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Monday, April 25, 2016

By Meeri Kim, PhD
Allogeneic hematopoietic stem cell transplantation, while proven as an effective therapy for a number of cancers and other diseases, remains limited by high morbidity and mortality after the procedure. Although patient outcomes have improved over the past few decades, possible adverse effects still include graft-versus-host disease, infection, and relapse.

A new retrospective study has found the immune cell content of granulocyte colony-stimulating factor-mobilized peripheral blood stem cell grafts may have an effect on how well transplants are tolerated by patients. The analysis indicated a higher dose of invariant natural killer T (iNKT) cells is associated with an improved outcome in terms of graft-versus-host disease and relapse/progression.

Allogeneic stem cell transplantation from an HLA-matched or unrelated donor was performed in 80 patients whose diagnoses included both myeloid and lymphoid malignancies. The researchers identified subsets of immune cells in each graft to determine any possible influence in terms of patient outcomes. A larger number of donor iNKT cells in the graft, while having no impact on overall survival, did correlate with improved graft-versus-host disease-free, progression-free survival.

The study, published online by the journal Blood in February, suggests new interventions could be developed to manipulate the graft content to up the dose of iNKT cells and lower the risk of complications related to allogenic stem cell transplantation.

"Our observation is quite strong and could pave the way for future developments. For instance, you can start tailoring the graft that you are going to infuse to the patient, which we know from the technological point of view is feasible," said study author Mohamad Mohty, MD, PhD, Professor of Hematology and Head of the Hematology and Cellular Therapy Department at the Saint-Antoine Hospital and University Pierre & Marie Curie. "If these iNKT cells have a positive impact, then why not tailor the graft to make it richer in terms of this substance?"

Continued Study

Although researchers have begun to scratch the surface, much still remains unknown about how much immune cell subsets matter in terms of allogenic hematopoietic stem cell transplantation. Also, most studies tend to focus on a specific immune cell population rather than a large collection of immune subpopulations. For instance, one recent publication found higher graft CD8 dose predicts better survival in patients undergoing reduced-intensity conditioned transplant.

"This is quite a long ago idea I had, along with many investigators in this field, that the content of the graft that we infuse to patients should be able to modulate or at least have some significant influence on the outcome of the patient—whether that be disease relapse, survival, or complications," said Mohty.

In 2004, Mohty and his colleagues discovered that a greater dose of CD8+ T cells in the allograft was associated with a higher likelihood of developing acute graft-versus-host disease in 57 patients who received allogenic peripheral blood stem cells from HLA-identical siblings. As an updated version of that study, they decided to use today's advanced technology to look deeper into the content of allografts and build upon that previous work.

The study used frozen aliquots of peripheral blood stem cell grafts from 80 patients who underwent allogeneic transplantation at the University Hospital of Nantes in France between 2010 and 2013. Their diagnoses included acute myeloid leukemia (n = 33), non-Hodgkin lymphoma (n = 17), acute lymphoblastic leukemia (n = 8), myelodysplastic syndrome (n = 8), and other myeloid or lymphoid malignancies.

A wide range of immune cells were analyzed by either flow cytometry or intranuclear staining techniques: naïve and memory T- cell subsets, B cells, regulatory T cells, iNKT cells, natural killer cells, and dendritic cell subsets.

For a median follow-up of 37 months among surviving patients, two-year overall survival was 58 percent. However, none of the cell subsets—including iNKT cells—had a significant association with this endpoint. The researchers also calculated another primary endpoint called graft-versus-host disease-free and progression-free survival (GPFS), defined as survival with no evidence of relapse/progression, grade III-IV acute graft-versus-host disease, and systemic therapy-requiring chronic graft-versus-host disease. The two-year GPFS was significantly higher for patients who received greater than the median number of iNKT cells (49%) versus those who did not (22%).

"To go away from the traditional cells, we analyzed cell subsets that had not been very much looked at in the literature," he said. "We were very keen on including iNKT cells because this is a very rare subset of cells which is not very easy to identify and quantify, but there was some strong scientific rationale that they would have an effect on patient outcome."

