Novel Antibodies Homing in on New Targets for AML, MDS

Patients with acute myeloid leukemia (AML) who have long-term disease-free survival following an allogeneic stem cell transplant (allo-SCT) may benefit from a robust immune response involving cytotoxic antibodies directed to a previously unsuspected cellular target, investigators from the Netherlands report.

The presence of these and other antibodies to newly identified cellular targets suggests new therapeutic strategies for treatment of AML and, potentially, for myelodysplastic syndromes (MDS), according to Mette D. Hazenberg, MD, PhD, from the Academic Medical Center in Amsterdam.

“What we learned from nature, from studying patients who through an allogeneic stem cell transplantation cured their leukemia, is that these patients make leukemia-specific antibodies to really unexpected targets,” she said in a report at the 2016 European Hematology Association (EHA) Congress in Copenhagen, Denmark.

By isolating and cloning anti-AML antibodies from long-term post-transplant survivors, Hazenberg and colleagues have identified antibodies that target small nuclear ribonucleoprotein U5 subunit 200 (U5 snRNP200). This large (250 kilodalton) protein is one of five major components of the spliceosome complex normally found within the nucleus of human and other eukaryotic cells, but which, as the team discovered, appears to be expressed on the cellular membrane of AML.

Long-term Survivors

“It has been shown that allogeneic stem cell transplants can mount immune responses, and we wondered whether antibodies could be involved in this,” Hazenberg said.

She and her colleagues selected three patients with high-risk leukemia who were alive for at least 5 years with no evidence of disease following an allo-SCT, whose longevity indicated a strong graft-versus-leukemia response. The investigators identified and isolated antibody-generating memory B cells from peripheral blood and cultured them in medium, screening the supernatant of the cultures and searching for antibodies that bind to AML.

The team then cloned the AML-binding B cells and identified 17 candidate monoclonal antibodies that bind to AML in cell lines, but do not bind to peripheral blood monocytes, fibroblasts, or other healthy cells. Of the 17 antibodies, seven recognized the same target: U5 snRNP200.

“We screened a few more patients, and found that four out of five patients had mounted such antibodies, so apparently that is a target that is often recognized by the immune system of the donor,” Hazenberg said. “The second striking observation was that these antibodies, when they interact with this protein on the cell membrane, actually kill the leukemic cells.”

Non-Apoptotic Process

The direct killing effect was seen both in AML cells in vitro, and in a human AML mouse model in vivo. The cell death occurred despite the absence of either cytotoxic leukocytes or of complement. Instead, the antibodies appear to induce AML death through a non-apoptotic process that relies on destabilization of the cytoskeleton. The nature of the cell death mechanism was supported by further experiments showing that AML cell death could be blocked when target cells were treated with cytochalasin D, an inhibitor of actin polymerization.

Furthermore, observation that the anti-U5 snRNP200 antibodies retained their cytotoxic abilities at both 4° and 37° C suggests that the cell death is induced through a passive process. This observation was further supported by the fact that the interaction of the antibodies with their target cells did induce calcium flux, the investigators noted.

 “Now with immunotherapy these days, investigators decide which target should be attacked by the immune system, and we try to help the immune system by making antibody drug conjugates, for example, but here we let nature decide what the target would be, and the target was, amongst others, this 200 subunit complex,” Hazenberg explained.

“And interestingly, when these antibodies bind to this complex they actually kill the target cell; they kill the leukemic cells. This is a novel phenomenon — we didn’t know this before — and we think we can develop this further into novel therapies.”

In a separate presentation at the EHA meeting, Hazenberg and colleagues reported on a second, novel tumor-specific target expressed on both AML and MDS blasts. The team first identified an immunoglobulin G1 antibody, labeled AT14-013, from the memory B lymphocytes of a patient with a robust graft-versus-leukemia response. This antibody homed in on a sialylated epitope of CD43 that is both uniquely and widely expressed on all types of AML.

Hazenberg and colleagues assert that antibodies targeted against onco-sialylated CD43 and U5 snRNP200 have significant potential as novel therapies for AML and MDS, either as “naked” antibodies or in combination in an antibody-drug conjugate, bispecific T-cell engager, or chimeric antigen receptor T cell (CAR-T) construct.

