Heinz-A. Horst

Targeted Therapy With the T-Cell–Engaging Antibody Blinatumomab of Chemotherapy-Refractory Minimal Residual Disease in B-Lineage Acute Lymphoblastic Leukemia Patients Results in High Response Rate and Prolonged Leukemia-Free Survival

PURPOSE Blinatumomab, a bispecific single-chain antibody targeting the CD19 antigen, is a member of a novel class of antibodies that redirect T cells for selective lysis of tumor cells. In acute lymphoblastic leukemia (ALL), persistence or relapse of minimal residual disease (MRD) after chemotherapy indicates resistance to chemotherapy and results in hematologic relapse. A phase II clinical study was conducted to determine the efficacy of blinatumomab in MRD-positive B-lineage ALL. PATIENTS AND METHODS Patients with MRD persistence or relapse after induction and consolidation therapy were included. MRD was assessed by quantitative reverse transcriptase polymerase chain reaction for either rearrangements of immunoglobulin or T-cell receptor genes, or specific genetic aberrations. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dose of 15 μg/m2/24 hours. RESULTS Twenty-one patients were treated, of whom 16 patients became MRD negative. One patient was not evaluable due to a grade 3 adverse event leading to treatment discontinuation. Among the 16 responders, 12 patients had been molecularly refractory to previous chemotherapy. Probability for relapse-free survival is 78% at a median follow-up of 405 days. The most frequent grade 3 and 4 adverse event was lymphopenia, which was completely reversible like most other adverse events. CONCLUSION Blinatumomab is an efficacious and well-tolerated treatment in patients with MRD-positive B-lineage ALL after intensive chemotherapy. T cells engaged by blinatumomab seem capable of eradicating chemotherapy-resistant tumor cells that otherwise cause clinical relapse.

IDH1 and IDH2 Mutations Are Frequent Genetic Alterations in Acute Myeloid Leukemia and Confer Adverse Prognosis in Cytogenetically Normal Acute Myeloid Leukemia With NPM1 Mutation Without FLT3 Internal Tandem Duplication

PURPOSE To analyze the frequency and prognostic impact of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations in acute myeloid leukemia (AML). PATIENTS AND METHODS We studied 805 adults (age range, 16 to 60 years) with AML enrolled on German-Austrian AML Study Group (AMLSG) treatment trials AML HD98A and APL HD95 for mutations in exon 4 of IDH1 and IDH2. Patients were also studied for NPM1, FLT3, MLL, and CEBPA mutations. The median follow-up for survival was 6.3 years. RESULTS IDH mutations were found in 129 patients (16.0%) -IDH1 in 61 patients (7.6%), and IDH2 in 70 patients (8.7%). Two patients had both IDH1 and IDH2 mutations. All but one IDH1 mutation caused substitutions of residue R132; IDH2 mutations caused changes of R140 (n = 48) or R172 (n = 22). IDH mutations were associated with older age (P < .001; effect conferred by IDH2 only); lower WBC (P = .04); higher platelets (P < .001); cytogenetically normal (CN) -AML (P< .001); and NPM1 mutations, in particular with the genotype of mutated NPM1 without FLT3 internal tandem duplication (ITD; P < .001). In patients with CN-AML with the latter genotype, IDH mutations adversely impacted relapse-free survival (RFS; P = .02) and overall survival (P = .03), whereas outcome was not affected in patients with CN-AML who lacked this genotype. In CN-AML, multivariable analyses revealed a significant interaction between IDH mutation and the genotype of mutated NPM1 without FLT3-ITD (ie, the adverse impact of IDH mutation [RFS]; P = .046 was restricted to this patient subset). CONCLUSION IDH1 and IDH2 mutations are recurring genetic changes in AML. They constitute a poor prognostic factor in CN-AML with mutated NPM1 without FLT3-ITD, which allows refined risk stratification of this AML subset.

RUNX1 mutations in acute myeloid leukemia are associated with distinct clinico-pathologic and genetic features.