Previous research had suggested iNKT cells could play a role in reducing the incidence of graft-versus-host disease or even the incidence of immunological conflict between the donor and recipient. Because these can initiate complications after transplant, Mohty hopes his findings on iNKT cell dose could reduce adverse effects without favoring disease relapse.

After additional confirmatory studies, possible interventions that would harness these findings include tailoring the graft to include a higher dose of iNKT cells, or giving a pill after transplant that would spur an in vitro proliferation and expansion of iNKT cells.

Monday, April 25, 2016

Researchers at The University of Texas MD Anderson Cancer Center, Houston, announced a new method for detecting DNA mutations in a single cancer cell versus current technology that analyzes millions of cells which they believe could have important applications for cancer diagnosis and treatment. The results are published in the April 18 online issue of Nature Methods.

Existing technology, known as next-generation sequencing (NGS), measures genomes derived from millions of cells versus the newer method for single-cell sequencing, called Monovar. Developed by MD Anderson researchers, Monovar allows scientists to examine data from multiple single cells. The study was, in part, funded by MD Anderson's Moon Shots Program.

"NGS technologies have vastly improved our understanding of the human genome and its variation in diseases such as cancer," said Ken Chen, PhD, Assistant Professor of Bioinformatics and Computational Biology and article co-author. "However, because NGS measures large numbers of cells, genomic variations within tissue samples are often masked."

This led to development of newer technology, called single cell sequencing (SCS), that has had a major impact in many areas of biology, including cancer research, neurobiology, microbiology, and immunology, and has greatly improved understanding of certain tumor characteristics in cancer.  Monovar improves further on the new SCS's computational tools which scientists found "lacking" by more accurately detecting slight alterations in DNA makeup known as single nucleotide variants (SNVs).

"To improve the SNVs in SCS datasets, we developed Monovar," said Nicholas Navin, PhD, Assistant Professor of Genetics and co-author of the paper. "Monovar is a novel statistical method able to leverage data from multiple single cells to discover SNVs and provides highly detailed genetic data."

Chen and Navin state that Monovar will have significant translational applications in cancer diagnosis and treatment, personalized medicine and prenatal genetic diagnosis, where the accurate detection of SNVs is critical for patient care.

This refinement of an existing technology could very well boost studies in many biomedical fields other than just cancer. The researchers believe it is a major advance for assessing SNVs in SCS datasets—crucial information for a variety of diseases.

"With the recent innovations in SCS methods to analyze thousands of single cells in parallel with RNA analysis which will soon be extended to DNA analysis, the need for accurate DNA variant detection will continue to grow," said Chen. "Monovar is capable of analyzing large-scale datasets and handling different whole-genome protocols, therefore it is well-suited for many types of studies.

Monday, April 25, 2016

Findings from an investigator-sponsored preclinical study have been released indicating that pacritinib, an inhibitor of JAK2, FLT3, IRAK1 and CSF1R, may be effective in reducing survival of myelofibrosis and acute myeloid leukemia (AML) repopulating cells. Further, this study also demonstrated the combination of pacritinib at low nanomolar concentrations with dasatinib may eliminate self-renewing leukemia stem cells in blast crisis of chronic myeloid leukemia (CML) with minimal toxicity toward normal progenitors. In myeloid leukemias, these leukemic stem cells can evade initial treatment and hide within the bone marrow microenvironment, develop resistance to current therapies, self-renew and eventually cause relapse.

These findings were presented by Larissa Balaian, PhD, from the Moores Cancer Center, University of California San Diego, in a poster presentation (Abstract 3338) titled: "Pacritinib Reduces Human Myeloid Leukemia Stem Cell Maintenance in a Defined Niche," during the American Association of Cancer Research Annual Meeting held April 16-20 in New Orleans.