AML: Conjugate Produces High Remission Rates in Older Patients

Early promising results for antibody-drug conjugate delivered with hypomethylating agents.

Older adults who are newly diagnosed with acute myeloid leukemia (AML) are often not sufficiently fit to withstand the rigors of remission induction therapy with cytarabine and an anthracycline such as daunorubicin or idarubicin. Other patients may decline to have intensive therapy due to frailty or concerns about toxicities. For these patients, clinicians in the United States often prescribe lower-intensity therapy with the hypomethylating agents decitabine and/or azacitdine, but these agents are both associated with low response rates and limited clinical benefits, according to treatment information from theNational Cancer Institute.

However, an investigational therapy consisting of a conjugated monoclonal antibody combined with hypomethylating agents (HMAs) has been shown in early clinical trials to induce high complete or near-complete remission rates in older adults with AML.

At the 2016 annual congress of the European Hematology Association, Amir T. Fathi, MD, from Massachusetts General Hospital Cancer Center in Boston, reported data from a phase I study of older adults with AML who were treated with a combination of the monoclonal antibody drug conjugate vadastuximab talirine (33A; Seattle Genetics) and either azacitdine or decitabine.

Among 49 patients evaluable for efficacy, the combined rate of complete remissions (CR) or CR with incomplete recovery of counts (CRi) was 71%.

“The high remission rate in this traditionally high-risk group and difficult-to-treat population is very compelling,” Fathi said. “Response rates were higher, and were achieved more quickly than would be expected from historical data associated with HMA therapy alone.”

Target: CD33

33A is a highly potent antibody-drug conjugate designed to deliver a cytotoxic agent to myeloid leukemia cells. The agent is targeted to CD33 receptors that are expressed on leukemic blasts in nearly all cases of AML. The antibody is conjugated to two molecules of a pyrrolobenzodiazepine (PBD) dimer. Upon binding to the receptors, the conjugate is internalized and transported to cellular lysosomes where the PBD dimer is released via proteolytic cleavage of the linker, resulting in a crosslinking of DNA, and leading to cell death.

The cell-killing activity of this agent had been shown in preclinical studies to be enhanced when delivered in combination with hypomethylating therapy, Fathi noted.

For the phase I trial, the combination of 33A and a hypomethylating agent was tested in 53 adults with a median age of 75. The patients all had CD33-positive AML, and all had declined to undergo an intensive chemotherapy induction regimen. Five patients had previously received low-intensity therapy for myelodysplastic syndromes, and the remaining 48 patients had not received any prior therapy for AML.

Enrollment criteria included an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1. Nineteen of the patients had adverse cytogenetic-risk disease, and 30 had intermediate-risk disease.

The patients received 33A in intravenous infusions of 10 mcg/kg delivered in an outpatient setting every 4 weeks on the last day of a hypomethylating therapy regimen — either azacitidine at 75 mg/m2 for 7 days, or decitabine at 20 mg/m2 for 5 days. Patients who had clinical benefit could be continued on treatment until disease relapse or unacceptable toxicity.

Responses were assessed by investigators according to the International Working Groupfor Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia (IWG). CRi was defined as either a platelet count of ≥100,000/µL or neutrophils of ≥1,000/µL.

The combined CR/CRi rate among the 49 patients evaluable for response at the time of data cutoff was 71%, with no difference in the rate of complete or near-complete remissions between patients treated with azacitidine or decitabine.

The overall response rate (CR, CRi, and partial responses) was 76%. Encouragingly, Fathi said, many higher-risk patients had responses, including 15 of 18 patients with adverse cytogenetics, and 16 of 22 with underlying myelodysplasia.

Eight of the 19 patients who had a CR met the criteria for minimal residual disease, as did 5 of 15 who achieved a CRi.

The overall survival results were ongoing at the time of the presentation. After a median follow-up of 12.58 months, the estimated median overall survival for the first 25 patients enrolled in the study was 12.75 months; and as of the most recent follow-up, 27 were alive and remained on study.

