Therapy for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) has changed modestly in the past 4 decades. The prognosis for AML and MDS remains poor, especially in patients with adverse prognostic factors, older age, or relapsed disease. An increased understanding of tumor biology and immune regulation has led to the development of novel molecular, epigenetic, and immune therapies for these patients. This review focuses on immunotherapeutic approaches in AML and MDS, with a special focus on the rationale for checkpoint-based therapies including antibodies to programmed cell-death protein-1 (PD-1/ PD-L1) and cytotoxic T-lymphocyteassociated antigen 4 (CTLA-4), an overview of clinical experience with these agents, and future considerations and combinations of these agents. Overexpression of various checkpoint receptors on T cells and ligands on AML blasts, especially those regulating the major inhibitory checkpoint pathways such as CTLA-4 and PD-1, interferes with effective T-cell antitumor response and is associated with leukemic progression in preclinical models. Novel monoclonal antibodies targeting CTLA-4 (eg, ipilimumab) or PD-1/PD-L1 (eg, nivolumab, pembrolizumab, and atezolizumab) can reverse immune suppression by unleashing the patient&rsquo;s T cells to attack leukemic cells. Results of numerous, ongoing early clinical trials evaluating these agents in monotherapy or in combination with the hypomethylating agents in relapsed AML and frontline posthypomethylating MDS are showing promising activity and safety. A phase I clinical trial with ipilimumab showed disease stabilization in 45% of patients with high-risk MDS. Ipilimumab also produced complete responses in 42% of patients with previously heavily treated AML who relapsed after stem cell transplantation in a phase I trial. A phase I/II trial with nivolumab, in combination with hypomethylating agent azacitidine, in patients with relapsed AML (median 2 prior therapies), demonstrated an overall response rate of 34%, lower early mortality, and the responses appear to be durable. These clinical trials will likely help us answer a number of critical issues regarding the utility of immune checkpoint therapy in AML and MDS, including the optimal dose and schedule of these agents, the efficacy and safety when used as a monotherapy or in combinations, the ideal agents to combine in the next wave of immune&ndash;immune or immune&ndash;molecular combinations, the management of checkpoint specific toxicities, and the identification and implementation of baseline and dynamic biomarkers of response to best select patients suited for these modalities.

Treatment for acute myeloid leukemia (AML), the most prevalent acute leukemia among adults, has improved modestly over the last 4 decades. Standard frontline treatment (&ldquo;7+3&rdquo;), introduced in the 1970s

, provides long-term overall survival (OS) in 40% of young adults. Outcomes of older patients, patients with adverse karyotype or TP53 mutation, or relapsed disease remain very poor with <10% long-term survivors.

A number of new molecular and epigenetic therapies have shown positive results in phase II/III studies and will likely change our current approaches to AML therapy in the near future.

Beyond molecular therapies, in the last year, immune therapies have emerged as an area of intense investigation in AML and myelodysplastic syndrome (MDS). The significant role of the immune system in producing durable remissions in hematologic malignancies has long been demonstrated by the success of allogeneic stem cell transplantation (aSCT) in producing long-term remissions in patients with leukemia, lymphoma, and multiple myeloma.

A better understanding of cancer immunology and availability of improved therapeutic tools including checkpoint antibodies, chimeric antigen receptor (CAR)-T cells, vaccines, and adoptive T-cell approaches, coupled with the success of these therapies in solid (melanoma, lung, head, and neck cancers) and liquid (Hodgkin lymphoma, multiple myeloma) tumors in recent years, has further fueled efforts to explore the efficacy, tolerability, and optimal approach to using these therapies in hematologic malignancies including AML, MDS, myelo brosis (MF), and chronic lymphocytic leukemia (CLL).

Immune homeostasis is normally maintained by balanced interactions between numerous costimulatory and coinhibitory signals. Tumor-mediated deregulation of T-cell responses leads to evasion by tumor cells of T-cell immune response.

The best- known receptors regulating T-cell activation or inhibition are the co-stimulatory receptors CD28, 4-1BB, CD27, CD80, and CD86, and co-inhibitory receptors cytotoxic T-lymphocyte-associated-protein 4 (CTLA- 4) and programmed cell-death protein (PD-1) with its ligands (PD-L1, PD-L2), respectively. Since the discovery of the inhibitory CTLA-4 receptor on the surface of T cells and the ability to unleash T cells by blocking the inhibitory CTLA-4 receptor,

a large number of clinical trials with checkpoint inhibitors, mainly targeting CTLA-4, PD-1 or PD-L1, or both receptors, have been conducted in solid tumors. CTLA- 4 is expressed predominantly on the T cells in the lymph nodes and regulates early T-cell activation, primarily by downregulation of T-helper cells and upregulation of T-regulatory cells,

whereas the PD-1 pathway regulates in ammatory responses in the peripheral tissues by inhibiting effector T cells.

(Figure 1). The fact that CTLA-4 and PD-1/ PD-L1 work at different levels in the immune system likely explains the demonstrated synergy when these agents are combined (Figure 2).

The evaluation of immunotherapy in leukemia is supported by the observation that various clinically targetable checkpoint receptors and ligands are overexpressed on T cells in ltrating the leukemic microenvironment, as opposed to T cells in the bone marrow aspirates of healthy individuals.

Daver et al evaluated 74 AML patients (36 untreated AML, 38 relapsed AML) to identify the degree and nature of T-cell infiltrate and the expression of a variety of checkpoint receptors and ligands on the T-lym- phocytes and blasts, respectively, in the bone marrow and peripheral blood in patients with AML, as compared with healthy donors.

Among a variety of checkpoint receptors evaluated, they noted that the bone marrow aspirates from patients with AML had significantly more PD-1positive T cells (including increased PD-1 CD8+, PD-1 CD4+FoxP3+CD127- negative, and CD4+FoxP3-CD127lo/+ T cells) as compared with healthy donors. The other checkpoint of interest that was identified was OX40, wherein bone marrow aspirates in patients with AML had significantly increased OX40-postive T cells. It has been reported that overexpression of PD-1 and CTLA-4 is associated with more aggressive leukemia

and that PD-1 expression is increased at progression from MDS to AML or AML relapse. PD-1 blockade to enhance the antileukemia response, reduce leukemia organ infiltration and increase survival in murine models of AML.

A phase I study of the PD-1 inhibitor pidilizumab in hematological malignancies including 9 patients with AML/MDS, showed safety, yet minimal response as a single agent with only 1 patient achieving a stable disease response.

CTLA-4 inhibitor ipilimumab showed disease stabilization in 45% of patients with high-risk MDS in a phase I study (5 of 11), 4 of whom had a durable response >6 months. Grade 3 immune related adverse events (AEs) were noticed in 7 patients (63%) and were easily managed with steroids.

