Evolving Therapies in Relapsed and Refractory Hodgkin Lymphoma

Approximately 20% of Hodgkin Lymphoma (HL) patients do not achieve a durable remission or fail to respond to front-line chemotherapy. Despite the attempt to improve clinical outcomes by using the risk adaptive therapy, a significant number of patients die as a results of relapsed/refractory (rel/ref) disease.1 Advances in understanding the etiology and molecular biology of HL are leading the development of novel therapeutic strategies that could be applied to improve clinical outcome of rel/ref HL patients. The pathologic features of HL reflect a defect in immune responses resulting from various cytokines and chemokines secreted partially by Hodgkin Reed-Sternberg (HRS) cells. HRS cells are unique in the way that they lost typical B cell gene expression pattern but retain the expression of surface molecules involving in antigen presentation (tumor necrosis factor receptor (CD30, CD40), CD80, MHC class II, and CD86). Aberration of Notch signaling pathway may contribute to their reprogramming.2,3 Multiple genetic lesions, deregulated signaling pathway and transcription factors play important role in pathogenesis of HL including constitutive activation of nuclear factor kappa B (NFκB) and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway.4-6 Moreover, the role of the microenvironment in HL has been increasingly recognized. The majority of the cell population in HL-affected tissue is composition of the inflammatory cellular infiltrate, not the HRS cells that represents only small population (<1%). Understanding the complex relationship between the HRS cells and the microenvironment and chemokines milieu involved in its formation is crucial for the development of new therapeutic strategies.


Introduction
Approximately 20% of Hodgkin Lymphoma (HL) patients do not achieve a durable remission or fail to respond to front-line chemotherapy. Despite the attempt to improve clinical outcomes by using the risk adaptive therapy, a significant number of patients die as a results of relapsed/refractory (rel/ref) disease. 1 Advances in understanding the etiology and molecular biology of HL are leading the development of novel therapeutic strategies that could be applied to improve clinical outcome of rel/ref HL patients. The pathologic features of HL reflect a defect in immune responses resulting from various cytokines and chemokines secreted partially by Hodgkin Reed-Sternberg (HRS) cells. HRS cells are unique in the way that they lost typical B cell gene expression pattern but retain the expression of surface molecules involving in antigen presentation (tumor necrosis factor receptor (CD30, CD40), CD80, MHC class II, and CD86). Aberration of Notch signaling pathway may contribute to their reprogramming. 2,3 Multiple genetic lesions, deregulated signaling pathway and transcription factors play important role in pathogenesis of HL including constitutive activation of nuclear factor kappa B (NFκB) and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. [4][5][6] Moreover, the role of the microenvironment in HL has been increasingly recognized. The majority of the cell population in HL-affected tissue is composition of the inflammatory cellular infiltrate, not the HRS cells that represents only small population (<1%). Understanding the complex relationship between the HRS cells and the microenvironment and chemokines milieu involved in its formation is crucial for the development of new therapeutic strategies.
In addition, Epstein Barr Virus (EBV) infection may plays a role in the pathogenesis of HL since it can influence the expression of certain chemokines (i.e. CXCL9, CXCL10, CCL3, and CCL5) that are highly expressed in HRS cells and the EBV gene encoding the latent membrane protein 1 (LMP1) can mimic constitutively active TNF receptor (via a CD40/CD40L interaction) and promote IκB turnover leading to activation of NFκB and downstream signaling events. 7 Ongoing efforts are focused in developing adaptive or adoptive immune therapies targeting EVB related proteins in rel/ref HL patients. Gr

CD40
CD40 is a member of TNFR family (TNFRSF5) that is constantly expressed in HRS cells. In addition, CD40+ HRS cells are surrounded by CD40L-expressing T-cell lymphocytes. The CD40/CD40L interaction contributes to the pathobiology of HL possibly by NFκB activation and increased autocrine growth factor CCL5 resulting in inhibition of apoptosis, increased proliferation and microenvironment formation. [17][18][19] Lucatumumab is a monoclonal antibody targeting the transmembrane receptor CD40.