We evaluated the frequency, genetic architecture, clinico-pathologic features and prognostic impact of RUNX1 mutations in 2439 adult patients with newly-diagnosed acute myeloid leukemia (AML). RUNX1 mutations were found in 245 of 2439 (10%) patients; were almost mutually exclusive of AML with recurrent genetic abnormalities; and they co-occurred with a complex pattern of gene mutations, frequently involving mutations in epigenetic modifiers (ASXL1, IDH2, KMT2A, EZH2), components of the spliceosome complex (SRSF2, SF3B1) and STAG2, PHF6, BCOR. RUNX1 mutations were associated with older age (16–59 years: 8.5%; ⩾60 years: 15.1%), male gender, more immature morphology and secondary AML evolving from myelodysplastic syndrome. In univariable analyses, RUNX1 mutations were associated with inferior event-free (EFS, P<0.0001), relapse-free (RFS, P=0.0007) and overall survival (OS, P<0.0001) in all patients, remaining significant when age was considered. In multivariable analysis, RUNX1 mutations predicted for inferior EFS (P=0.01). The effect of co-mutation varied by partner gene, where patients with the secondary genotypes RUNX1mut/ASXL1mut (OS, P=0.004), RUNX1mut/SRSF2mut (OS, P=0.007) and RUNX1mut/PHF6mut (OS, P=0.03) did significantly worse, whereas patients with the genotype RUNX1mut/IDH2mut (OS, P=0.04) had a better outcome. In conclusion, RUNX1-mutated AML is associated with a complex mutation cluster and is correlated with distinct clinico-pathologic features and inferior prognosis.

Secondary genetic lesions in acute myeloid leukemia with inv(16) or t(16;16): a study of the German-Austrian AML Study Group (AMLSG).

In this study, we evaluated the impact of secondary genetic lesions in acute myeloid leukemia (AML) with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11. We studied 176 patients, all enrolled on prospective treatment trials, for secondary chromosomal aberrations and mutations in N-/KRAS, KIT, FLT3, and JAK2 (V617F) genes. Most frequent chromosomal aberrations were trisomy 22 (18%) and trisomy 8 (16%). Overall, 84% of patients harbored at least 1 gene mutation, with RAS being affected in 53% (45% NRAS; 13% KRAS) of the cases, followed by KIT (37%) and FLT3 (17%; FLT3-TKD [14%], FLT3-ITD [5%]). None of the secondary genetic lesions influenced achievement of complete remission. In multivariable analyses, KIT mutation (hazard ratio [HR] = 1.67; P = .04], log(10)(WBC) (HR = 1.33; P = .02), and trisomy 22 (HR = 0.54; P = .08) were relevant factors for relapse-free survival; for overall survival, FLT3 mutation (HR = 2.56; P = .006), trisomy 22 (HR = 0.45; P = .07), trisomy 8 (HR = 2.26; P = .02), age (difference of 10 years, HR = 1.46; P = .01), and therapy-related AML (HR = 2.13; P = .14) revealed as prognostic factors. The adverse effects of KIT and FLT3 mutations were mainly attributed to exon 8 and tyrosine kinase domain mutations, respectively. Our large study emphasizes the impact of both secondary chromosomal aberrations as well as gene mutations for outcome in AML with inv(16)/t (16;16).

Long-term survival and T-cell kinetics in relapsed/refractory ALL patients who achieved MRD response after blinatumomab treatment