A recent study highlighted the ef cacy of ipilimumab as a single agent in post allogeneic stem cell transplant (post-ASCT) relapsed AML. Twenty- eight patients were treated, including 12 patients with relapsed AML (including 3 patients with leukemia cutis). Complete responses were seen in 5 (42%) patients with AML, including 3 patients with leukemia cutis, and 4 patients who had durable responses for >1 year. This compares favorably to historical outcomes with post-ASCT relapsed AML, where expected response rates are 15% to 20% and <10% are expected to be alive at 1 year. Immune-related AEs were observed in 21% of patients (n = 6), including 1 death and graft versus host disease precluding further administration of ipilimumab in 14% (n = 4).

Epigenetic agents are a standard therapy for front-line and salvage in patients with AML and MDS.

The epigenetic agent, azacitidine, has been shown to upregulate PD-1, PD-L1, and PD-L2 (&ge;2-fold) in >50% of 61 evaluable patients with AML/MDS, which may be associated with emergence of resistance to azacitidine.

Therefore, concomitant inhibition of the PD-1/PD-L1 axis may prevent or overcome resistance. Trials combining epigenetic agents with PD-1/PD-L1 based therapies have recently started enrollment for AML and/or MDS including azacitidine with the PD-1 inhibitor nivolumab (NCT02397720), azacitidine with or without PD-L1 inhibitor durvalumab (NCT02775903), azacitidine with or without the PD-L1 inhibitor atezolizumab (NCT02508870). Results from the first to open of these studies, a phase IB/II trial of nivolumab with 5-azacytidine, are encouraging. In an interim analysis, 35 evaluable patients with a median follow-up of 3.6 months showed an overall response rate of 34% including 6 complete remissions and/or complete remission with insuficient count recovery (CR/ CRi);(18%); 5 hematologic improvements including 2 patients who had concomitant >50% blast reduc- tion (16%).

This compares favorably with a historic response rate of 15% to 20% in a similar patient treated with azacitidine alone or decitabine alone.

Additionally, 5 patients (23%) had a >50% blast reduction and 2 had stable disease (7%). The patients had a significantly improved overall response rate, 8-week mortality, and median progression-free survival as compared with historical outcomes with azacitidine-based salvage protocols from the same institution, but the follow-up at this time is short. Grade 2 to 4 immune adverse events (iAEs) were observed in 11 (31%) patients with a time to onset of 4 days to 3.5 months. The pattern of iAEs was different from that seen in solid tumors with most common iAEs of pneumonitis, nephritis, colitis, and dermatitis as opposed to endocrine, skin, and liver iAEs typically seen in solid tumors. Also different from solid tumor experiences, all iAEs responded quickly to steroids with successful rechallenge with nivolumab in all, but one case.

Table 1 provides details on currently ongoing combinational trials of PD-1/CTLA-4 inhibitors with epigenetic, cytotoxic agents, or tumor specific vaccines for AML and MDS.

In summary, emerging data suggest potential efficacy of checkpoint inhibitors in AML, especially in combination with hypomethylating agents. A number of trials evaluating these combinations and comparing their efficacy and tolerability to a single hypomethylating agent in both AML and MDS have been initiated and will help us better answer these questions. Other immune-based approaches for patients with AML/MDS, include conjugated antibodies targeted to leukemia specific antigens that deliver toxic payload to leukemic cells (SGN 33A, SGN 123, IMGNCD33 and CD123), bispecific T-cell engaging antibodies (AMG CD3 CD33, JNJ CD3 CD123, XmAb CD3 CD123), DARTs, and CAR T-based approaches targeting CD123 or CLEC12A. These agents may eventually be rational choices to combine with checkpoint inhibitors to develop immuneimmune strategies for AML and MDS or in combination with epigenetic and cytotoxic therapies. The ongoing clinical trials will likely help answer a number of critical issues regarding the utility of immunotherapy in leukemia including accurate timing and dynamics of response to such therapies, identification of additional novel checkpoint targets in AML, biomarkers to allow for selection of candidates with highest propensity to response, awareness and management of immune toxicities, and mechanisms of immune modulation and resistance.

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Articles

Clinical Articles

The researchers discuss the rationale for checkpoint-based therapies including antibodies to PD-1/PD-L1 and CTLA-4, an overview of clinical experience with these agents, and future considerations and combinations of these agents.

Immune Checkpoint Approaches in AML and MDS: A Next Frontier?

Immune checkpoint inhibitors targeting the cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and programmed cell death 1 (PD-1) receptors have recently made their way into the oncology clinics, producing substantial clinical benefits for certain tumor types and patients. Data are rapidly emerging for multiple malignancies, with numerous additional molecules and antibodies in development. Considering advanced colorectal cancer, the early signals of activity are largely involving PD-1 inhibition, specifically against a subset of colorectal cancer (CRC), microsatellite instability high (MSI-H) tumors. This has prompted multiple studies and an even stronger push for universal MSI testing. Unfortunately, the colon cancers which are not MSI-H (MSS), those which make up the majority of the tumors in clinical practice, have not seen benefit through single agent PD-1/PD-L1 inhibition. Recently, a combination of MEK inhibition and PD-L1 inhibition demonstrated highly intriguing, but preliminary evidence of clinical efficacy, prompting the initiation of a phase III trial (NCT02788279). Numerous additional efforts are underway evaluating immunomodulatory therapeutic combinations that may favorably alter the tumor microenvironment. This article presents a brief review of the immune environment of CRC, recently reported studies of immunotherapy in CRC, and a survey of ongoing efforts.

Largely attributable to the development of immune checkpoint inhibitors, specifically monoclonal antibodies targeting cytotoxic T-lymphochyte associated protein-4 (CTLA-4), programmed cell death (PD-1), or PD-L1, there has recently been an &lsquo;immunotherapy revolution&rsquo; in the oncology community. PD-1 is ubiq- uitously expressed in T cells, B cells and NK cells and is similarly upregulated in an inflammatory milieu. PD-1 binds to its ligands PD-L1 and PD-L2 and induces T effector cell exhaustion and conversion of T effector cells to regulatory T (Treg) cells. PD-L1 is expressed in multiple tissues, including tumor cells and antigen presenting cells, while PD-L2 is selectively expressed on hematopoietic cells.

Tumors that have not been traditionally considered immune responsive have now been shown to be susceptible to immune modulation of the tumor microenvironment. Conclusive benefit has been demonstrated in nonsmall cell lung cancer (NSCLC), melanoma, renal cell cancer, urothelial bladder cancer, and head and neck cancer among others, resulting in approval of multiple PD-1 and/or PD-L1 inhibitors by the FDA. In the meantime, intriguing results are emerging in multiple other tumor types; recently, benefit has been demonstrated in mismatch repair (MMR)-deficient (or microsatellite instability high [MSI-H]) colorectal cancer (CRC).

A hallmark of neoplastic transformation and subsequent tumor survival in the host milieu is immune evasion. The prevailing mechanism by which this occurs is termed cancer immunoediting, a process involving 3 phases.

