Optimal sequencing of ibrutinib, idelalisib, and venetoclax in chronic lymphocytic leukemia: results from a multicenter study of 683 patients.

Background: Ibrutinib, idelalisib, and venetoclax are approved for treating CLL patients in the United States. However, there is no guidance as to their optimal sequence. Patients and methods: We conducted a multicenter, retrospective analysis of CLL patients treated with kinase inhibitors (KIs) or venetoclax. We examined demographics, discontinuation reasons, overall response rates (ORR), survival, and post-KI salvage strategies. Primary endpoint was progression-free survival (PFS). Results: A total of 683 patients were identified. Baseline characteristics were similar in the ibrutinib and idelalisib groups. ORR to ibrutinib and idelalisib as first KI was 69% and 81%, respectively. With a median follow-up of 17 months (range 1-60), median PFS and OS for the entire cohort were 35 months and not reached. Patients treated with ibrutinib (versus idelalisib) as first KI had a significantly better PFS in all settings; front-line [hazard ratios (HR) 2.8, CI 1.3-6.3, P = 0.01], relapsed-refractory (HR 2.8, CI 1.9-4.1, P < 0.001), del17p (HR 2.0, CI 1.2-3.4, P = 0.008), and complex karyotype (HR 2.5, CI 1.2-5.2, P = 0.02). At the time of initial KI failure, use of an alternate KI or venetoclax had a superior PFS when compared with chemoimmunotherapy. Furthermore, patients who discontinued ibrutinib due to progression or toxicity had marginally improved outcomes if they received venetoclax (ORR 79%) versus idelalisib (ORR 46%) (PFS HR .6, CI.3-1.0, P = 0.06). Conclusions: In the largest real-world experience of novel agents in CLL, ibrutinib appears superior to idelalisib as first KI. Furthermore, in the setting of KI failure, alternate KI or venetoclax therapy appear superior to chemoimmunotherapy combinations. The use of venetoclax upon ibrutinib failure might be superior to idelalisib. These data support the need for trials testing sequencing strategies to optimize treatment algorithms.

PD-1(hi)TIM-3(+) T cells associate with and predict leukemia relapse in AML patients post allogeneic stem cell transplantation.

Prognosis of leukemia relapse post allogeneic stem cell transplantation (alloSCT) is poor and effective new treatments are urgently needed. T cells are pivotal in eradicating leukemia through a graft versus leukemia (GVL) effect and leukemia relapse is considered a failure of GVL. T-cell exhaustion is a state of T-cell dysfunction mediated by inhibitory molecules including programmed cell death protein 1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3). To evaluate whether T-cell exhaustion and inhibitory pathways are involved in leukemia relapse post alloSCT, we performed phenotypic and functional studies on T cells from peripheral blood of acute myeloid leukemia patients receiving alloSCT. Here we report that PD-1(hi)TIM-3(+) cells are strongly associated with leukemia relapse post transplantation. Consistent with exhaustion, PD-1(hi)TIM-3(+) T cells are functionally deficient manifested by reduced production of interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). In addition, these cells demonstrate a phenotype consistent with exhausted antigen-experienced T cells by losing TN and TEMRA subsets. Importantly, increase of PD-1(hi)TIM-3(+) cells occurs before clinical diagnosis of leukemia relapse, suggesting their predictive value. Results of our study provide an early diagnostic approach and a therapeutic target for leukemia relapse post transplantation.

BU and CY as conditioning regimen for autologous transplant in patients with multiple myeloma.

High-dose melphalan is considered the current standard of care among the preparative regimens used in peripheral blood autologous SCT (ASCT) for multiple myeloma (MM). We report the results of a single ASCT in 79 MM patients using the BU/CY conditioning regimen, with BU 1 mg/kg p.o. or 0.8 mg/kg i.v. every 6 h x 16 doses, and CY 60 mg/kg per day i.v. for 2 days. ASCT was carried out in first (62%) or subsequent remission/refractory disease (38%). For an overall RR of 86%, 48 and 20 patients achieved PR and CR, respectively. At a median follow-up of 41 months (range 2-132 months), the estimated median OS and PFS were 45 months (95% confidence interval (CI)=38-92) and 20 months (95% CI=15-25), respectively. The BU/CY regimen was well tolerated, and transplant-related mortality was 4%. Clinical outcomes of the BU/CY regimen are not superior to those obtained in historical controls with high-dose melphalan followed by a single ASCT. Therefore, considering even the greater complexity of administration of the BU/CY regimen compared with that of single-agent melphalan, we believe the latter should remain the conditioning regimen of choice for ASCT in MM.

