection of its exceptionally high t(11;14) frequency. In summary, the subclone patterns in PC-AL and PC–non-AL are closely related, implying that subclone formation depends on the main cytogenetic categories and is independent of disease entity and stage.

Background

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure1. The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion2,3. However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)4–8. Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention. Individuals who are at high risk of developing acute myeloid leukaemia can be identified years before diagnosis using genetic information from blood samples.

In cancer of unknown primary (CUP), metastases are clinically and histologically confirmed, but the primary tumor site remains elusive after extensive work-up. CUPs make up for 2–3% of all epithelial malignancies. The two prevailing histologies are adenocarcinomas and undifferentiated carcinomas, whereas squamous cell carcinomas, neuroendocrine carcinomas and rare histologies account for the remaining 10%.

Chromosomal instability is a hallmark of cancer and correlates with the presence of extra centrosomes, which originate from centriole overduplication. Overduplicated centrioles lead to the formation of centriole rosettes, which mature into supernumerary centrosomes in the subsequent cell cycle. While extra centrosomes promote chromosome missegregation by clustering into pseudo-bipolar spindles, the contribution of centriole rosettes to chromosome missegregation is unknown. We used multi-modal imaging of cells with conditional centriole overduplication to show that mitotic rosettes in bipolar spindles frequently harbor unequal centriole numbers, leading to biased chromosome capture that favors binding to the prominent pole. This results in chromosome missegregation and aneuploidy. Rosette mitoses lead to viable offspring and significantly contribute to progeny production. We further show that centrosome abnormalities in primary human malignancies frequently consist of centriole rosettes. As asymmetric centriole rosettes generate mitotic errors that can be propagated, rosette mitoses are sufficient to cause chromosome missegregation in cancer.

Metaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non–core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P< .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features.

Metaphase karyotyping is an established diagnostic standard in acute myeloid leukemia (AML) for risk stratification. One of the cytogenetic findings in AML is structurally highly abnormal marker chromosomes. In this study, we have assessed frequency, cytogenetic characteristics, prognostic impact, and underlying biological origin of marker chromosomes. Given their inherent gross structural chromosomal damage, we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. In 2 large consecutive prospective, randomized, multicenter, intensive chemotherapy trials (AML96, AML2003) from the Study Alliance Leukemia, marker chromosomes were detectable in 165/1026 (16.1%) of aberrant non–core-binding-factor (CBF) karyotype patients. Adverse-risk karyotypes displayed a higher frequency of marker chromosomes (26.5% in adverse-risk, 40.3% in complex aberrant, and 41.2% in abnormality(17p) karyotypes, P < .0001 each). Marker chromosomes were associated with a poorer prognosis compared with other non-CBF aberrant karyotypes and led to lower remission rates (complete remission + complete remission with incomplete recovery), inferior event-free survival as well as overall survival in both trials. In multivariate analysis, marker chromosomes independently predicted poor prognosis in the AML96 trial ≤60 years. As detected by array comparative genomic hybridization, about one-third of marker chromosomes (18/49) had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of marker chromosome-negative complex aberrant karyotypes (1/34). The chromothripsis-positive cases were characterized by a particularly high degree of karyotype complexity, TP53 mutations, and dismal prognosis. In conclusion, marker chromosomes are indicative of chromothripsis and associated with poor prognosis per se and not merely by association with other adverse cytogenetic features.

Electron microscopy is the gold standard to characterize cellular ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. Here, we describe a semi-automated high-throughput strategy using single-axis serial section electron tomography to investigate and analyze centriole ultrastructure in bone-marrow-derived, primary human CD138posplasma cells. The protocol comprises steps for EM sample preparation, semi-automated transmission electron microscopy screening, and screening evaluation for cells of interest. Thereafter, we detail tomography acquisition, data reconstruction, and joining.

Electron microscopy is the gold standard to characterize centrosomal ultrastructure. However, production of significant morphometrical data is highly limited by acquisition time. We therefore developed a generalizable, semi-automated high-throughput electron tomography strategy to study centrosome aberrations in sparse patient-derived cancer cells at nanoscale. As proof of principle, we present electron tomography data on 455 centrioles of CD138posplasma cells from one patient with relapsed/refractory multiple myeloma and CD138negbone marrow mononuclear cells from three healthy donors as a control. Plasma cells from the myeloma patient displayed 122 over-elongated centrioles (48.8%). Particularly mother centrioles also harbored gross structural abnormalities, including fragmentation and disturbed microtubule cylinder formation, while control centrioles were phenotypically unremarkable. These data demonstrate the feasibility of our scalable high-throughput electron tomography strategy to study structural centrosome aberrations in primary tumor cells. Moreover, our electron tomography workflow and data provide a resource for the characterization of cell organelles beyond centrosomes.