In the initial phase, there is activation of the adaptive and innate immune response to tumor neoantigens, characterized by trafficking of NK cells, along with B and T lymphocytes. This phase is also associated with secretion of cytokines: IFN-&alpha;, IFN-&gamma;, TNF, and IL-12. If the neoplastic tissue survives this, an equilibrium phase follows, involving a balance between tumor cell destruction by the adaptive immune system, namely activated CD4+ and CD8+ cells. Here, malignant clones persist but an active immune response prevents overall tumor growth and produces dormancy. Ultimately, the clones are able to adapt and escape, thus resulting in immune evasion.

Immunologic escape can occur via multiple mechanisms, including loss of antigen presenting ability, release of inhibitory cytokines, and expression of immune checkpoint molecules such as PD-1 and PD-L1 that would serve to suppress effector T- cell activity.

In addition, many signaling pathways that are believed to be drivers of oncogenesis have been noted to have secondary immune modulating effects. Activation of the WNT/&beta;-catenin pathway in metastatic melanoma is correlated with an absence of T cells in the tumor microenvironment.

This is of great relevance, as this pathway is activated in the majority of CRCs.

CRCs are believed to develop through a stepwise process of mutations in key genes such as APC, KRAS, P53, and SMAD4, first delineated more than 2 decades ago.

Genetic alterations have been firmly linked to tumorigenesis for many years. Immune evasion was not part of the originally proposed 6 hallmarks of cancer, but it has now been added to this list as a core feature of tumorigenesis.

Inflammation, on the other hand, has been long associated with cancer, with a link to chronic inflammatory conditions that appear to facilitate neoplastic transformation, as with inflammatory bowel disease and CRC. In fact, epidemiologic data and some randomized data support a role for nonsteroidal anti-inflammatory drugs (NSAIDs) in decreasing the formation of both polyps and CRCs.

These points are raised to highlight the delicate interplay between immune activation, the healthy body, and cancer. Disruption of the delicate balance of the immune system, hyperactivation, inappropriate activation, or immunosuppression can all lead to development of cancer.

About a decade ago, evidence came to light suggesting that subsets of effector immune cells are important in CRC. Using markers to evaluate numbers of all T cells (CD3+), cytotoxic T cells (CD8+), and memory T cells (CD45RO+) in the tumor core as well as the invasive margin, Galon et al demonstrated superior outcomes for those patients with tumors containing more dense T-cell infltration, compared with those which were relatively T-cell depleted.

In fact, both total T cells (CD3) and memory T cells (CD45RO+) proved superior to TNM staging in predicting outcomes. Multiple other studies have evaluated the predictive capacity of lymphocyte subsets in CRC, with a more robust inflammatory infiltrate generally conferring a more favorable prognosis.

Recently, the Immunoscore methodology, which assesses CD3+ and CD8+ cellular density in various compartments, has been demonstrated to be predictive of response as well as prognosis in locally advanced rectal adenocarcinomas undergoing neoadjuvant chemoradiation.

Thus, immune activation and immune effector subsets are important on their own, but also potentially relevant in the efficacy of standard anticancer therapies.

At the most basic, clinically relevant level, CRC can be segregated by site of origin, by RAS and BRAF mutational status, and by microsatellite status (MSI- H or microsatellite stable [MSS]). Microsatellite instability is seen in 15% to 20% of early-stage CRCs and in 3% of metastatic CRCs.

These tumors are more commonly right-sided, mucinous, poorly differentiated, and characterized by lymphocytic infiltration. They also more commonly possess BRAF mutations. The observed MSI is due to infidelity of DNA replication, owing to dysfunction of one of the MMR proteins (MLH1, MSH2, MSH6, PMS2), in line with the other common terminology: deficient mismatch repair (dMMR). While MSI/dMMR has been linked to Lynch syndrome, it is important to note that MSI-H status is most often due to an epigenetic alteration (hypermethylation of the MLH1 promoter), which silences the MLH1 protein. Epigenetic MLH1 silencing represents approximately 80% of all MSI-H CRCs, usually sporadically occurring in the patient. This underscores an important point: most MSI-H tumors are not Lynch syndrome related.

Initially, MSI testing emerged as a means to screen for Lynch syndrome, although it was later found to have prognostic and predictive importance.

Patients with early-stage CRC and MSI-H tumors appear to have improved prognoses, whereas those with stage IV MSI-H tumors may fare worse.

It has been further demonstrated that patients with stage II MSI-H CRC treated with uorouracil (5-FU)- based therapy derive little to no benefit. In fact, such patients potentially face worse outcomes if their tumors are MSI-H, compared with MSS.

Further retrospective data suggest that the addition of oxaliplatin may overcome this observed resistance to 5-FU alone, of relevance for stage III CRC.

Importantly, CRCs with MSI-H are characterized by greatly elevated mutational rates. It is widely believed that the resultant mutations may serve as tumor neoantigens, triggering an immune response. Recent data suggest a correlation between frameshift mutation frequency and the density of tumor-infiltrating CD8+ T cells, supporting this hypothesis that the mutational landscape is directly related to tumor antigenicity.

Finally, it is worth noting that MSI-H tumors, which would otherwise seem to exist in a microenvironment that is more innately hostile to neoplasms, are characterized by highly upregulated expression of multiple immune checkpoints, including PD-1, PD-L1, CTLA-4, LAG-3, and IDO.

This implies that MSI-H CRCs are a tumor type characterized by a robust population of immune effector cells, but also possessing an immune exhaustion phenotype within the microenvironment.

Immune Checkpoint Inhibitors in Colorectal Cancer

As noted, immunotherapy is not a new concept in CRC. Multiple previous efforts have been undertaken. CRC, too, has been the subject of studies involving CTLA-4, PD-1, and PD-L1 inhibition. Relevant completed studies and those with informative interim analyses are described in Table 1. Key planned trials are detailed in Table 2. A narrative of the developments follows.

Tremelimumab is a fully human immunoglobulin G2 (IgG2) monoclonal antibody (mAb) that binds to the CTLA-4 receptor. Because of its initial activity in melanoma, as well as supporting preclinical data, a phase 2 study of tremelimumab was undertaken in patients with refractory metastatic CRC, at a dose of 15 mg/kg every 90 days. In this multicenter study, 49 patients with chemorefractory metastatic CRC were treated, with a median duration on study of 2.3 months. The 6-month progression-free survival (PFS) was a meager 2.1%. The sole patient who received more than 1 dose of therapy experienced a partial response by Response Evaluation Criteria In Solid Tumors (RECIST), continuing without disease progression for 14.5 months and surviving for 19 months from the start of therapy. Toxicities were as expected, with an 11% incidence of grade 3 diarrhea.

Although 1 patient appeared to derive substantial benefit, the limited response rate did not suggest general applicability. As such, this therapy has not been further pursued. Unfortunately, the tumor or host characteristics that enabled the responding patient to benefit have not emerged to date.

