Recent data indicate a role for a pathway involved in controlling cell proliferation as a potential site for therapeutic intervention in breast cancer (BC). The concept of intervening in cell cycle control in cancer is not new, but has been borne out in preclinical studies, and is now offering real possibilities for treatment in women with BC who fail a commonly used endocrine therapy.

Unrestrained cell proliferation is a defining feature in cancer, and it is understood that, under normal conditions, this process is tightly regulated at many levels, one of which is the cell cycle.

In a critical cell cycle &lsquo;checkpoint&rsquo; mitogenic signals cause upregulation of cyclin D1, which binds to, and activates, cyclindependent kinases (CDKs) 4 and 6. CDKs 4 and 6, when activated, phosphorylate the retinoblastoma (Rb) protein, an event which causes its release from the E2F transcription factor.

The release of E2F results in the transcriptional regulation of genes involved in the subsequent transition from the G1 phase of the cycle to the DNA synthesis (S) phase.

Additional control is provided by inhibitors of CDK 4/6, such as p21 and p16.

Mateusz Opyrchal, MD, PhD, assistant professor of oncology at the Roswell Park Cancer Institute, Buffalo, New York, noted the considerable effort under way to examine the role of CDKs in oncogenesis and progression of cancers.

&ldquo;CDKs play an important role in regulating cell division, and they guide many processes before and during the cell division, making this a highly regulated process. In cancers, aberrations in CDK function, and specifically in the CDK 4/6 and Rb pathway, which regulates initiation of gene transcription, lead to increased cell proliferation and tumor growth, as well as resistance to treatments,&rdquo; Opyrchal said. Early evidence for a role of the CDK 4/6 pathway in BC came from key findings, such as cyclin D1 overexpression and dysregulation of Rb function in BC.

Breast cancers are grouped by hormone receptor status (ie, estrogen receptor [ER]) and human epidermal growth factor receptor 2 (HER2) expression, and these classifications guide many treatment options (eg, tamoxifen, aromatase inhibitors for ER+ disease; trastuzumab for HER2+ disease).

Therapies that antagonize activity in the ER, such as tamoxifen and aromatase inhibitors (in postmenopausal women), are the mainstay for patients with ER+ disease, but resistance to these therapies is common.

We now know that an important component of efficacy for antiestrogen therapies is cell cycle control by the Rb-E2F pathway, and this could be at least one mechanism for resistance.

Initial data on the potential benefit of targeting CDK 4/6 in BC were reported in 2009 by Finn et al, who evaluated the effect of palbociclib (PD0332991), an orally active, specific inhibitor of CDK 4 and 6, to inhibit the growth of a range of ER+ BC cell lines.

They found that luminaltype ER+ breast cancers were most sensitive to growth inhibition by palbociclib, inclusive of those that were HER2 amplified, while those of the nonluminal/basal subtype were largely resistant. Moreover, by comparing large-scale gene expression profiles between palbociclib-sensitive and palbociclib-resistant cells, some 450 differentially expressed genes were identified; interestingly, p16 expression was decreased, and phosphorylated Rb (pRb) and cyclin D1 were upregulated in the most sensitive cell lines.

Consistent with its mechanism of action, pRb was decreased in the cell lines that were sensitive to palbociclib inhibition, and cells sensitive to palbociclib were arrested in the G0/G1 phase of the cell cycle. Most notably, palbociclib acted synergistically with both tamoxifen (in ER+ cell lines), as well as with trastuzumab (in HER2 amplified cell lines), and cells conditioned to be resistant to ER antagonists (ie, tamoxifen) could be partly resensitized by cotreatment with palbociclib.

Further preclinical findings were reported by Thangavel et al in 2011, who demonstrated an uncoupling of cell cycle control and the functional (active/inactive) state of the ER in BC cell lines.

They found that luminal B cancers, which exhibit poor durability in response to endocrine therapy, exhibited elevated expression of a gene signature indicative of loss of Rb function. Thus, despite retaining Rb gene expression, there is a disruption in the function of Rb in this BC subtype.