The median relapse-free survival was 7.7 months; 30- and 60-day mortality rates were 2% and 8%, respectively. There were no treatment-related deaths reported.

Safety Profile

Patients generally tolerated the therapy well, Fathi said. Grade 3 or greater adverse events reported in at least 20% of patients included, in order of frequency, febrile neutropenia (47% of patients), thrombocytopenia (42%), anemia (34%), and neutropenia (28%).

Other common treatment-emergent adverse events were fatigue, nausea, constipation, decreased appetite, and peripheral edema.

Fathi noted that a phase III trial to evaluate 33A in combination with hypomethylating agents in previously untreated older AML patients is now open for enrollment.

Myeloma Patients at Risk for AML, Other Second Primaries

Patients with multiple myeloma are living longer than ever before, thanks to major advances in treatment. However, an unintended and unwelcome consequence of longer survival for these patients, investigators say, is a steady rise in the incidence of second primary cancers.

Since the 1960s, oncologists have been aware that alkylating agents such as melphalan, commonly used in high-dose regimens for treatment and pre-transplant conditioning of patients with multiple myeloma, are associated with increased risk for treatment-related leukemia.

Now, drawing on comprehensive European cancer registries, investigators in Sweden, Germany, and the United States have found that patients with multiple myeloma have an approximately two- to five-fold higher risk for developing acute myeloid leukemia (AML), as well as elevated risks for kidney cancer and nervous system cancers compared with the general population.

“Although their exact underlying biologic mechanisms have not been well characterized, treatment-related factors may contribute in addition to inherited genetic predisposition and shared non-genetic factors,” wrote Tianhui Chen, MD, PhD, from the German Cancer Research Centre in Heidelberg, Germany, in Scientific Reports.

The investigators, which included the GEKID Cancer Survival Working Group, looked at data on patients age 15 and older at the time of a multiple myeloma diagnosis. Chen et al identified survivors enrolled in Sweden’s nationwide cancer registry and 12 German cancer registries. For each cohort, standardized incidence ratios (SIRs) were calculated to estimate the relative risks that patients would develop specific second primary malignancies compared with the general population in their region.

In all, 752 of the 18,735 myeloma survivors in the German registries developed a second primary cancer, as did 349 of 7,560 survivors in the Swedish registry. In each country, prostate cancer was the most common second primary cancer, followed by colorectal cancer. Except for a higher frequency of stomach cancer in Germany than in Sweden, and more frequent nervous system cancers in Sweden than in Germany, the rankings of various cancers were generally similar, the authors found.

When they looked at the risks for specific malignancies both in the overall population and by period of diagnosis, they found that in Germany, the only significantly elevated SIR in the overall population was for leukemia (SIR 1.7, 95% CI, 1.2-2.4).

In Sweden, significantly higher SIRs were seen for all second primary cancers combined (SIR 1.3, 95% CI, 1.2-1.4); kidney cancer (SIR 2.3, 95% CI, 1.3-3.7); nervous system cancers (SIR 1.9, 95% CI, 1.1-3.1); and leukemia (SIR 1.6, 95% CI, 1.0-2.4).

The investigators noted that the increased SIRs for leukemia were accounted for by AML: In Germany, the SIR for AML was 4.9, 95% CI, 3.2-7.3, and the SIR in Sweden was 2.3, 95% CI, 1.2-4.0).

The researchers also found that the elevated risk for AML was greater for earlier periods of diagnosis, suggesting a possible effect of changes in treatment practice. For example, in Germany the SIR for AML for patients diagnosed with multiple myeloma from 1997 through 2003 was 9.7 (95% CI, 4.2-19), compared with 3.5 (95% CI, 1.5-6.9) for those diagnosed from 2004 through 2010. The respective SIRs in Sweden by time period were 3.8 (95% CI, 1.4-8.3) compared with 2.2 (95% CI, 0.3-7.8).