Nivolumab is a fully humanized IgG4 mAb that binds and inhibits PD-1. Nivolumab (Opdivo) garnered broad attention when the initial phase 1 data were presented in 2012.

In the study, 296 patients were treated at various doses (1 mg/kg, 3 mg/kg,10 mg/ kg) every 2 weeks. Responses were noted in melanoma (28%), NSCLC (18%), and RCC (27%). Responses were durable, with up to 1 year or more in 20 of 31 initially responding patients. No clear doseresponse relationship was demonstrated. In 42 tumor samples that underwent immunohistochemical analysis for PD-L1 expression, a 36% objective response was noted in PD-L1&ndash;positive tumors and 0% response rate in PD-L1&ndash;negative tumors. In this study, 17 patients had prostate cancer and 19 patients had CRC. No objective responses were observed in either cohort.

A later report describing several patients from the initial nivolumab study was published in 2013. That study noted that 1 patient with CRC experienced a complete response (CR) to therapy, ongoing at 3 years. This patient was a 71-year-old male with MSI- H CRC, refractory to prior treatment, and notably with membranous PD-L1 expression on tumor-infiltrating macrophages and lymphocytes as well as rare tumor cells associated with in ltrating PD-1+ and CD-3+ T cells.

Thus, significant benefit was possible with this drug, but only in the rare patient.

Pembrolizumab (Keytruda) is an IgG4 mAb that binds and inhibits PD-1. Pembrolizumab has received great attention recently in CRC, attributable to the highly encouraging results of a multicenter phase II study initially presented at the 2015 ASCO Annual Meeting. In this study, patients with chemo-refractory MSI-H or MSS metastatic CRC, as well as MSI-H cancers of other origin, were included. The trial was based on the rationale described above: previous activity in 1 patient with MSI-H CRC, elevated immunogenic tumor neoantigens, and the inflamed tumor environment often seen in MSI-H cancers. A standardized polymerase chain reaction (PCR)- based test was used to assess MSI, with pembrolizumab was administered every 2 weeks at a dose of 10 mg/kg.

At the time of the initial presentation, no responses were recorded among the mismatch repair (MMR) proficient (pMMR or MSS) CRC patients, with 2 (11%) achieving stable disease at 12 weeks. However, of the 10 evaluable MSI-H patients with CRC, 4 (40%) experienced a re- sponse, with 7 of 9 (78%) patients achieving stable disease. In parallel, 5 (71%) of the 7 evaluable MSI-H patients with tumors of non-CRC origin experienced a response, with 1 (14%) achieving a CR.

Updated results were presented at the 2016 ASCO Annual Meeting. There were 83 evaluable patients with refractory tumors described: MSS CRC (28 patients), MSI-H CRC (25 patients), and MSI-H cancers of other origin (30 patients). Among the MSI-H patients with CRC, 16 (57%) experienced a response, with 3 (11%) having a CR and 9 (32%) patients achieving stable disease. Median (PFS) was not reached (NR) for MSI-H CRC and 2.4 months for MSS CRC (HR, 0.135; 95% CI, 0.043-0.191; P &le;.0001). Median overall survival (OS) was NR for MSI-H CRC versus 6 months for MSS CRC (HR,0.247;95%CI,0.117-0.589;P=.001).For MSI-H CRC, the PFS rate was 61% at 24 months and the OS rate was 66% at 24 months. Similarly, among patients with non-CRC MSI-H tumors, 16 (53%) experienced a response, with 9 (30%) patients achieving a CR; a pro-portionate response was observed to the mutational load of each tumor.

These data are exciting and, while preliminary, have resulted in the FDA granting a priority review to pembrolizumab for MSI-H metastatic CRC. Further, given the multiple ongoing studies, these data support the recommendation to more broadly screen patients for MSI, even in stage IV disease without a high suspicion for Lynch syndrome. An ongoing trial, KEYNOTE-164, is seeking to confirm these results, using a lower dose of pembrolizumab, 200 mg IV every 21 days (NCT02460198). This is a single-arm phase II study that enrolled 2 cohorts: those with refractory disease and those who have received at least 1 line of therapy. KEYNOTE-177 is an additional important study that is enrolling untreated patients with MSI-H or dMMR metastatic CRC (NCT02563002). In a 1:1 fashion, with a crossover design, this phase III study is assigning patients to standard of care chemotherapy or single-agent pembrolizumab. The primary endpoint is PFS, with secondary endpoints of overall response rate (ORR) and OS.

Durvalumab (MEDI4736) is a humanized mAB directed against PD-L1. An ongoing investigation is evaluating durvalumab in a phase II study of locally advanced or metastatic MSI-H CRC with an elevated density of tumor-infiltrating lymphocytes (TILs) on biopsy (NCT02227667). This study is unique in that an assessment of tumor microenvironment is being utilized for inclusion.

Because CTLA-4 inhibition (ipilimumab) and PD-1 inhibition (nivolumab) appear to offer improved response rates and PFS compared with either therapy alone in melanoma, there is hope that the combination might also offer broadened benefit in CRC. In CRC syngeneic mouse models, combined PD-1 and CTLA-4 inhibition achieves success where neither agent was capable of consistently doing so on its own.

The phase I/II CheckMate 142 study compared use of nivolumab monotherapy arm (3 mg/kg q2wk) with nivolumab (3 mg/kg q2wk) + ipilimumab (1 mg/kg x 4 doses) in patients with recurrent and metastatic MSI-H and MSS CRC (NCT02060188). Preliminary results were presented at the ASCO 2016 meeting. Seventy patients were treated in the nivolumab monotherapy arm, including 47 evaluable patients who had been on therapy with at least 12 weeks follow-up. Here, 12 (25.5%) patients achieved a partial response, with an additional 14 (30%) with stable disease, a disease control rate of 55.5%. The median duration of response had not yet been reached (range, 0  15.2 mos).

Thirty patients with MSI-H CRC were treated with combined nivolumab and ipilimumab. The results for the 27 evaluable patients with at least 12 weeks follow-up were presented, with 9 (33%) demonstrating a partial response and 14 (52%) with stable disease. This translates to an 85% disease control rate. The investigator assessed 6-month PFS appeared slightly better than nivolumab monotherapy: 66.6% versus 45.9%, respectively. Initially, the re- sponse rates and disease control appear superior to nivolumab monotherapy, but the small numbers and very limited follow-up time preclude any firm conclusions as to differential efficacy. Since responses with ipilimumab can be delayed, it will be highly interesting to see the mature follow-up data to establish whether there is a meaningful difference in activity which would support the further development and use of CTLA-4/PD-1 doublets despite concerns of increased toxicity.

Of 20 evaluable patients with MSS CRC who received combination therapy, a partial response was observed in 1 patient (5%). However, for the combination therapy group, PFS was poor, with the median standing at less than 3 months.