"The potential ability for pacritinib to eradicate therapy resistant leukemia stem cells in relapse AML as a single-agent, as well as eliminate self-renewing stem cells in CML, when used in combination with standard of care therapy, demonstrates that targeting niche-dependent signaling with pacritinib could represent a new approach to treating patients with refractory acute myeloid leukemia and blast crisis of CML," said Balaian.

Tuesday, April 19, 2016

A microdevice implanted into a tumor could release up to 100 individual cancer therapeutics or combinations, and upon retrieval from the tumor and analyses, could identify the best treatment option for that tumor, according to preclinical research presented at the AACR Annual Meeting 2016, April 16-20.

The research team that developed this microdevice has initiated a clinical trial to test the safety and feasibility of placing and removing the microdevice in patients with early-stage HER-2 positive or triple-negative breast cancer.

 "Currently, there are about 150 cancer drugs approved by the U.S. Food and Drug Administration, and many cancer patients have different drugs to choose from to treat their specific disease. However, patients respond differently to different drugs, and often, no two patients have the same response to a specific drug. It has been a major challenge to determine which drug or combination of drugs to give to which patient," said Oliver Jonas, a postdoctoral fellow appointed jointly in the laboratories of Robert S. Langer, ScD, the David H. Koch Institute Professor, and Michael Cima, PhD, a David H. Koch Professor of Engineering, at Massachusetts Institute of Technology.

 "Being able to identify the right therapy that will work optimally for every patient will be a major advance," he added.

 Jonas and colleagues engineered a small implantable microdevice that is less than a millimeter in diameter and about 4 millimeters in length, which has multiple reservoirs to hold single agents and combination therapies. The device is implanted into the tumor through a small biopsy needle and left there for about 24 hours. The implant is then extracted along with a layer of surrounding tumor tissue, and the tumor tissue is analyzed to determine the effect of each of these drugs and combinations on the tumor.

"We published a study last year in Science Translational Medicine, in which we demonstrated that we can implant this microdevice into mouse tumors and that we can test 16 different therapies. We've increased that number to 100 since then," Jonas said. "We showed that the local readout that we can get from many agents in a single tumor is actually predictive of the drug sensitivity."

The researchers engineered the cylindrical microdevice, using biocompatible plastics. When implanted in a mouse tumor, the therapeutics, loaded in specific reservoirs within the device, were released in such a way that crosstalk between the different drugs was eliminated by taking into account the chemistry of drug and formulating the drugs appropriately to separate the reservoirs from each other. The researchers can also adjust the drug-release rates to mimic the concentration of the drug achieved by standard systemic delivery, Jonas explained.

In addition to further developing the device to hold up to 100 different drugs and combinations, they are now able to study the tumor at different time points while the device is still in place, by real-time imaging using optical fibers attached to each reservoir, Jonas added. "We are now able to measure how tumors adapt and change when they are treated locally or systemically, and how their sensitivity to different drugs changes when switched from standard-of-care therapy," Jonas said.

"Our study points to two important aspects," Jonas said.

He explained that traditionally, there are two approaches to systems biology; one approach is to study the effects of multiple drugs on one type of cells in the laboratory, and the other is to conduct whole mouse studies where one could only test one or two agents at a time, which takes a long time and comes at a high cost. "We are showing that there is a middle ground where one can work in vivo, so that we take into account the effects of the microenvironment, but can also screen 100 or more compounds efficiently and rapidly in a single tumor, such that we can prioritize therapy to a patient, in addition to being able to understand the cancer in a functional way that we haven't been able to do before," Jonas said.

As proof of principle, Jonas and his colleagues at MIT's Koch Institute for Integrative Cancer Research have so far tested the effects of many drugs in patient-derived xenograft (PDX) mouse models of melanoma, and prostate and breast cancer. In recent studies, they tested the sensitivity of estrogen receptor-positive breast cancer to single agents and combinations that target the ER, CDK4/6, PI3K, and other pathways.

"This microdevice also enables us to investigate the mechanisms behind drug resistance. Our results demonstrate new patterns of therapy evasion by cancer cells and adaptive signaling mechanisms, which offer clues for effective combination therapy," Jonas said.