Alkylators Suspected

Patient exposure to alkylating agents such melphalan and cyclophosphamide, either with or without immunomodulators such as thalidomide, were likely to have been more common in the earlier diagnosis periods, prior to the advent of proteasome inhibitors and other therapies, which could explain the higher risk for AML, the investigators speculated.

“Therefore, these patients might have been exposed to high doses of alkylating agents in the earlier years of diagnosis. However, after the dawn of the new millennium, alkylating agents might not have been used as intensively and frequently as in earlier diagnosis periods because of the introduction of the novel agents. In the later periods, survival from multiple myeloma diagnosis improved markedly, and long-term survivors from both diagnosis periods accumulated. Therefore, AML as a long-term side effect from alkylating agents, as described from other cancer entities such as testicular and ovarian cancers, was more likely to evolve on long-term follow-up.”

Looking at the issue from another angle, Lindsay M. Morton PhD, from the Division of Cancer Epidemiology and Genetics at the National Cancer Institute in Bethesda, Md., and colleagues reported at the 2015 annual meeting of the American Society of Hematologythat among 746,007 adults with various cancers who were initially treated with chemotherapy from 2000 through 2012 and survived for at least 1 year after diagnosis, the SIR for treatment-related AML or myelodysplastic syndromes (MDS) was 4.1 (95% CI, 3.9-4.2), and that the risk for treatment-related AML/MDS for patients diagnosed with multiple myeloma was six-fold compared with general population (SIR 6.3, 95% CI. 5.1-7.6).

Normal enzyme aids a mutant one to fuel blood cancer’s growth.

The combination of these enzymes also linked to resistance to treatment

Reinforcing the need to look beyond genomic alterations to understand the complexity of cancer, researchers from Dana-Farber/Boston Children’s Cancer and Blood Disorders Center report that a normal enzyme called SYK pairs with FLT3, the most commonly mutated enzyme found in acute myelogenous leukemia (AML), to promote progression of the disease. This molecular partnership also promotes AML cells’ resistance to treatment with FLT3-blocking drugs, potentially explaining the relatively poor showing of FLT3 inhibitors in multiple clinical studies. In an animal model of AML, treatment with a combination of FLT3- and SYK-inhibiting drugs was significantly more effective than treatment with either drug alone.

The findings, published Feb. 10 in the journal Cancer Cell, raise hopes that treatment strategies that focus on both enzymes simultaneously could help bring molecularly targeted treatments to AML, a common blood cancer. The study also may have broader implications for how clinicians approach the development of such treatments and understand mechanisms behind resistance to treatment in other cancers.

Approximately 14,600 Americans are expected to be diagnosed with AML this year; children account for about 18 percent of patients. The overall outlook for AML patients is mixed; while the majority of patients with AML achieve remission with treatment, many relapse. The cancer cells of some 20 percent of adult and 15 percent of childhood AML patients harbor a genomic alteration called FLT3-ITD, in which segments of the FLT3 enzyme are duplicated again and again, making the enzyme overactive.

“Patients whose AML cells express FLT3-ITD are among the highest risk group of patients with AML,” says the study’s senior author, Kimberly Stegmaier, MD, of Dana-Farber/Boston Children’s Hematologic Malignancies Center. “Their AML is particularly difficult to treat.”

FLT3 is a kinase, a molecular switch that routs signals for growth, division and other processes within cells. Many cancers harbor mutations or other alterations that leave kinases stuck in the “on” position. This knowledge laid the foundation for the targeted cancer treatment revolution inaugurated by imatinib (Gleevec®), which has made another blood cancer, chronic myelogenous leukemia, a controllable, chronic disease for many patients.

However, patients with AML have not yet benefitted from that revolution. Researchers and pharmaceutical companies are actively working on FLT3 inhibitors, but to date these efforts have been hampered by low efficacy and concerns about drug resistance.

In 2009, Stegmaier’s laboratory discovered that SYK, a kinase that had attracted attention for its role in other malignancies, could be a potential drug target in AML. Unlike other cancer-associated kinases, SYK rarely undergoes mutations or other genomic alterations in cancer cells, remaining in its normal or “wild-type” form.