The story for MSI-H tumors appears promising. As we wait on further data, there is hope that the activity spectrum of checkpoint inhibitors can be expanded in CRC. Given the highly immunosuppressive tumor milieu, combination approaches or alternative agents would appear to be necessary for broader application. A selection of additional investigational approaches and initial results, when applicable, is described in the sections that follow.

Cobimetinib (Cotellic) is a selective inhibitor of mito-gen-activated protein kinase kinase (MEK). MEK is a dual specificity kinase, a key component of the RAS/ RAF/MEK/ERK pathway that is constitutively activated in a wide variety of tumors. Atezolizumab (Tecentriq) is an IgG1 mAb targeting PD-L1. In preclinical models, targeted inhibition of MEK leads to upregulation of MHC I on tumor cells, induces intratumoral T-cellin lnfiltration and synergizes with PD-1/L1 inhibition.

Results of a phase Ib clinical trial using the combination cobimetinib and atezolizumab in patients with advanced solid tumors were reported at the 2016 ASCO Annual Meeting (NCT01988896). Twenty-three patients enrolled had metastatic CRC (mCRC), 20 of whom compromised a KRAS-mutant CRC expansion cohort. Thirty percent of the patients were MSS or MSI-low (MSI-L), while 70% had unknown MSI status. Of great interest, 4 (17%) patients demonstrated objective response. Three of four were pMMR (MSS equivalent) and for 1 patient the status remains unknown. Six (26%) patients were on therapy for >12 months. Although median PFS was 2.3 months, 6-month OS was 72%. This compares favorably with regorafenib (Stivarga) and TAS-102, where the median OS stands at 6.4 and 7.1 months, respectively.

Of note, MEK inhibition alone has not previously demonstrated significant clinical activity in CRC, suggesting that MEK inhibition may be able to prime an immune response. This opens the door to related priming strategies. On the heels of these encouraging results, a phase III study has been launched, which is randomizing patients with refractory metastatic CRC to 1 of 3 arms: atezolizumab + cobimetinib, atezolizumab monotherapy, or regorafenib (NCT02788279). In addition, an ongoing phase I study is evaluating the efficacy of the VEGF-TRAP, anflibercept, combined with pembrolizumab, in patients with recurrent or mCRC. Multiple additional trials are underway examining combinations of targeted therapy with PD-1/PD-L1 inhibition (Table 3).

Chemotherapy is capable of immunomodulatory effects on its own. There are preclinical data to support 5-FU and gemcitabine as being toxic to suppressive myeloid-derived suppressor cells (MDSCs), with FOLFOX decreasing levels of peripherally circulating MDSCs in patients with CRC.

Drugs that target VEGF may have beneficial effects by reducing regulatory T cells.

However, scant clinical data have been presented on combined chemotherapeutics and checkpoint inhibitors across all cancers, and particularly in CRC. Thus, forgetting toxicity concerns for a moment, it remains uncertain clinically whether there will be a beneficial or detrimental impact of combination with chemotherapy.

Initial safety data from a multiarm study of atezolizumab were presented at the 2015 GI Cancers Symposium meeting (NCT01633970). In arm A, 14 patients with refractory CRC were treated with bevacizumab and atezolizumab. One (7%) partial response was seen, with 9 (64%) patients achieving stable disease, which lasted at least 24 weeks in 2 patients. In arm B, 30 patients (70% chemotherapy naïve) were treated with FOLFOX plus bevacizumab and atezolizumab. In this arm, of the 23 first-line patients, a 48% response rate was achieved with an 87% disease control rate (response or stable disease). Immune activation was demonstrated in the tumor biopsies and peripheral blood.

However, at this interim analysis, it was not clear whether or not there is an additional benefit conferred by adding PD-L1 blockade to standard therapy; the outcomes looked strikingly similar to those achieved via FOLFOX and bevacizumab alone. Similarly, it cannot be determined with certainty whether or not the observed immune activation differs from that which would be seen with use of chemotherapy alone. Of course, given the lack of single-agent antiPD-1 activity in patients with unselected CRC, such combinations might not improve response rates, but they may be capable of deepening or prolonging responses. Therefore, further follow-up analyses, including data on PFS and OS would be useful.

Additional studies are evaluating PD-1/PD-L1 inhibition and chemotherapy combinations in CRC. Pembrolizumab is being tested in combination with mFOLFOX6 in 2 ongoing clinical studies, one specifically for CRC (NCT02375672, NCT02268825).

Atezolizumab is being evaluated in a biomarker-driven multi-arm trial of various maintenance strategies in treatment-naïve metastatic CRC outside of the United States (NCT02291289). One arm will evaluate the combination of 5-FU, bevacizumab, and PD-L1 inhibition after initial induction therapy with mFOLFOX6 and bevacizumab. In refractory disease,the BACCI study will seek to evaluate the impact of adding atezolizumab to capecitabine and bevacizumab (NCT02873195). and other metastatic solid tumors (NCT02298959).

Ali Maawy, MD, and Patrick M. Boland, MD, provide a brief review of the immune environment of colorectal cancer (CRC) and recently reported studies of immunotherapy in CRC.

Immune Checkpoint Inhibitors in CRC

A dysregulated cell cycle can lead to cancer. The retinoblastoma (RB1) gene is a tumor suppressor gene and its corresponding protein Rb has an integral role in cell-cycle regulation. Dephosphorylated Rb halts the cycle while phosphorylated Rb restarts it. Cyclin-dependent kinase (CDK) 4 and 6 are responsible for phosphorylating Rb, D-type cyclins, which are expressed in response to stimulatory mitogens, are CDK activators while p16INK4a encoded by the CDKN2A gene is an innate CDK inhibitor. CDKs have a vital role in unleashing cell cycle arrest and are targeted by a class of antitumor drugs called CDK inhibitors.

Inactivation of Rb pathway is seen in more than 70% of nonsmall cell lung cancer (NSCLC). The most common event for inactivation is the loss of CDK2NA gene. Moreover, this inactivation occurs frequently in patients with mutant KRAS lung adenocarcinomas. Early-phase studies have shown that CDK inhibition can induce immediate senescence of NSCLC with KRAS mutation. In addition, CDK4 and cyclin D1 amplifications have also been associated with lung cancer, specifically squamous cell lung cancer. Thus, there has been a significant interest in investigating the role of CDK inhibitors in lung cancer. Various broad spectrum nonselective first generation CDK inhibitors encountered obstacles in clinical development due to poor pharmacokinetic profiles and toxicities. This review article will focus on the therapeutic relevance of newer generation selective CDK inhibitors which have recently progressed the furthest in clinical development in lung cancer.

Cell-cycle dysregulation is one of the hallmarks of cancer.