This study was funded by the National Institutes of Health and the Koch Institute Frontier Research Program. Jonas declares no conflict of interest.


Tuesday, April 19, 2016

Many patients with advanced Merkel cell carcinoma (MCC), an aggressive type of skin cancer, who received the immunotherapeutic pembrolizumab as first-line therapy in a phase II clinical trial had durable responses, and responses were seen in those whose cancers were driven by a virus as well as those whose cancers were induced by exposure to ultraviolet (UV) light, according to research presented at the AACR Annual Meeting 2016, April 16-20.

This study is being simultaneously published in The New England Journal of Medicine.

"In this clinical trial, patients with metastatic Merkel cell carcinoma who received pembrolizumab had an objective response rate of 56 percent, which is similar to chemotherapy outcomes, but the duration of response to pembrolizumab appears to be significantly longer than that for chemotherapy," said Paul Nghiem, MD, PhD, affiliate investigator of the Clinical Research Division at Fred Hutchinson Cancer Research Center in Seattle and Professor of Medicine, Division of Dermatology at the University of Washington School of Medicine. "While the study is still ongoing, the vast majority of patients [86 percent] who responded to pembrolizumab are still experiencing excellent disease control more than six months after starting therapy."

MCC is a rare, aggressive type of skin cancer, and Merkel polyomavirus (MCPyV) is the driving factor in about 80 percent of MCC cases, Nghiem explained. About 2,000 new cases of MCC are diagnosed in the United States per year. MCC is 35-fold less common than melanoma, but on average, it is about three times more likely to kill a patient than melanoma. Response to chemotherapy is typically quite brief and half of patients develop progressive disease within three months of initiating treatment, he added.

Nghiem and colleagues enrolled 26 patients with advanced/metastatic MCC who had received no prior systemic therapy in this single-arm, open-label trial. Of them, 17 had MCPyV-positive disease. All patients received 2 mg/kg body weight of pembrolizumab every three weeks and responses were assessed every nine to 12 weeks. At the time of data analysis, patients had received four to 49 weeks of therapy.

The overall response rate was 63 percent in patients with virus-positive MCC and 44 percent in those with virus-negative (UV-induced) MCC. Four patients, three with virus-positive disease, had complete responses (CR), and 10 patients, seven with virus-positive disease, had partial responses (PR).

Adverse events in this trial were similar to other anti-PD-1 trials and were largely managed with steroid treatment and stopping the study drug, Nghiem added. The condition of two patients who developed severe drug-related toxicities improved with corticosteroid treatment and discontinuation of pembrolizumab. "Importantly, both these patients have ongoing antitumor responses many months after discontinuation of pembrolizumab," he noted.

"We believe that the immune system is likely 'seeing' different targets in the virus-positive and virus-negative patients," Nghiem said. He explained that the virus-positive tumors produce the viral proteins needed for the tumors to grow. These viral proteins may be readily seen by the immune system. In contrast, virus-negative MCC has extremely high numbers of mutations caused by sunlight. These mutations can change the normal cellular proteins so they no longer appear as "self" and the immune system can then see and attack these tumors.

Pembrolizumab acts by removing the "brakes" present on tumor-specific immune cells called T cells, thereby allowing the T cells to kill the cancer cells.

"Currently there are no FDA-approved drugs for the treatment of MCC. We are expanding this trial to recruit additional patients and we hope that these data will contribute to meaningful new therapeutic options becoming available for these patients," Nghiem said.

"It was initially challenging to partner with pharmaceutical companies because of the rarity of MCC. We are very thankful that the National Cancer Institute (NCI)'s Cancer Therapy Evaluation Program (CTEP), the Cancer Immunotherapy Trials Network (CITN), Merck, and multiple clinical sites came together to carry out this challenging study, which we believe is providing significant hope for MCC patients," he added.

Nghiem is a consultant for EMD Serono, Inc., and receives funding from Bristol-Myers Squibb to perform biomarker studies in MCC clinical trials.

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