To better understand SYK’s role in AML, in the current study, Stegmaier, lead author Alexandre Puissant, PhD, of Dana-Farber/Boston Children’s, and their collaborators screened AML cell lines to reveal the full scope of the enzyme’s molecular interactions. They found evidence of strong interactions between wild-type SYK and mutated FLT3, in particular FLT3-ITD.

“We wanted to understand the cooperative oncologic effects by which SYK contributes to AML,” Stegmaier explains. “The concept of a normal enzyme aiding a mutant one has not yet been widely explored, and so we were both surprised and pleased to see FLT3-ITD come up as a high-priority hit in our screens.”

Through experiments in cell lines, primary patient samples and animal models, the research team found that SYK and FLT3-ITD’s interactions are a key ingredient in the progression of myeloproliferative disorder, a related blood cell disorder, into AML. AML cells’ continued growth after turning malignant also relied on these interactions.

Additionally, the team found that SYK’s hyperactivated form can promote resistance to the FLT3-targeting drug quizartinib (AC220, Ambit Biosciences). They could overcome this resistance with a combination of quizartinib and the SYK-blocking molecule PRT062607 (Portola Pharmaceuticals), significantly increasing survival and reducing signs of disease in an FLT3-ITD AML mouse model.

Highlighting their findings’ clinical relevance, the researchers found strong SYK activity in cells from FLT3-ITD AML patients. The cells were also highly sensitive to SYK inhibition.

“These data affirm that SYK is an important target in AML,” Stegmaier states. “They also suggest that interactions between oncologic kinases and SYK or other wild type enzymes may contribute to resistance of kinase inhibitors more broadly.”

Stegmaier notes that over the course of this research, the team has developed a suite of tools that could prove invaluable for future clinical studies of treatments with SYK inhibitors or SYK inhibitors in combination with FLT3 inhibitors.

“We have not only identified SYK as a candidate treatment target in AML, but we have also identified a specific population of patients with AML more likely to respond to SYK inhibitors: patients with FLT3 mutations,” she says. “Moreover, we have developed tools for identifying patients with high levels of SYK and FLT3 activation and can monitor these two targets while patients are receiving treatment. Predictive biomarkers of response are becoming increasingly important in the development of effective clinical trials of targeted therapies.”

The study was supported by the National Cancer Institute (grant number R01CA140292), the American Cancer Society, the Starr Cancer Consortium, Project Cupid and One Mission, the Swedish Research Council and Swedish Cancer Society and the Leukemia and Lymphoma Society.

Treatment of polycythemia vera with hydroxyurea and pipobroman: final results of a randomized trial initiated in 1980.

The overall impact of hydroxyurea (HU) or pipobroman treatments on the long-term outcome of patients with polycythemia vera (PV) has not been assessed in randomized studies. We report final analyses from the French Polycythemia Study Group (FPSG) study, which randomly assigned HU versus pipobroman as first-line therapy in 285 patients younger than age 65 years.
PATIENTS AND METHODS: The full methodology has been described previously. FPSG results were updated with a median follow-up of 16.3 years. Statistical analysis was performed by using competing risks on the intention-to-treat population and according to main treatment received.
RESULTS: Median survival was 17 years for the whole cohort, 20.3 years for the HU arm, and 15.4 years for the pipobroman arm (P = .008) and differed significantly from that in the general population. At 10, 15, and 20 years, cumulative incidence of acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) was 6.6%, 16.5%, and 24% in the HU arm and 13%, 34%, and 52% in the pipobroman arm (P = .004). Cumulative myelofibrosis incidence at 10, 15, and 20 years according to main treatment received was 15%, 24%, and 32% with HU versus 5%, 10%, and 21% with pipobroman (P = .02).
CONCLUSION: Data from this unique randomized trial comparing HU with another cytoreductive drug in PV showed that (1) survival of patients with PV treated with conventional agents differed from survival in the general population, (2) evolution to AML/MDS is the first cause of death, (3) pipobroman is leukemogenic and is unsuitable for first-line therapy, and (4) incidence of evolution to AML/MDS with HU is higher than previously reported, although consideration should be given to the natural evolution of PV.