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SYNERGISTIC SCIENCE

Original research at the OSUCCC–James leads to innovative clinical trials such as OSU-10080

BY DARRELL E. WARD

Women diagnosed with triple-negative breast cancer face significant challenges.

Triple-negative breast cancer (TNBC) is characteristically more aggressive and has fewer treatment options than other types of breast cancer. The reason is biological. These tumors lack estrogen (ER) and progesterone receptors (PR), and they do not overexpress the human epidermal growth factor receptor (HER2). This “triple-negative” quality leaves them unresponsive to hormone therapy and to HER2-targeted therapy, which have greatly improved survival in women with ER/PR-positive and HER2-positive tumors. TNBC treatment is typically limited to chemotherapy, and the tumors tend to recur quickly.

TNBC accounts for about one-fifth of the 1 million breast-cancer cases that occur annually worldwide. It mainly affects women age 40 and younger, especially African-American women and women who inherit mutated BRCA1 and BRCA2 (BRCA1/2) genes.

“These are devastating cancers, and new therapies are badly needed for them,” says breast oncologist Bhuvana Ramaswamy, MD, MRCP, an assistant professor in the Division of Medical Oncology at Ohio State, and co-medical director of the Clinical Trials Office at Ohio State’s Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute (OSUCCC — James).

When the National Cancer Institute (NCI) issued a request for proposals to evaluate a new targeted therapy that might improve the treatment of TNBC, Ramaswamy gathered with collaborators at the OSUCCC – James and developed a protocol that the NCI approved in 2012.

Titled “A Phase 1 Dose-escalation Study of ABT-888 (veliparib) in Combination with Carboplatin in HER-2-negative Metastatic Breast Cancer,” OSU-10080 evaluates the
safety and identifies the maximum tolerated dose of the PARP inhibitor veliparib in combination with the chemotherapy drug carboplatin in women with metastatic TNBC (see the sidebar for an overview of the trial).

“Other centers chosen by the NCI to evaluate this agent designed trials that combine veliparib with two or three chemotherapy drugs, but we chose instead to use one
chemotherapy agent and a higher dose of veliparib,” says Ramaswamy, principal investigator for the trial.

OSU-10080 also incorporates original OSUCCC – James research that makes this PARP inhibitor trial particularly innovative:


• An immunofluorescence assay developed by Miguel Villalona, MD, Wenrui Duan, PhD, and colleagues to detect tumors with functional Fanconi Anemia defects enabled the
trial to include certain patients with ER/PR-positive breast tumors;

• FLT-PET imaging with an experimental marker of proliferation to predict tumor response, led by Michael Knopp, MD, PhD, director of Ohio State’s Wright Center of Innovation;

• Monitoring response to therapy in real time using circulating tumor cells (CTCs) from patient blood samples and technology developed by Jeffrey Chalmers, PhD, in Ohio State’s College of Engineering and by Maryam Lustberg MD, assistant professor of Medical Oncology in the College of Medicine;

• Applying microRNA research by Carlo M. Croce, MD, to learn if changes in microRNA expression influence response to treatment. First and foremost, OSU-10080 was designed to evaluate the safety of the PARP inhibitor veliparib combined with the chemotherapy drug carboplatin and to identify the maximum tolerated dose in women with metastatic TNBC. But like most clinical trials, correlative studies were added to answer other questions related to the drug.

Following is a look at the agent being evaluated by OSU-10080 and at the OSUCCC – James research that sets this trial apart from other veliparib trials.

OSU-10080 SYNOPSIS

ClinicalTrials.gov identifier: NCT01251874

The trial was open to patients with

  • Triple-negative breast cancer
  • ER/PR+, HER2- metastatic breast cancer with defects in Fanconi anemia pathway
  • BRCA1/2 germline mutations

Trial objectives

  • Determine a safe, effective dose for a phase II trial, toxicity and preliminary efficacy of
  • veliparib in combination with carboplatin
  • Evaluate pharmacodynamic markers possibly associated with PARP inhibition in tumor, including
  • FLT-PET changes, circulating tumor cells (CTCs) for the induction of the histone variant gamma
  • H2AX, and PAR levels in peripheral blood mononuclear cells
  • Evaluate biomarkers in primary tumor that could predict antitumor response following PARP
  • inhibitor treatment