Their findings also suggested that Rb transcriptional control is a key component of the response to therapies that antagonize the ER. Moreover, in models of acquired antiestrogen resistance, there was a failure of therapies such as tamoxifen to active Rb-mediated transcriptional repression, with only sensitive cell lines exhibiting a downregulation of cyclin D1 RNA and protein levels. Treatment with palbociclib resulted in dephosphorylation of Rb, the attenuation of downstream target gene transcription, and inhibition of cell cycle progression.

Clinical InvestigationsPhase II and Beyond

Collectively, these findings suggest that CDK 4/6 inhibition is a means by which sensitivity to antiestrogen therapy can be restored in tumors failing endocrine therapy through a dysregulation of Rb phosphorylation.

A further intriguing finding from this study was that, unlike antiestrogen therapy, prolonged exposure to palbociclib-induced cell cycle arrest and a phenotype consistent with cellular senescence, a state whereby cells remain growth arrested but are capable of long-term survival.

A phase II clinical trial (PALOMA-1) has investigated the efficacy of palbociclib, with or without letrozole, an aromatase inhibitor, in postmenopausal women with ER+/HER2 BC. Findings of the study, initially presented at the San Antonio Breast Cancer Symposium in 2012, showed a remarkable improvement in progression-free survival (PFS), the primary endpoint, for patients randomized to the combination versus letrozole alone (26.2 vs 7.5 months; hazard ratio [HR] = 0.32;

<.001). The treatment was well tolerated, with neutropenia, leukopenia, anemia, and fatigue being the most common events in the combination arm of the trial.

In a recent panel discussion, Hope S. Rugo, MD, professor of medicine and director of the Breast Oncology, Clinical Trials, and Education Program, at the University of California San Francisco Comprehensive Cancer Center, commented on the findings of PALOMA-1, noting, &ldquo;We&rsquo;ve all been very excited because the PFS was [some] 3 1/2 times longer in the experimental arm&hellip;&rdquo;

PALOMA-2 is a phase III study evaluating the use of palbociclib in combination with letrozole versus letrozole alone in postmenopausal women with ER+, HER2 BC who have not received treatment for advanced disease, with the primary outcome of PFS.10 The study will also evaluate potential tumor tissue biomarkers of response, and/ or resistance to treatment. Speaking on the use of ER positivity as a selection criteria for PALOMA-2, Richard S. Finn, MD, from the division of hematology/oncology at the Jonsson Comprehensive Cancer Center at the University of California in Los Angeles, noted: &ldquo;We are not looking at any other selection marker other than that, because the data we have to date from the phase II study show that the best predictive marker is just being ER positive.&rdquo;11

Looking toward second-line treatment, the phase III PALOMA-3 trial is investigating palbociclib in combination with endocrine therapy (fulvestrant) versus placebo + fulvestrant in women with ER+/HER2 BC that has progressed after a prior endocrine therapy, with the primary outcome of PFS.12 Nicholas Turner, PhD, consultant medical oncologist at The Royal Marsden Hospital in London, stated: &ldquo;In the PALOMA-3 trial, we&rsquo;re specifically targeting women who progressed on their first-line hormone therapy. There are a number of options [in this setting], but one of the drugs that has the best data is fulvestrant, given at the 500-mg dose, and that&rsquo;s why we chose to partner that with palbociclib&hellip; in this study.&rdquo;

According to Turner, there was no evidence of any drug interaction between palbociclib and fulvestrant, and very strong data that endocrine-resistant cancers can still be sensitive to palbociclib, possibly extending the length of time that the cancer can be controlled.

In addition to palbociclib, 2 other inhibitors of the CDK 4/6 pathway are undergoing clinical evaluation, LEE011 and LY2835219.

Studies are evaluating the use of these agents in advanced breast cancer, either alone or in combination with hormonal agents.

As with many other targeted therapies, however, there is a need to identify prospectively those patients most likely to benefit from these therapies, and those who will not, or are no longer benefitting.

&ldquo;The one disappointing result of the PALOMA-1 trial was that it failed to identify biomarkers to select patients who are best suited for the therapy,&rdquo; Oprychal added, noting that several rationally chosen biomarkers (eg, amplified cyclin D1 and/or loss of p16) &ldquo;did not have any influence on whether the patients received benefit from palbociclib.&rdquo; Efforts will therefore continue to identify potential biomarkers in breast and other cancers, and their potential for use in combination with other targeted therapies.