Various genes and their corresponding proteins, interact with each other, external stresses, hormones, and cytokines to regulate the cell cycle. Alteration in the regulatory pathways may lead to the dysregulation of cell cycle and tumorigenesis. Cyclin-dependent kinases (CDKs) are serine/threo- nine kinases that by definition require activation by cyclins or cyclin-related proteins and have been implicated in unleashing the cell-cycle arrest, and thus, they have been targeted by a class of antitumor drugs called CDK inhibitors. Excluding the related CDK1 (also known as CDC2)-like kinases, there are currently 20 members of the CDK family, several of which function in noncell cycle processes such as transcription. Various broad-spectrum, nonselective first-generation CDK inhibitors, such as avopiridol, encountered obstacles in clinical development due to a poor pharmacokinetic profile and/or toxicities. We will focus our discussion on the therapeutic relevance of newer generation selective CDK inhibitors that have recently progressed the furthest in clinical development in lung cancer.

The retinoblastoma (RB1) gene is a tumor-suppressor gene that encodes for the retinoblastoma protein, pRb. The Rb protein undergoes intermittent phosphorylation as the cell traverses the cell cycle. A dephosphorylated Rb protein acts as a brake in the cell-cycle progression. Rb is dephosphorylated (activated) as the cell exits mitosis, is hyperphosphorylated (inactivated) toward the end of G1 phase, and remains hyperphosphorylated until the cell goes to mitosis phase.

CDKs regulate both the cell cycle and transcription. CDKs 1, 2, 4, and 6 are required for the correct timing and order of the events of the cell-division cycle. CDK7 is a component of the CDK-activating complex that contributes to the assembly of CDK1/cyclin B. In addition, CDKs 7, 8, and 9 function as transcriptional CDKs.3 CDK4 and CDK6 phosphorylate the Rb protein, thus releasing the cell-cycle arrest during the G1 phase. CDK4 and CDK6 are activated by D-type cyclins, which are expressed in response to various extracellular signals such as stimulatory mitogens, inhibitory cytokines, cellcell contact, and other spatial cues.

These CDKs are inhibited by phosphorylation, ubiquitination, and binding of the endogenous cellular inhibitor p16INK4a (Figure 1). p16INK4a is encoded by CDKN2A (also known as ARF or INK4a) and its expression is induced by variety of hyperproliferative stress signals.

Thus, competition between stress-activated p16INK4a and mitogen-activated D-type cyclins to bind with CDKs determines whether the cell stays in G1 arrest or proceeds to S phase.

Regulation of CDKs by p16INK4a is often disrupted in cancer cells, causing increased activity of CDKs. Additionally, p16INK4a expression suppresses tumorigenic effects in cancer cell lines.

Many studies identify the CDKN2A gene as a frequent target of inactivating mutations and deletions in many human cancers, and show that the loss-of-function alterations in genes encoding p16INK4a and pRb are mutually exclusive events in tumor cells.

There are enough preclinical data suggesting the role of CDKs in tumorigenesis. Hence, CDKs have been investigated as antitumor drug targets. CDK expression and assembly with D-type cyclins depend on the activation of the RAS-dependent kinase cascade involving the sequential activation of RAF1, MEK1, MEK2, and ERKs. Similarly, a separate RAS signaling pathway involving PI3k and AKT also prevents phosphorylation of D-type cyclins. Cancer-specific mutations affecting receptor tyrosine kinases (RTKs), RAS, RAF, PI3K, or PTEN can enhance D-type cyclin-dependent CDK4/6 activities, thus promoting oncogenesis. Conversely, inhibitors of these intracellular signaling cascades such as antiproliferative cytokines and antagonists of hormones and interleukins can decrease the activity of CDK4 and CDK6, inducing cell-cycle arrest.

CDK4/6 inhibitors have a cytostatic effect by inhibiting the cell cycle. Thus, they are primarily expected to provide disease control rather than objective responses even though preclinical studies have shown that CDK4/6 inhibition can produce rapid tumor regression and decrease in tumor burden in multiple human tumor xenograft models at high doses. Tumor cell kill was evident with high doses of palbociclib (Ibrance), a selective CDK4/6 inhibitor against human xenograft models of colon and breast cancer in a study by Fry et al.9 However, recent clinical trials have failed to replicate objective tumor responses seen in these preclinical experiments. But, significant disease control has been shown consistently as benefits in progression-free survival (PFS) owing to the cytostatic nature of CDK4/6 inhibitors. Palbociclib demonstrated remarkable PFS benefit in the PALOMA 1 trial in combination with letrozole compared with letrozole only (20.2 vs 10.2 months) among postmenopausal women with estrogen receptor (ER)positive, HER2-negative, advanced breast cancer. There was a modest benefit in objective response (43% vs 27%).

In the PALOMA 3 trial, palbociclib in combination with fulvestrant compared with fulvestrant alone in postmenopausal women with ER positive, HER2-negative, advanced breast cancer who have progressed on prior endocrine therapy showed significant PFS benefit (9.2 vs 3.8 months). There was no significant benefit in objective response rate.

Among patients with CDK4-amplified liposarcomas that have progressed despite systemic therapy, treatment with palbociclib was associated with a favorable PFS in a phase II study. Of 29 patients evaluated, PFS was 66% (90% CI, 51-100) at 12 weeks, significantly exceeding the primary endpoint of PFS >40% at 12 weeks.

Majority of patients had stable disease (SD), with only 1 objective response noted among 57 evaluable patients. Similarly, in a phase III randomized trial, ribociclib, another specific CDK4/6 inhibitor, in combination of letrozole compared with letrozole plus placebo, yielded significant benefits in PFS. The median duration of PFS was not reached in the ribociclib group (95% CI, 19.3 to not reached) versus 14.7 months (95% CI, 13.0-6.5) in the placebo group (hazard ratio, 0.56; 95% CI, 0.43-0.72; P <.001) for superiority). There was a modest difference in response rate (52.7% vs 37.1%, P <.001).

CDK Inhibition in NonSmall Cell Lung Cancer

Inactivation of the Rb pathway is seen in more than 70% of nonsmall cell lung cancer (NSCLC). The most common event for inactivation is the loss of the CD- KN2A gene. After multiple preclinical studies demonstrating frequent aberrations in p16INK4a among lung cancer cell lines, Gazzeri et al studied genetic changes of p16INK4a in 43 NSCLC samples.

Twenty-one of these samples exhibited one of the following genetic or epigenetic alterations affecting p16INK4a: homozygous deletion, frame-shift or missense mutation or methylation of exon 1 alpha. Moreover, its inactivation occurs frequently in patients with mutant KRAS lung adenocarcinomas. Mutant KRAS exerts its oncogenic activity through the dysregulation of several signaling networks. Among these, the most extensively characterized are the RAF/MAP-ERK kinase (MEK)/ex- tracellular signal-related kinase (ERK) and the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathways.

In approximately 30% of 323 lung adenocarcinoma cases from The Cancer Genome Atlas (TCGA) database with oncogenic KRAS mutations, 53% had concurrent inactivation of p16 by one of the mechanisms described above.