Hypothesis

  • Higher doses of veliparib will be better tolerated when combined with single-agent
  • carboplatin alone
  • FLT-PET uptake will reliably predict antitumor responses
  • Higher induction of gamma H2AX in CTCs will reflect higher doses of veliparib
  • Correlatives
  • Use of FLT-PET to assess tumor response (with FDG-PET-CT scan to evaluate
  • radiological response)
  • Isolation of peripheral blood mononuclear cells to assess PAR levels
  • Assess the induction of gamma H2AX in CTCs as a measure of veliparib to induce
  • DNA damage in a dose-dependent fashion
  • Assess primary tumor tissue for BRCA1/2 protein miR-155 expression

Parp Inhibition

Many chemotherapy drugs and ionizing radiation kill cancer cells by causing breaks in one or both strands of the DNA helix. Such breaks also occur commonly in healthy cells from natural causes. If these breaks are not repaired, the cell dies. Consequently, cells have acquired elaborate systems to stitch up damaged DNA. In cancer cells, ramped-up DNA repair can be an important component of drug resistance.

One important mechanism of DNA repair relies on an enzyme complex called poly (ADP-ribose) polymerase (PARP). Cancer cells often show high PARP-repair activity, which might enable them to survive DNA-damaging chemotherapy and radiation.

PARP inhibitors are a new class of drugs that block the PARP mechanism. “It’s believed that inhibiting PARP will sensitize tumor cells to DNA-damaging chemotherapy such as carboplatin and enhance the ability of these agents to kill them,” Ramaswamy says.

The agents should be particularly effective in cancer cells that have pre-existing damage to a DNA-repair mechanism such as BRCA1/2 mutations. BRCA1 is a component of a mechanism called homology-directed repair. That mechanism is crippled in cancer cells with BRCA1 mutations. These cells then rely on PARP for survival.

“TNBCs often have defective DNA-repair mechanisms, which is one reason we are hopeful that PARP inhibitors will help these patients,” Ramaswamy says, noting that some patients entering the trial do not have documented BRCA mutations. Tumor cells from these patients are therefore assessed for BRCA1/2 protein levels to determine if a tumor has a functional BRCA pathway.

“This data should give us additional biomarkers that can be used to identify tumor subtypes that will benefit from PARP inhibitor therapy,” Ramaswamy says.

The FATSI Assay

Women with ER/PR-positive breast cancer can also have damaged DNA-repair mechanisms and may be treatable with PARP inhibitors. About 20 percent of the women enrolled in OSU-10080 have defects in the Fanconi anemia DNA-repair pathway. The trial evaluates whether these tumors are susceptible to PARP inhibitors.

For all patients entering the trial, tumor tissue was tested for Fanconi anemia functional defects using the FA Triple Stain Immunofluorescence (FATSI) assay developed by OSUCCC – James researcher Miguel Villalona, MD, professor of Internal Medicine and of Pharmacology, and director of the Division of Medical Oncology, Wenrui Duan, PhD, who heads Villalona’s laboratory, and a group of collaborators. The assay is performed and interpreted in the
CLIA-certified molecular-pathology laboratory of Weiqian Zhao MD, PhD, a researcher in the OSUCCC – James Molecular Biology and Cancer Genetics program.

The FA network involves at least 15 genes and includes BRCA2. It regulates DNA-damage responses that help maintain genome integrity. In 2013, Villalona and his collaborators published the method in the journal Translational Research, using it to determine the functional status of the Fanconi anemia pathway in tumor cells (see
figures, page 26).

“Tumors that are dysfunctional in any component of the FA network might be susceptible to PARP inhibition,” Villalona says.

“In addition, FA patients have a high incidence of malignancy, and cells from these patients are highly sensitive to DNA cross-linking agents, such as mitomycin C and
cisplatin.”

Villalona is principal investigator on another NCI-approved trial (OSU-9100; NCI0117640) under way now at the OSUCCC – James. It evaluates veliparib as monotherapy
and in combination with mitomycin C in patients with solid tumors with deficiency in homologous recombination repair.

Because of the FATSI assay, four patients with ER-positive tumors have also been enrolled on this trial.

“No one else was using PARP in ER-positive tumors,” Ramaswamy says. “But including these tumors was a natural next step, and the FATSI test developed at Ohio State
made it possible. This also makes our trial different from what other centers were doing.”

Imaging

Another question that OSU-10080 seeks to answer relates to dose of the drug: Does veliparib cause dose-related cancer-cell death or does it plateau after a certain
dose?

Trial co-investigator Michael Knopp proposed using an experimental functional-imaging method called F-18 fluorothymidine and positron emission tomography
(FLT-PET) to answer the question.