American Cancer Society. Breast Cancer. Available at: http://www.cancer.org/acs/groups/ cid/documents/webcontent/003090-pdf.pdf Accessed July 20, 2014.

Lange CA, Yee D. Killing the second messenger: targeting loss of cell cycle control in endocrineresistant breast cancer.

Finn RS, Dering J, Conklin D, et al. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro.

Gillett C, Fantl V, Smith R, et al. Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining.

Bosco EE, Knudsen ES. RB in breast cancer: at the crossroads of tumorigenesis and treatment.

Thangavel C, Dean JL, Ertel A, et al. Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer.

Musgrove EA, Sutherland RL. Biological determinants of endocrine resistance in breast cancer.

Finn RS, Crown JP, Lang I, et al. Results of a randomized phase 2 study of PD 0332991, a cyclin-dependent kinase (CDK) 4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2- advanced breast cancer (BC).

OncLive Peer Exchange: CDK4/6 Inhibitors in Metastatic Breast Cancer. Available at: http:// www.onclive.com/peer-exchange/breast-cancertherapies/ CDK46-Inhibitors-in-Metastatic- Breast-Cancer Accessed: July 19, 2014.

Dr. Richard Finn Describes the Phase III Trial of Palbociclib for Breast Cancer. https://www. youtube.com/watch?v=aHp6LxPWCrQ Accessed: July 19, 2014.

Dr. Turner Discusses the PALOMA-3 Trial. https://www.youtube.com/watch?v=r6Km7IAocro Accessed: July 19, 2014.

Dickson MA. Molecular pathways: CDK4 inhibitors for cancer therapy.

Clinicaltrials.gov. http://clinicaltrials.gov/ ct2/results?term=LEE011+and+breast+cancer&S earch=Search Accessed July 20, 2014.

Clinicaltrials.gov. Available at: http://clinicaltrials. gov/ct2/results?term=LY2835219+and+b reast+cancer&Search=Search Accessed July 20, 2014.

Articles

Clinical Articles

Recent data indicate a role for a pathway involved in controlling cell proliferation as a potential site for therapeutic intervention in breast cancer.

Concept to Clinic: CDK 4/6 Inhibition in ER-Positive Breast Cancer

Five novel agents for the treatment of patients with advanced squamous cell carcinoma (SCC) of the lung will be evaluated in the recently launched Lung-MAP trial. This innovative, biomarker-driven study aims to improve the drug development process while exploring therapeutic options for this challenging malignancy.

The study, formally known as SWOG S1400, may cost up to $160 million and involve testing an additional 5 to 7 drugs beyond the agents initially selected. Plans call for enrolling up to 5000 patients through more than 200 medical centers during the next 5 years in randomized phase II substudies of the novel agents after participants have been screened through a master protocol.

The first round of agents to be tested include 4 targeted therapiesGDC- 0032, palbociclib, AZD4547, and rilotumumab&mdash; and the immunotherapy MEDI4736.

The logistics of the trial have been challenging to coordinate, but academic researchers, government regulators, and pharmaceutical companies have embraced the concept, according to Vassiliki A. Papadimitrakopoulou, MD, the principal investigator. Collaborators include 6 companies, the SWOG and the National Clinical Trials Network research cooperatives, and the National Cancer Institute (NCI).

&ldquo;Everybody sees the advantages in the process. We have an unprecedented willingness to participate,&rdquo; said Papadimitrakopoulou, a professor in the department of thoracic/head and neck medical oncology at The University of Texas MD Anderson Cancer Center in Houston.

&ldquo;There is a realization that we&rsquo;re doing things in such a fractional way in clinical research now that we&rsquo;re not really fulfilling our goals of getting safe and effective drugs to the patients as fast as possible,&rdquo; she said.

Papadimitrakopoulou discussed the Lung-MAP trial in August in Huntington Beach, California, during the 15th Annual International Lung Cancer Congress&reg;, where she explained the use of the master protocol design for implementing personalized medicine.