Of interest, p16 methylation was linked to KRAS mutation but mutually exclusive with EGFR mutation.

Loss of p16 appears to confer a worse clinical phenotype with increased metastasis and decreased survival.

Moreover, Puyol et al demonstrated that CDK4 ablation induces immediate senescence in NSCLC with KRAS-activating mutations.

Subsequent human xenograft models of NSCLC showed that tumors with KRAS mutations were more sensitive to CDK inhibitors relative to KRAS wild-type tumors.

The antitumor ef cacy of CDK inhibition against KRAS-mutant lung cancer also appears to be synergistic in combination with MEK inhibition, with combination therapy showing increased cytotoxicity than either agent alone, both in vitro and in vivo.

In addition to activating KRAS and loss of INK4a gene causing tumorigenesis, CDK4 ampli cation and cyclin D1 (CCND1) amplifications have also been associated with lung cancer, specifically squamous cell lung cancer (SqCLC). In TCGA, CCND1 amplification was seen in 12% of SqCLC, while only 1% of SqCLC is expected to have CDK4 amplification.

Thus, there has been a significant interest in exploring the role of CDK4/6 inhibitors, specifically in lung cancer.

The role of CDK inhibitors in lung cancer is not as established as it is in hormone receptorpositive breast cancer. Table 1 summarizes representative trials of CDK inhibitors currently being tested in lung cancer. Palbociclib and abemaciclib (LY2835219) are selective CDK4/6 inhibitors currently being investigated in NSCLC. Palbociclib has a half-life of 25.9 to 26.7 hours and is dosed at 125 mg daily (3 weeks, 1-week drug holiday) or 200 mg daily (2 weeks, 1-week drug holiday).

Abemaciclib has a half-life of 17 to 38 hours and is dosed at 200 mg twice daily, continuously. Abemaciclib crosses the blood brain barrier. The major dose-limiting toxicities with palbociclib are neutropenia and thrombocytopenia, while those of abemaciclib are fatigue and gastrointestinal (GI) events (diarrhea).

Ribociclib is another selective CDK4/6 inhibitor that recently received priority review from FDA as first- line therapy combination with letrozole in metastatic HRpositive breast cancer. It is dosed at 600 mg daily (3 weeks on, 1 week off), is being explored in other advanced solid tumors in phase I/II trials, and has limited data in NSCLC.

Patnaik et al evaluated a cohort of 68 patients with previously treated NSCLC, including 29 patients harboring KRAS mutations. The overall disease control rate (CR+PR+SD) with abemaciclib was 49% in the total study population, 55% in the KRAS-mutant cohort, and only 39% in the KRASwild type cohort. The 6-month PFS was 26% (95% CI, 16-38). The common KRAS mutation involving codons 12 and 13 and the less frequent KRAS mutations Q61H and A146V were associated with decrease in tumor size following single-agent abemaciclib treatment. SD lasting more than 24 weeks was seen in 31% of the KRAS mutant cohort compared with 12% in the wild-type cohort. The most common treatment-related adverse events included fatigue and gastrointestinal effects such as diarrhea, nausea, vomiting, anorexia, and weight loss. Only 1% of all the enrolled patients had grade 4 toxicity (neutropenia).

This study suggested that abemaciclib has promising activity against RAS-mutant NSCLC and that the recommended dose of 200 mg twice daily appears to be safe and tolerable.

JUNIPER (NCT02152631) is a randomized, phase III study of abemaciclib with best supportive care versus erlotinib with best supportive care in patients with stage IV NSCLC with a detectable KRAS mutation, whose disease has progressed after platinum-based chemotherapy. The primary outcome measures are PFS and overall survival (OS). The estimated study completion date is August 2019.

Gopalan et al conducted a phase II, single-arm study of palbociclib in 19 previously treated patients with recurrent or metastatic NSCLC who are negative for p16 expression by immunohistochemistry.

The primary endpoint was overall response rate. Of the 16 evaluable patients, there were no responses, thus the trial was closed to accrual. However, 8 patients with previously progressive disease had SD lasting 16 to 42 weeks. The remaining 8 patients reported progressive disease within 8 weeks. The median PFS was 12.5 weeks, comparable to other second-line chemotherapy in this setting. In addition to the modest clinical activity, 5 out of 16 patients developed grade 3 or 4 toxicities (transaminitis, rhab- domyolysis, neutropenia, and thrombocytopenia). This study required that tumors be negative for p16 expression. In contrast, the S1400C substudy of the LUNG MAP trial is investigating the role of palbociclib for patients with previously treated stage IV Sq- CLC (NCT02154490), whose tumors express gene amplification of CDK4, CCND1, 2, or 3, for eligibility. This study will evaluate the role of these cell-cycle gene alterations as a predictive biomarker for palbociclib in this patient population.

In terms of agents that inhibit CDKs other than CDK4/6, dinaciclib (MK-7965, formerly SCH 727965), a potent inhibitor of CDKs 1, 2, 5, and 9, had been evaluated in a randomized phase II study versus erlotinib in patients with previously treated NSCLC.

Preclinical studies have shown that dinaciclib is a potent inducer of apoptosis of various cell lines, including lung cancer.

In this trial, 17 patients were allocated into the dinaciclib arm while the erlotinib arm had 33 patients. Dinaciclib was administered at 50 mg/m2 over 1 to 2 hours IV infusion. The study showed that dinaciclib, as expected, is more toxic in terms of myelosuppression than erlotinib. A total of 78% patients in the dinaciclib arm had grade 3 or 4 AEs, including relatively higher rates of GI events such as diarrhea, nausea and vomiting, while only 29% of patients in erlotinib arm en- countered grade 3 or 4 adverse events, mainly dermatologic. The time to progression was 1.49 months (95% CI, 1.31-2.63) with dinaciclib while it was 1.58 months (95% CI, 1.38-2.83) with erlotinib. This study did not meet its primary endpoint in establishing activity of dinaciclib as monotherapy in this population. However, unlike more contemporary CDK4/6 inhibitor studies, this study was performed among molecularly unselected, advanced NSCLC patients.

Indeed, preclinical investigations of dinaciclib suggest that its activity is exerted through inhibition of MCL1, a BCL2 anti-apoptotic family member.

Additional data suggest that high ratio of MCL1 mRNA relative to BCL- XL or high MCL1 copy number may be a predictive biomarker for antitumor response to dinaciclib in solid malignancies, including lung cancer.

Several trials are currently exploring various combinations of CDK inhibitors with other agents. As described above, MEK is the downstream protein&nbsp;to RAS in the MAPK/ERK pathway (RAS-RAF-MEK- ERK pathway). Activity of D-type cyclins is highly dependent on RAS signaling. Preclinical studies showing synergistic effect of CDK inhibition and MAP kinase inhibition in melanoma, pancreatic cancer, neuroblastoma, as well as lung cancer have generated significant interest in this approach.