CD20
A pilot study evaluating rituximab monotherapy in rel/ref HL has shown that depletion of B lymphocytes may has therapeutic potential. 23 The rationale of using rituximab in classical HL (cHL) is based on several laboratory and clinical observations: HRS stem cells express CD20 even though HRS cells infrequently express CD20, elimination of CD20+ B lymphocytes supporting HRS cells may deprive survival signals and improve immune response against HRS cell. 24 Previously unknown factors influenced the negative results observed in such trials such as 1) the agonist effects of the antibodies initially tested, 2) the modulation of CD30 in HRS cells (i.e. antigen shedding and/or internalization), and/or 3) impaired immune effector function in heavily pre-treated patients with HL. Two therapeutic approaches (i.e. development of drug conjugates or antibody re-engineering) had been explored clinically with significant improvement in clinical activity.  (Table 3)

NFκB activity
As previously noted, constitutive activation of NFκB is one of the most important events in pathogenesis of HL and the result of multiple mechanisms.   52 However, their clinical activity in HL remains unknown.

Deacetylation of histone and other cellular proteins in HL biology: The therapeutic role of deacetylase inhibitors (DACi)
Gene expression profiling studies had demonstrated that HL is characterized by the silencing of key regulatory genes involved in B-cell maturation (i.e. CD79a, CD19, CD20, etc). In general gene expression is a tightly regulated process that is influenced by the 1) DNA/mRNA sequence, 2) expression/activity of transcription factors, 3) epigenetics (including the DNA, chromatin, and histone modifications); and 4) messenger RNA stability.
Post-transcriptional histone modification plays an important role in regulating gene transcription and is mediated by two groups of enzymes: histone acetyltransferase (HATs) and histone deacetylase (HDACs). The balance between HATs and HDACs is crucial in regulating the expression/function of several proteins involved in cell proliferation, cell cycle, apoptosis, angiogenesis, and immune regulation. Altering balance between HATs and HDACs had been found to be associated with various malignancies including HL.
To date, 18 HDACs have been identified in humans. HDACs are grouped in two major categories and four classes; zinc-dependent HDACs (Class I, II and IV) and NAD-dependent HDACs (Class III). Class I includes HDAC 1, 2, 3, 8, and 11; Class II includes HDAC 4, 5, 6, 7, 9, and 10; Class III includes homologues of yeast SIRT 1-7, and Class IV, which includes only HDAC 11. As a group, HDAC are known to regulate several key cellular functions such cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. The activity spectrum of each HDAC is yet to be defined and there is overlap between the function of different HDAC regardless of their group or class.
HATs and HDACs interact also with non-histone proteins such as transcription factors (i.e.   61 Panobinostat was administered at a dose of 40 mg thrice weekly in 21-day cycle until disease progression. The update results showed encouraging clinical activity of panobinostat with 1 patient achieved CR and 10 pateints achieved PR. 62 Moreover, disease control rate (CR+PR+SD) was 79%. Panobinostat was well tolerated and reversible thrombocytopenia was managed by dose delay or dose reduction. The interim results for this phase II study continues to demonstrate encouraging activity of panobinostat. 63 The final results are currently not available. As previously demonstrated with other DAC inhibitors, a decrease in serum TARC levels was observed in panobinostat treated patients achieving an objective response (i.e. PR or CR). 64 The safety and efficacy of the oral agent belinostat (PXD-101) was evaluated in patients with rel/ref NHL or cHL by Zain et al. 65 Tumor size reduction found in 1/3 of patients with HL using the recommended dose-schedule for patients with solid tumors (750 mg daily, D1-14 every 21days).