The Lung-MAP trial seeks to recruit patients with advanced or incurable stage IIIB/IV SCC who have progressed after receiving one front-line platinum-containing chemotherapy regimen for metastatic disease (Table). Detailed criteria for assignment to a substudy group and then to a study drug also have been developed.

To screen for biomarkers, investigators will conduct genomic profiling on either archived or freshly obtained tumor specimens using the Foundation- One test, which analyzes 182 genes.

After genomic evaluation, patients will be channeled into 1 of 5 substudies, depending on their biomarker status, and then randomized within that study. The substudies are:

Participants whose tumors do not harbor any of the targeted alterations will be randomized to receive either MEDI4736, a monoclonal antibody directed against the programmed death ligand-1 (PD-L1), or the standard chemotherapy docetaxel. MedImmune, a research arm of AstraZeneca, is developing the immunotherapy agent.

Patients whose tumors test positive for a PI3KCA mutation will receive either GDC-0032 or docetaxel. GDC-0032, a beta-isoform-sparing PI3K inhibitor, is an oral agent from Genentech.

Palbociclib versus docetaxelPatients whose tumors test positive for CDK4/6 or CCDN1/2/3 amplification will be randomized for treatment with either palbociclib or docetaxel. Palbociclib is a small molecule from Pfizer that has gained Breakthrough Therapy status in breast cancer.

Patients whose tumors harbor alterations in FGFR1/2/3 will be given either AZD4547 or docetaxel. AZD4547 is a tyrosine kinase inhibitor that AstraZeneca is developing.

Rilotumumab plus erlotinib versus erlotinib

Patients whose tumors have HGF/c-MET mutations will receive rilotumumab intravenously on day 1 and erlotinib daily, versus erlotinib daily. Rilotumumab is a monoclonal antibody in Amgen&rsquo;s pipeline that inhibits hepatocyte growth factor activity. Erlotinib (Tarceva) is an epidermal growth factor receptor (EGFR) inhibitor approved in several lung cancers, including as a secondline treatment for patients who progress after chemotherapy.

Papadimitrakopoulou said 16 pharmaceutical companies competed to have their drugs included in the trial in a rigorous process that included presenting data before a selection committee.

The primary endpoint of the phase II and phase III trials is progression-free survival (PFS) as measured by Response Evaluation Criteria in Solid Tumors (RECIST). Secondary endpoints include response rates and toxicity frequencies. Researchers also will be evaluating the percentages of screened patients who register in a specific substudy and receive at least one dose of the study drug in an effort to track successful registration and to screen failure rates.

Agents that demonstrate sufficient efficacy to graduate from phase II to phase III could be considered for registration by the US Food and Drug Administration (FDA), while agents that do not, will be replaced by new agents or combinations.

Squamous cell carcinomas represent 25% to 30% of all lung cancers and constitute the second-largest subtype of non-small cell lung cancer (NSCLC) after adenocarcinoma,

but no targeted therapies have been developed yet for patients whose tumors have this histology.

&ldquo;Despite a lot of impressive therapeutic advances that we have seen in lung cancer in the last few years, most of these advances are in reference to lung adenocarcinomas in specific mutational subsets, and they do not pertain to the squamous subset of patients for whom standard therapy still remains chemotherapy,&rdquo; said Papadimitrakopoulou. &ldquo;In many respects, this particular subset of lung tumors remains an orphan setting.&rdquo;

This situation has persisted even though genomic alterations in SCC lung cancer were fully characterized 2 years ago in research conducted through The Cancer Genome Atlas (TCGA) Network,

In the Lung-MAP trial, researchers are using the database from Foundation Medicine, which developed the genomic profiling test being used in the study, for insight into alterations in refractory disease, as well as TCGA data derived from early-stage disease specimens.

Table. Key Eligibility Criteria for Lung-MAP

Squamous-cell NSCLC confirmed by tumor biopsy and/or fine-needle aspiration

Primary squamous-cell diagnosis based on H & E slides with or without specific defined IHC characteristics

Mixed histologies containing &ge;50% squamous component

Patients must have progressed after exactly 1 front-line platinum-containing metastatic chemotherapy regimen

Adequate tumor tissue available, defined as &ge;20%

Zubrod performance status &le;2 within 28 days prior to screening registration

Patient must provide prior smoking history

Patient consent for submitting tissue for biomaker analysis for the trial and for banking for future specimen use

H&E indicates hematoxylin and eosin; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer. Source: www.ClinicalTrials.gov, NCT02 154490.