Therapeutic potential of leukemia-derived dendritic cells: preclinical and clinical progress.

Human leukemia-derived dendritic cells show potential as tools for therapy. Leukemic cells of patients with chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and chronic myelomonocytic leukemia (CMML) will all undergo substantial differentiation toward dendritic cells (DC) and may be used to drive autologous T cells to acquire anti-leukemic cytotoxicity. This article describes the use of these human leukemia-derived dendritic cells for stimulation of allogeneic donor lymphocytes and presents a clinical trial of autologous CML DC-stimulated lymphocytes.

Apoptosis. Molecules and mechanisms.

This review of the molecules and pathways involved in programmed cell death (apoptosis) discriminates triggers of apoptosis (e.g. chemotherapy, radiation, Fas ligation), modulators of apoptosis (e.g. Bcl-2 family members, Bcl-2 interacting proteins, Apafs, IAPs, and Fas/FasL modulators including FLICE and FLIPs), effectors (caspases 1-13) and cleavage substrates (e.g. PARP). Special consideration is given to the structure-function relationship of Bcl-2 family members and to their post-transcriptional modification. Brief references are made to the role of apoptotic pathway in leukemias and lymphomas and to strategies of modulating apoptotic pathways.

Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses.

We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor-alpha or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.

Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: evidence for multiple critical loci.

Complete or partial deletions of the long arm of chromosome 7 (7q- and -7) are nonrandom abnormalities seen in primary and therapy-induced myelodysplasia (MDS) and acute myelogenous leukemia (AML). Monosomy 7, occurring as the sole cytogenetic anomaly in a small but significant number of cases, may denote a dominant mechanism involving critical tumor suppressor gene(s). We have determined the extent of allele loss in cytogenetically prescreened MDS and AML patients for microsatellite markers from chromosome 7q22 and 7q31. Whereas >80% of these cases revealed allele loss for the entire region, a rare case of the 7q- chromosome showed allele loss for only the proximal 7q31.1 loci flanked by the markers D7S486 and D7S2456, and a case of monosomy 7 revealed allele loss for loci at both 7q31 and 7q22 with retention of sequences between these sets of loci. Furthermore, a case of AML with no cytogenetic anomaly of chromosome 7 revealed a submicroscopic allelic imbalance for a third distal locus, D7S677. These findings suggest the presence of three distinct critical loci that may contribute alone or in combination to the evolution of MDS and AML. The data also provide molecular evidence for unbalanced translocation with noncontiguous deletions, as an alternate mechanism underlying monosomy 7.

Allogeneic peripheral-blood progenitor-cell transplantation for poor-risk patients with metastatic breast cancer.

PURPOSE: To evaluate the feasibility of allogeneic peripheral-blood progenitor-cell (PBPC) transplantation and to assess graft-versus-tumor effects in patients with metastatic breast cancer. PATIENTS AND METHODS: Ten patients with metastatic breast cancer that involved the liver or bone marrow were treated with high-dose chemotherapy and allogeneic PBPC transplantation. The median age was 42 years (range, 29 to 55). The median number of metastatic sites was three (range, one to five). The conditioning regimen was cyclophosphamide (6,000 mg/m2), carmustine (BCNU; 450 mg/m2), and thiotepa (720 mg/m2) (CBT regimen). Patients received graft-versus-host disease (GVHD) prophylaxis using cyclosporine- or tacrolimus-based regimens. RESULTS: All patients had engraftment and hematologic recovery. Three patients developed grade > or = 2 acute GVHD and four patients had chronic GVHD. After transplantation, one patient was in complete remission (CR), five achieved a partial remission (PR), and four had stable disease (SD). In two patients, metastatic liver lesions regressed in association with skin GVHD after withdrawal of immunosuppressive therapies. The median follow-up time was 408 days (range, 53 to 605). The median progression-free survival duration was 238 days (range, 53 to 510). CONCLUSION: We conclude that allogeneic PBPC transplantation is a feasible procedure for patients with poor-risk metastatic breast cancer. The regression of tumor associated with GVHD provides suggestive clinical evidence that graft-versus-tumor effects may occur against breast cancer. Compared with autologous transplantation, allogeneic PBPC transplantation is associated with the additional risks of GVHD and related infections. Allogeneic transplantation should only be performed in the context of clinical trials and its ultimate role requires demonstration of improved progression-free survival.