The impact of nutritional status on outcome of allogeneic stem cell transplantation (alloSCT) is controversial. This study investigates the influence of pretransplant weight loss and serologic indicators of nutritional homeostasis on relapse and death of acute myeloid leukemia (AML) after alloSCT.

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL−/−mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL−/−cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novogeneration of centrioles in STIL−/−cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.

Treatment outcomes for older patients with acute myeloid leukemia (AML) have remained dismal. This randomized, phase 2 trial in AML patients not considered suitable for intensive induction therapy compared low-dose cytarabine (LDAC) with or without volasertib, a highly potent and selective inhibitor of polo-like kinases. Eighty-seven patients (median age 75 years) received LDAC 20 mg twice daily subcutaneously days 1-10 or LDAC + volasertib 350 mg IV days 1 + 15 every 4 weeks. Response rate (complete remission and complete remission with incomplete blood count recovery) was higher for LDAC + volasertib vs LDAC (31.0% vs 13.3%; odds ratio, 2.91; P= .052). Responses in the LDAC + volasertib arm were observed across all genetic groups, including 5 of 14 patients with adverse cytogenetics. Median event-free survival was significantly prolonged by LDAC + volasertib compared with LDAC (5.6 vs 2.3 months; hazard ratio, 0.57; 95% confidence interval, 0.35-0.92; P= .021); median overall survival was 8.0 vs 5.2 months, respectively (hazard ratio, 0.63; 95% confidence interval, 0.40-1.00; P= .047). LDAC + volasertib led to an increased frequency of adverse events that was most pronounced for neutropenic fever/infections and gastrointestinal events; there was no increase in the death rate at days 60 + 90. This study was registered at www.clinicaltrials.govas #NCT00804856.

Treatment outcomes for older patients with acute myeloid leukemia (AML) have remained dismal. This randomized, phase 2 trial in AML patients not considered suitable for intensive induction therapy compared low-dose cytarabine (LDAC) with or without volasertib, a highly potent and selective inhibitor of polo-like kinases. Eighty-seven patients (median age 75 years) received LDAC 20 mg twice daily subcutaneously days 1-10 or LDAC + volasertib 350 mg IV days 1 + 15 every 4 weeks. Response rate (complete remission and complete remission with incomplete blood count recovery) was higher for LDAC + volasertib vs LDAC (31.0% vs 13.3%; odds ratio, 2.91; P = .052). Responses in the LDAC + volasertib arm were observed across all genetic groups, including 5 of 14 patients with adverse cytogenetics. Median event-free survival was significantly prolonged by LDAC + volasertib compared with LDAC (5.6 vs 2.3 months; hazard ratio, 0.57; 95% confidence interval, 0.35-0.92; P = .021); median overall survival was 8.0 vs 5.2 months, respectively (hazard ratio, 0.63; 95% confidence interval, 0.40-1.00; P = .047). LDAC + volasertib led to an increased frequency of adverse events that was most pronounced for neutropenic fever/infections and gastrointestinal events; there was no increase in the death rate at days 60 + 90. This study was registered at www.clinicaltrials.gov as #NCT00804856.

Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9–driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts.

Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9–driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts.

Wolf: Celgene: Honoraria, Research Funding. Bug:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hexal: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Eurocept: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz: Honoraria; Neovii: Other: Travel; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Sanofi: Other: Travel. Götze:Celgene: Research Funding. Stelljes:Amgen: Consultancy, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Subklewe:Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Seattle Genetics: Research Funding; Morphosys: Research Funding; Janssen: Consultancy; AMGEN: Consultancy, Honoraria, Research Funding; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding. Haenel:Amgen, Novartis, Roche, Celgene, Takeda, Bayer: Honoraria. Rollig:Amgen, Astellas, BMS, Daiichi Sankyo, Janssen, Roche: Consultancy; Abbvie, Novartis, Pfizer: Consultancy, Research Funding. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria. Thiede:AgenDix GmbH: Other: Co-owner and CEO.Off-label: treatment with azacitidine to prevent or substantially delay an overt relapse in MRD-positive patients with MDS or AML