Perhaps the predominantly cytostatic effects of CDK4/6 inhibition could be reprogrammed to induce apoptosis in response to drugs targeting RTK and/or downstream MAPK signaling pathways that are essential for cell viability.

Indeed, a recent screen of drug combination in ALK-mutated neuroblastoma showed synergistic efficacy for the combination of ceritinib, an oral ALK inhibitor, with ribociclib, compared with each drug alone.

A phase Ib/II study is evaluating safety and efficacy of this combination in patients with ALK-positive NSCLC (NCT02292550).

As described previously, combination of MEK and CDK inhibition was synergistic in KRAS-mutant NSCLC. A phase I study is ongoing to determine the maximally tolerated dose and recommend phase II dose for the combination of palbociclib and MEK inhibitor PD-0325901. Subsequently, the randomized phase II portion of the study will then further analyze the response rate and PFS with the combination regimen (palbociclib with PD-0325901) compared with single-agent palbociclib and single-agent PD- 0325901 (NCT02022982) for this lung cancer genotype. The estimated study completion date is December 2020.

Data for CDK inhibition in small cell lung cancer (SCLC) are sparse. CDK4/6 inhibitors require a functioning Rb gene to exercise their antitumor property. There is a loss-of-function mutation of the Rb gene in about 90% of SCLC, so CDK4/6 inhibitors are theoretically ineffective.

Roniciclib (BAY 1000394) inhibits the activity of CDKs 1, 2, 3, and 4, and of transcriptional CDKs 7 and 9. It has demonstrated antitumor activity among various solid tumor cell lines. Siemeister et al showed in a preclinical study that roniciclib strongly improved the efficacy of the standard-of-care combination treatment with the DNA-damaging agents cisplatin and etoposide in the Rb-negative NCI-H82 SCLC xenograft model without further worsening the tolerability of the therapy.

Subsequently, the randomized, phase II CONCEPT- SCLC trial was conducted to investigate the safety, efficacy, and clinical benefit of roniciclib versus placebo, when given in combination with cisplatin/carbopla-

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This manuscript focuses on the latest clinical developments of newer generation selective CDK inhibitors in lung cancer.

Clinical Development of Cyclin-Dependent Kinase Inhibitors in Lung Cancer

Results from the phase I portion of the first trial testing axicabtagene ciloleucel (KTE-C19), an autologous CD3&zeta;/CD28-based chimeric antigen receptor (CAR) T-cell therapy, indicate that the regimen can safely be administered and lead to durable complete remission (CR) after more than 12 months in patients with refractory diffuse large B-cell lymphoma (DLBCL). Frederick Locke, MD, a medical oncologist at Moffitt Cancer Center, Tampa, Florida, and colleagues reported an overall response rate of 71% (5 of 7 patients) including 4 patients who developed a rapid CR within 1 month of treatment. The treatment was durable, with 43% (3 patients) remaining in complete remission after one year. The results of this phase 1 study led to the initiation of the pivotal ZUMA-1 phase 2 registration trial.

&ldquo;The overall and complete response rate in this small group of patients is remarkable, as the expected complete response rate for such patients is 8% with conventional chemotherapies,&rdquo; said Locke in a statement. &ldquo;This is truly an exciting time for the oncology community and our patients. Engineered immune cell therapies are one step closer to widespread availability,&rdquo; added Locke, who is director of Research for Moffitt&rsquo;s Immune Cell Therapy Clinical Trial Group.

Axicabtagene ciloleucel, developed by Kite Pharma, is an autologous CAR T-cell therapy in which T cells are isolated from a patient&rsquo;s blood and engineered to target the CD19 protein that is found on lymphoma cells. The re-targeted T cells are then infused back into the same patient.

The researchers demonstrated that the conditioning regimen of cyclophosphamide at 500 mg/m2 and fludarabine at 30 mg/m2 followed by axicabtagene ciloleucel is safe for further study with manageable toxicity. In the multicenter ZUMA-1 phase 1 study, patients received chemotherapy with concurrent cyclophosphamide and fludarabine chemotherapy (500 mg/m2) for 3 days, followed by KTE-C19 at a target dose of 2 x 106 CAR T cells/kg.

Eligible patients had ECOG performance status of 0 or 1, absolute neutrophil count of &ge;1,000/mL, and platelet count of &ge;50,000/mL. Patients must have had all of the following: (1) histologically confirmed B-cell non Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma, or transformed follicular lymphoma (TFL); (2) chemotherapy refractory disease; (3) prior therapy must have included an anti-CD20 monoclonal antibody-containing regimen and an anthracycline-containing chemotherapy regimen; for patients with TFL, prior chemotherapy for follicular lymphoma and subsequent refractory disease after transformation to DLBCL; (4) at least 1 measurable lesion; (5) no evidence of central nervous system lymphoma by magnetic resonance imaging; and (6) &ge;2 weeks since prior radiation therapy or systemic therapy at the time of leukapheresis.

The researchers also concluded that cryopreserved KTE-C19 is associated with robust CAR T-cell expansion and durable clinical responses that are similar to those observed with fresh anti-CD19 CAR T-cell therapy in studies conducted at single institutions. Three patients were refractory to second-line or later lines of therapy, and 4 patients had relapsed post autologous stem cell transplantation (ASCT) within 1 year. Two of the 4 patients who relapsed post ASCT received another regimen before enrollment: patient 4 had stable disease after 3 cycles of R-GEMOX and patient 6 had progressive disease after 2 cycles of ipilimumab and lenalidomide therapy, meeting the definition of refractoriness as defined in the protocol.

The investigators reported that all patients experienced KTE-C19-related adverse events of any grade, with grade 3 and 4 events reported in 4 (57%) and 1 (14%) patient(s), respectively. No patient had a grade 5 KTE-C19-related event. B-cell aplasia and hypogammaglobulinemia was observed in 3 patients.

This is the first multicenter study of a CAR-T therapy that was produced at a centrally located facility. The manufacturing process for axicabtagene ciloleucel (KTE-C19) was successful for all of the patients and was completed within approximately 2 weeks.

Results from the phase I portion of the first trial testing axicabtagene ciloleucel, an autologous CD3&zeta;/CD28-based CAR T-cell therapy, indicate that the regimen can safely be administered and lead to durable CR after more than 12 months in refractory diffuse large B-cell lymphoma.

2017 February | The Journal of Targeted Therapies in Cancer

Immune Checkpoint Approaches in AML and MDS: A Next Frontier?

Immune Checkpoint Inhibitors in CRC

Clinical Development of Cyclin-Dependent Kinase Inhibitors in Lung Cancer

Axicabtagene Ciloleucel Demonstrates Safety and Efficacy in Lymphoma

Missing Tumor-Specific Mutations Lead to Increased Resistance to Checkpoint Inhibitors