Human leukemia-derived dendritic cells: ex-vivo development of specific antileukemic cytotoxicity.

The human myeloid leukemias are a diverse group of disorders characterized by massive clonal expansion of myeloid cells showing variable degrees of differentiation block. Leukemic dendritic cells were generated in culture from chronic myelogenous leukemia (CML). These were used to stimulate autologous T cells to develop leukemia-specific cytotoxicity. Available data suggest that the cells responsible for the cytolytic activity are at least in part CD8+ and HLA restricted in their function. Additional data suggest that some anti-CML cellular activity may be Fas mediated. T-cell receptor studies provide evidence for an oligoclonal response implying a recognition of a limited number of antigens. We have used culture techniques similar to those used for CML to study the ability of AML cells to differentiate toward dendritic cells. Four of five patients have shown acute leukemia-derived dendritic cells. This work offers an avenue for the development of novel strategies for the control of human myeloid leukemias.

CBF beta-SMMHC, expressed in M4Eo AML, reduced CBF DNA-binding and inhibited the G1 to S cell cycle transition at the restriction point in myeloid and lymphoid cells.

CBF beta-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBF beta-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBF beta-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBF beta-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBF beta. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBF beta-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBF beta-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBF beta-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor alpha or beta subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBF beta-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBF beta-SMMHC by allowing its increased expression.

Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia.

The success of adoptive immunotherapy for the treatment of leukemia depends on the generation of T cells that can specifically react with malignant cells. Dendritic cells (DCs) are important antigen-presenting cells in the development of antileukemic T-cell responses. In this study, we generated DCs from peripheral blood cells of patients with chronic myelogenous leukemia (CML). CML cells incubated concurrently with granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha in vitro developed morphologic and phenotypic characteristics of DCs. Fluorescence in situ hybridization showed the presence of t(9;22) in the nuclei of these cells, indicating that they were leukemic in origin. These cells were potent stimulators of lymphocyte proliferation in specific in vitro assays for DC function. Autologous T cells stimulated with in vitro-generated, leukemic DCs displayed vigorous cytotoxic activity against CML cells but low reactivity to major histocompatability complex-matched normal bone marrow cells. Cytotoxic activity against CML targets was fourfold to sixfold higher using DC-stimulated autologous T cells than with autologous T cells expanded by culture with interleukin-2 alone. DC-stimulated T cells also inhibited growth of CML clonogenic precursors in colony-forming assays in vitro. These results suggest that cytokine-driven in vitro differentiation of CML cells results in generation of DCs with potent T-cell stimulatory function. In vitro-generated DCs can be effectively used as antigen-presenting cells for the ex vivo expansion of antileukemic T cells.

The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.

The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1/MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the alpha subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the beta subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AML1 (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression prevented tumor growth. These results suggest that expression of AML1/EAP and AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line.

PML/RARalpha, a fusion protein in acute promyelocytic leukemia, prevents growth factor withdrawal-induced apoptosis in TF-1 cells.

A unique mRNA produced by the t(15;17) (q22-24;q11-21) translocation in the leukemic cells of acute promyelocytic leukemia patients encodes a chimeric protein, PML/RARalpha, which is formed by the fusion of the retinoic acid receptor alpha (RARalpha) and the promyelocytic locus gene (PML). This translocation is often the only visible karyotypic aberration present which is detected in almost 100% of acute promyelocytic leukemia patients. As an initial step to study the role of PML/RARalpha in leukemogenesis, we attempted to express the fusion protein in hematopoietic cells through retrovirus-mediated gene transfer of the retroviral vector, pGPRCHT, which contains the PML/RARalpha cDNA. Transduction of the PML/RARalpha cDNA fragment used in this vector, which extends from the position 31 bp to the position 2638 bp in a transcription unit driven by the Moloney murine sarcoma virus LTR, was found to abrogate the growth factor dependence of TF-1 cells. In addition, introduction of PML/RARalpha into TF-1 cells can protect these cells from the apoptosis usually induced in TF-1 cells by growth factor withdrawal, as measured by three assays for apoptosis: morphology, DNA ladder formation, and end labeling of nicked DNA with fluorescent-conjugated nucleotide precursors followed by a fluorescence-activated cell sorting assay. These data suggest that the PML/RARalpha fusion protein may inhibit programmed cell death in myeloid cells.