This long-term follow-up analysis evaluated overall survival (OS) and relapse-free survival (RFS) in a phase 2 study of the bispecific T-cell engager antibody construct blinatumomab in 36 adults with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL). In the primary analysis, 25 (69%) patients with relapsed/refractory ALL achieved complete remission with full (CR) or partial (CRh) hematologic recovery of peripheral blood counts within the first 2 cycles. Twenty-five patients (69%) had a minimal residual disease (MRD) response (<10(-4) blasts), including 22 CR/CRh responders, 2 patients with hypocellular bone marrow, and 1 patient with normocellular bone marrow but low peripheral counts. Ten of the 36 patients (28%) were long-term survivors (OS ≥30 months). Median OS was 13.0 months (median follow-up, 32.6 months). MRD response was associated with significantly longer OS (Mantel-Byar P = .009). All 10 long-term survivors had an MRD response. Median RFS was 8.8 months (median follow-up, 28.9 months). A plateau for RFS was reached after ∼18 months. Six of the 10 long-term survivors remained relapse-free, including 4 who received allogeneic stem cell transplantation (allo-SCT) as consolidation for blinatumomab and 2 who received 3 additional cycles of blinatumomab instead of allo-SCT. Three long-term survivors had neurologic events or cytokine release syndrome, resulting in temporary blinatumomab discontinuation; all restarted blinatumomab successfully. Long-term survivors had more pronounced T-cell expansion than patients with OS <30 months.

Molecular heterogeneity of sporadic adult Burkitt-type leukemia/lymphoma as revealed by PCR and cytogenetics : correlation with morphology, immunology and clinical features

Chromosomal translocations involving the MYC oncogene are a hallmark of Burkitt lymphoma but they are only found in a varying frequency in mature Burkitt-type acute lymphoblastic leukemia (B-ALL). We have investigated samples of 56 sporadic Burkitt leukemia/lymphoma patients for the translocations t(8;14)(q24;q32), t(2;8)(p11;q24) and t(8;22)(q24;q11). Long PCR was used for detecting the immunoglobulin heavy chain (IgH) translocation and cytogenetics and/or fluorescence in situ hybridization for detecting the ‘variant’ MYC translocations. A total of 29 samples (51.8%) were t(8;14)-positive by long PCR. Approximately one-third had a chromosomal breakpoint in the IgH joining region while the others had breakpoints in the IgH switch regions. Among them were two cases with a previously unreported MYC translocation into the IgE switch region. Long PCR was more reliable compared to conventional cytogenetics for detecting the t(8;14). Epstein–Barr virus was detected in high copy number in two (3.6%) t(8;14)-positive cases by real-time quantitative PCR. Human herpesvirus 8 was not detected in any case by nested PCR. A typical L3 or L3-compatible cytomorphology was highly predictive (>80%) but not specific of a MYC translocation. A total of 34 patients were treated according to the GMALL B-ALL therapy protocols and there was no significant difference in overall survival between patients with or without t(8;14).

Immunocytology of plasmacytoid T cells: Marker analysis indicates a unique phenotype of this enigmatic cell

Clusters of plasmacytoid T cells (PTC) were detected in axillary lymph nodes draining an invasive ductal breast cancer in a 64-year-old woman. Immunocytology of PTC revealed a remarkable antigenic profile. Analysis with a broad spectrum of monoclonal antibodies demonstrated that PTC bear the CD4 surface antigen (Leu-3a+ and OKT4+), the transferrin receptor (OKT9+), and components of the HLA class-II antigens (TU35+, TU39+, Leu-10+). Surprisingly, PTC were stained by two monoclonal antibodies recognizing monocytes and macrophages (Ki-M6 and Ki-M7). Finally, Leu-8, which detects most mature T lymphocytes, also identified the PTC, and all pan T-cell markers (Leu-1, UCHT 1, and Lyt 3) were constantly negative. The cytogenesis and the functional properties of PTC remain a matter of discussion.

Valproic acid in combination with all-trans retinoic acid and intensive therapy for acute myeloid leukemia in older patients