&ldquo;This is the first attempt to systematically go after the alterations that we have sufficient knowledge of, and I think this is going to open up the path for additional alterations that can be targeted,&rdquo; said Papadimitrakopoulou. &ldquo;This trial is going to provide the largest repository of squamous lung cancer materials from patients from the refractory setting.&rdquo;

The impetus for organizing the Lung- MAP trial stems from many factors, including a mandate from the NCI and the Institute of Medicine to improve clinical trial designs.

&ldquo;The rationale is mostly efficiency exploring novel therapeutics in a setting where we haven&rsquo;t seen any approvals of novel therapeutics that are targeting alterations so far, and trying to bring into our trials next-generation sequencing, which is a clinical reality but hasn&rsquo;t been generalized in its use in other studies,&rdquo; said Papadimitrakopoulou.

The trial has mobilized a network of resources throughout the United States in research, advocacy, and industry. The study is being launched through SWOG, formerly the Southwest Oncology Group, and cooperative groups represented by substudy chairs and cochairs throughout the country will lead the various substudies.

The cochairs for the study are David R. Gandara, MD, of the University of California Davis Comprehensive Cancer Center, who is SWOG&rsquo;s Lung Committee chair, and Roy S. Herbst, MD, PhD, of the Yale Cancer Center in New Haven, Connecticut, who is leading the Lung-MAP steering committee.

Also playing leading roles are Fred R. Hirsch, MD, PhD, of the University of Colorado Cancer Center in Denver, Philip C. Mack, PhD, of UC Davis Comprehensive Cancer Center, and Mary W. Redman, PhD, of the Fred Hutchinson Cancer Research Center in Seattle, Washington.

Other key players in the Lung-MAP cooperative include the Friends of Cancer Research, a Washington, DC-based advocacy group, led by Ellen V. Sigal, PhD, and the Foundation for the National Institutes of Health&rsquo;s Biomarker Consortium. Among industry partners, the collaboration brings together the companies whose drugs are being evaluated and Foundation Medicine.

&ldquo;I think what is enticing to the companies is, first of all, the established mechanism for screening patients up front, using the resources and the patient populations that exist within the cooperative groups,&rdquo; said Papadimitrakopoulou. &ldquo;It&rsquo;s quite a unique resource because each company would have to recruit its own network to run similar clinical trials targeted toward any of the alterations in squamous lung cancer.&rdquo;

Moreover, the FDA was directly involved in oversight and advisement regarding the design of the trial, which brings the regulatory agency into the process before any potential filings for new drug approvals.

Ultimately, the groundbreaking design is intended to steer clinical trials away from the model in which large numbers of patients are screened to find a relatively small number of individuals who may benefit from the study drug. Such trials are not only expensive and time-consuming for industry, but are also &ldquo;wasted opportunities for patients,&rdquo; said Papadimitrakopoulou. TT For further information about the trial, visit www.Lung-MAP.org.

Groundbreaking collaborative clinical trial launched [news release]. Lung-MAP collaborative; June 16, 2014. http://www.Lung-MAP.org/media/press/groundbreakingcollaborative- clinical-trial-launched. Accessed July 24, 2014.

NIH Clinical Trials registry. www.ClinicalTrials. gov website. Identifier number: NCT02154490.

American Cancer Society. What is non-small cell lung cancer? http://www.cancer .org/cancer/lungcancer-non-smallcell/ detailedguide/non-small-cell-lung-cancer-whatis- non-small-cell-lung-cancer. Updated April 30, 2014. Accessed July 24, 2014.

The Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers [published online September 9, 2012].

Five novel agents for the treatment of patients with advanced squamous cell carcinoma (SCC) of the lung will be evaluated in the recently launched Lung-MAP trial. 

September 2014 | Targeted Therapies in Oncology

Concept to Clinic: CDK 4/6 Inhibition in ER-Positive Breast Cancer

Extensive Lung-MAP Trial to Study 5 Novel Drugs