Epifluorescent image of a circulating tumor cell showing the presence of gamma-H2AX (red), a marker of DNA damage. Only the cell nucleus is visible,
colored blue by the fluorescent stain DAPI.

FLT-PET is a whole-body scan that can reveal multiple metastatic tumors.

FLT works as follows: As proliferating cancer cells make DNA, they take up the radiolabeled FLT as a source of thymidine.

Tumors with proliferating cells will appear brighter on the PET image; in tumors with less proliferation,
the image will appear dimmer.

Fluorodeoxyglucose PET (FDGPET) scans, with CT scans, were also used for more traditional radiological screening and measurement of response using RECIST criteria.
During the trial, patients received FLT-PET scans prior to treatment and again after seven days and 14 days of treatment.

“That was the original schedule; it called for 14 days of treatment,” Ramaswamy says. “Then we went to 21 days of treatment because at 14 days, many patients were
showing greater tumor reduction by FLT-PET, so we decided to add another week of treatment.

“That was particularly rewarding she says, “because we’d chosen to use FLT-PET, which, as a research-imaging modality, was not covered by trial participants’
insurance, and it was expensive. But the choice proved to be a good one because it enabled us to make an important clinical decision.”

“Our hope is that FLT-PET will become a tool to guide dosing regimens during clinical trials,” adds Knopp, who is professor and vice chair of Radiology.

Circulating Tumor Cells

There is another question OSU-10080 is investigating: Should veliparib be administered to patients continuously or intermittently?

Here, Ramaswamy turned to OSUCCC – James researcher Jeffrey Chalmers, PhD, professor of Engineering and director of the Analytical Cytometry Shared Resource. Chalmers had developed a method to isolate CTCs using a negative-selection technology based on immunomagnetic tagging and removal of cells that are positive for CD45, the leukocyte common antigen, which is expressed on most/all hematopoietic cells.

At the Analytical Cytometry Shared Resource, the CTCs are isolated from samples of peripheral blood collected before treatment and during the trial. An immunofluorescence assay is used to detect a marker of DNA damage called gamma-H2AX.

H2AX is a histone protein and a building block of nucleosomes, molecular spools involved in DNA folding and gene expression. When DNA sustains a double-strand break, the H2AX in the nucleosomes undergoes a chemical change; it is phosphorylated. This phosphorylated form is called gamma-H2AX, and it is a marker of DNA damage.

If the veliparib-carboplatin combination inflicts DNA damage and blocks repair as expected, CTCs should show higher levels of gamma-H2AX with increasing doses of veliparib.

“We hope to learn if we can detect changes in CTCs and whether the drug is really killing the cancer cells,” Chalmers says.

The researchers are also monitoring CTCs for the appearance of HER2 markers in patients with TNBC. “We’re not the first group to do this, but we are pushing technology to look at this more closely,” Chalmers says.

MicroRNA

Are there still other breast cancer patients whom PARP inhibitors might help? Work by OSUCCC – James researcher Carlo Croce, MD, professor and chair, Molecular Virology, Immunology and Medical Genetics, and director of Human Cancer Genetics, suggests, yes, and they might not be difficult to identify.

Croce has led laboratory and animal studies that link high levels of microRNA-155 (miR-155) to B cell lymphoma and to breast cancer and other solid tumors. The work indicates that overexpression of miR-155 results in the loss of function of several genes involved in DNA repair.

“This suggests that cancer cells that overexpress miR-155 should be extensively sensitive to PARP inhibition,” Ramaswamy says. To help answer that question, OSU-10080 includes a correlative study to evaluate primary tumors of patients for miR-155 expression, which will be correlated with tumor responses to therapy.

“Those findings should help us learn if high miR-155 expression in tumor cells is a marker for breast-cancer patients who might benefit from a PARP inhibitor,” Ramaswamy says.

In the end, OSU-10080 accrued 44 women with breast cancer; four patients were ER/PR-positive cases identified using the FATSI assay. Overall, 21 percent of tumors tested so far did have defective Fanconi anemia pathway. Since the trial has just completed accrual, the findings of the correlative studies won’t be known for some time, although the preliminary toxicity and FLT results were presented in the European Society of Medical Oncology Breast Cancer conference (IMPAKT) in Brussels and at the 2013 ASCO annual meeting.

“Our ultimate goal is to live in a cancer-free world,” Ramaswamy says. “OSU-10080 trial is an example of the imaginative, collaborative research and innovative team-science at the OSUCCC – James that will help get us there.”


 
30-Jul-13
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