The outcome of patients with acute myeloid leukemia who are older than 60 years has remained poor because of unfavorable disease characteristics and patient-related factors. The randomized German-Austrian AML Study Group 06-04 protocol was designed on the basis of in vitro synergistic effects of valproic acid (VPA) and all-trans retinoic acid with chemotherapy. Between 2004 and 2006, 186 patients were randomly assigned to receive 2 induction cycles with idarubicin, cytarabine, and all-trans retinoic acid either with VPA or without (STANDARD). In all patients, consolidation therapy was intended. Complete remission rates after induction tended to be lower in VPA compared with STANDARD (40% vs 52%; P = .14) as a result of a higher early death rate (26% vs 14%; P = .06). The main toxicities attributed to VPA were delayed hematologic recovery and grade 3/4 infections, observed predominantly during the second induction cycle. After restricting VPA to the first induction cycle and reducing the dose of idarubicin, these toxicities dropped to rates observed in STANDARD. After a median follow-up time of 84 months, event-free and overall survival were not different between the 2 groups (P = .95 and P = .57, respectively). However, relapse-free-survival was significantly superior in VPA compared with STANDARD (24.4% vs 6.4% at 5 years; P = .02). Explorative subset analyses revealed that AML with mutated Nucleophosmin 1 (NPM1) may particularly benefit from VPA. This trial was registered at www.clinicaltrials.gov as #NCT00151255.

ASXL1 mutations in younger adult patients with acute myeloid leukemia: a study by the German-Austrian Acute Myeloid Leukemia Study Group.

We studied 1696 patients (18 to 61 years) with acute myeloid leukemia for ASXL1 mutations and identified these mutations in 103 (6.1%) patients. ASXL1 mutations were associated with older age (P<0.0001), male sex (P=0.041), secondary acute myeloid leukemia (P<0.0001), and lower values for bone marrow (P<0.0001) and circulating (P<0.0001) blasts. ASXL1 mutations occurred in all cytogenetic risk-groups; normal karyotype (40%), other intermediate-risk cytogenetics (26%), high-risk (24%) and low-risk (10%) cytogenetics. ASXL1 mutations were associated with RUNX1 (P<0.0001) and IDH2R140 mutations (P=0.007), whereas there was an inverse correlation with NPM1 (P<0.0001), FLT3-ITD (P=0.0002), and DNMT3A (P=0.02) mutations. Patients with ASXL1 mutations had a lower complete remission rate (56% versus 74%; P=0.0002), and both inferior event-free survival (at 5 years: 15.9% versus 29.0%; P=0.02) and overall survival (at 5 years: 30.3% versus 45.7%; P=0.0004) compared to patients with wildtype ASXL1. In multivariable analyses, ASXL1 and RUNX1 mutation as a single variable did not have a significant impact on prognosis. However, we observed a significant interaction (P=0.04) for these mutations, in that patients with the genotype ASXL1mutated/RUNX1mutated had a higher risk of death (hazard ratio 1.8) compared to patients without this genotype. ASXL1 mutation, particularly in the context of a coexisting RUNX1 mutation, constitutes a strong adverse prognostic factor in acute myeloid leukemia.

Characterization and frequency distribution of lymphoreticular infiltrates in axillary lymph node metastases of invasive ductal carcinoma of the breast.

One hundred and seventy‐five axillary lymph nodes containing metastatic deposits from 46 invasive ductal carcinomas of the breast were evaluated histologically and immunohistologically. The study yielded the following results: (1) tumor‐infiltrating lymphoreticular cells preferentially accumulated in the stromal bands; the tumor foci generally showed a considerably lower degree of infiltration; (2) in most cases, monocytes/macrophages (Mono 1+) represented the overwhelming majority of tumor‐infiltrating cells; (3) next in frequency were T‐lymphocytes (Leu‐1+), especially CD4+ lymphocytes (Leu‐3a+), while CD8+ lymphocytes (Leu‐2a+) mostly occurred only in moderate numbers; (4) B‐lymphocytes (To15+), plasma cells, natural killer cells (Leu‐7+), tissue mast cells, and T‐accessory reticulum cells (OKT 6+) were observed mostly in low or very low numbers, while eosinophils were nearly absent and B‐accessory reticulum cells (Ki‐M4+) were totally absent from the lymphoreticular infiltrates. Definite conclusions regarding the functional properties of the tumor‐infiltrating cells cannot be drawn from an immunohistologic analysis in situ alone, but the preferred localization of most tumor‐infiltrating cells in the stroma does not support an intensive interaction between the host defenses and the metastatic tumor.