Molecular characterization of 16p deletions associated with inversion 16 defines the critical fusion for leukemogenesis.

The inversion of chromosome 16 [inv(16)] in acute myeloid leukemia (AML) is associated with a p-arm deletion in a subset of patients. The inversion results in two fusion genes: 5'-CBFB/MYH11-3' on 16p and 5'-MYH11/CBFB-3' on 16q. We have studied cells from 42 patients with inv(16) (38 patients) or t(16;16) (four patients) to define the frequency and characteristics of the deletion further. Using fluorescence in situ hybridization (FISH) with probes from cosmids, cosmid contigs, and yeast artificial chromosomes (YACs), we found that six patients with inv(16) had a deletion of between 150 and 350 kb centromeric to the p-arm inversion breakpoint cluster region (p-ibc). This region was shown to contain the 5' portion of the myosin heavy chain (MYH11) gene. YACs containing the p-ibc, which had been useful as FISH probes in the diagnosis of inv(16), detected the inversion in deletion as well as nondeletion patient cells. Thus, the deleted region identified in patients is entirely contained within the human genomic content of the YACs. Southern blot experiments using probes flanking the p-ibc indicated that the deletion removes segments within 10 kb centromeric of the p-ibc. Reverse transcription-polymerase chain reaction (RT-PCR) using primers from the 5' region of CBFB and the 3' region of MYH11 (distal to the p-ibc) produced the 5'-CBFB/MYH11-3' chimeric transcript in inv(16)/del patients. These data confirm that the 5'-CBFB/MYH11-3' chimeric transcript, rather than the reciprocal 5'-MYH11/CBFB-3', is the critical product for chromosome 16-related leukemogenesis.

Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia.

Pericentric inversion of chromosome 16 [inv(16)(p13q22)] and the related t(16;16)(p13;q22) are seen in a subset of acute myelogenous leukemia (AML) phenotypically and prognostically differing from other cases. We have recently shown that inv(16) results in fusion of CBFB/PEBP2B, a gene encoded at 16q22 to MYH11, a smooth muscle myosin heavy chain gene encoded at 16p13. Chimeric transcripts consisting of upstream CBFB fused to downstream MYH11 coding sequences result from this fusion. In this study we have examined a series of 37 of these cases using reverse transcriptase-polymerase chain reaction (RT-PCR) to detect expression of a hybrid CBFB/MYH11 transcript. Chimeric cDNAs were detected in all but 1 of 37 leukemias with typical inv(16) or t(16;16). Such chimeric products were not seen in a case with inv(16)(p13q24) (ie, a variant q arm breakpoint) or any of 10 cases of AML without these chromosomal changes. Four different chimeric transcripts were found, representing differing fusion points within MYH11 spliced to position 495 of CBFB. Primer sets are described for efficient amplification of these different cDNA forms. Amplification of cDNA showed that all but 17 codons of the CBFB coding sequence are included in the abnormal transcripts. RT-PCR was shown to be highly sensitive and potentially useful for detection of leukemic cells during morphologic remission.

Smooth muscle myosin heavy chain locus (MYH11) maps to 16p13.13-p13.12 and establishes a new region of conserved synteny between human 16p and mouse 16.

The human smooth muscle myosin heavy chain locus (MYH11) was mapped by fluorescence in situ hybridization to the middle of the p arm of chromosome 16 using a genomic cosmid clone containing coding sequences of the gene as probe. Probe from coding sequence, when applied to Southern blots of a panel of hybrids containing different portions of human chromosome 16, localized the gene to 16p13.13-13.12. Coding sequence PCR primers, when used on the DNA from a CHO-mouse hybrid clone mapping panel informative for mouse chromosomes, showed that the gene was located on mouse chromosome 16. These results correct a recent assignment of MYH11 from 16q12.2 to the region of the 16p-arm inversion breakpoint seen in acute myelomonocytic leukemia (AMML) M4Eo and demonstrate that the conflicting data do not result from the presence of additional MYH genes on the q arm of the chromosome. Also, a new region of conserved synteny between human 16p and mouse 16 is established.

Identification of yeast artificial chromosomes containing the inversion 16 p-arm breakpoint associated with acute myelomonocytic leukemia.

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.