Entinostat (SNDX-275) is a class I isotype-selective HDAC inhibitor with longer half-life.
Pre-clinical data from Khaskhely et al demonstrated in vitro activity in HL-derived cell lines. 66 Entinostat induced cell death with an IC50 of 0.4 μM. At the molecular level, entinostat up-regulates p21 expression, increased H3 acetylation and down-regulates the anti-apoptotic X-linked inhibitor of apoptosis (XIAP) resulting in apoptosis. Moreover, the combination entinostat with gemcitabine or bortezomib has shown synergistic effects. Jóna et al found that entinostat down-regulates anti-apototic Bcl-2 and Bcl-xL expression without altering Mcl-1 or Bax levels and its effect was enhanced by two Bcl-2 inhibitors (ABT-737 and obatoclax). 67 Younes et al recently presented an update of a phase II clinical study evaluating the safety and efficacy of entinostat as a single agent in relapsed/refractory HL. 68  patients had failed HDC-ASCT, and 4 of those patients had also failed an alloSCT. 70 Givinostat was administered at a dose of 100 mg orally daily in three 4-week cycles. Seven of 13 patients (54%) whom completed at least one cycle of therapy were evaluable for response, SD was observed in 46% of the patients. Toxicity includes grade 1-2 thrombocytopenia, leukopenia, diarrhea and/or abdominal pain; nonetheless, twenty percent of patients had transient drug discontinuation due to prolonged QTc. Another phase II study evaluated the safety and clinical activity of givinostat in combination with meclorethamine in patients with rel/ref HL. 71 Anti-tumor activity was observed and correlated with a reduction in serum TARC levels.

Targeting the PI3K/AKT/mTOR pathway in HL
Everolimus (RAD001) binds to FK506-binding protein 12 (FKBP12) forming a complex that has mTOR kinase inhibition activity, inhibit tumor cell proliferation and angiogenesis by decreasing hypoxia-inducible factor 1a (HIF1a) expression. 72 Preclinical data from Georgakis et al showed temsirolimus (CCI-779) induced cell cycle arrest at G0/G1 phase and autophagy in HL-derived cell lines suggesting that this particular mTOR inhibitor may have therapeutic value in patients with HL. 76 Several phase I/II trials studying the safety and efficacy of single agent various mTOR inhibitors (i.e. temsirolimus or everolimus) monotherapy, or combination with lenalidomide or sorafinib are being conducted to test the concept of mTOR inhibition in treatment rel/ref HL.

Heat Shock Protein (HSP)
Heat shock protein acting as chaperones are essential in promoting cell survival by maintaining the structure and function of key regulatory proteins involved in cell cycle, proliferation and apoptosis. HSP over-expression had been demonstrated in several malignancies including HL. Inhibition of HSP is another attractive target in cancer therapeutics. Boll et al demonstrated the biological effects of a HSP90 inhibitor, BIIB021 on HL-derived cell lines. 77 The investigators demonstrated that, BIIB021 inhibited the constitutive activity of NFκB independent of IκB mutation status and increased susceptibility of HL cells for NK cell-mediated killing via inducing the expression of activating NK-cell ligands. Schoof et al showed that inhibition of HSP90 by either geldanamycin derivative 17-AAG or RNA interference in HL cells led to decrease cell proliferation and inhibition of STAT1, STAT3, STAT5, and STAT6 tyrosine phosphorylation possible secondary to reduced protein expression of Janus kinase (Jaks). 78 HSP90 may be a promising target in patients with rel/ref cHL.

Lenalidomide
Lenalidomide, a novel IMiDs  immunomodulatory drug is emerging as an attractive therapeutic option for patients with B-cell lymphoproliferative neoplasms, including HL. Studies in lymphoma and multiple myeloma (MM) models have demonstrated that lenalidomide exerts higher anti-tumor activity than thalidomide, has a unique capacity to enhance the innate immune system, enhance the anti-tumor activity of rituximab, and inhibit angiogenesis. 79,80 Abnormal immune response, increased angiogenesis, and apoptosis resistance, which contribute to the development of HL, support the scientific standpoint of evaluating lenalidomide in rel/ref HL. [81][82][83] Lenalidomide was evaluated in patients with relapsed/refractory HL in three phase II clinical trials (  Table 4. Clinical trials evaluating lenalidomide monotherapy in relapsed/refractory Hodgkin lymphoma patients.

EBV specific CTL therapy
In general, while chemotherapy agents, small molecule inhibitors, drug immunoconjugates or immunodulatory drugs exhibit promising anti-tumor activity in patients with rel/ref HL, the duration of response to each agent when reported by investigators is rather short. Giving the median age of patients with rel/ref HL the incorporation of therapeutic strategies with durable remissions is necessary. Two therapeutic approaches are been actively evaluated with promising results: 1) Autologous or allogeneic LMP2-specific cytotoxic T-cell lymphocytes (CTL) and 2) allogeneic bone marrow transplantation.
Approximately 30-40% of HL cases are associated with EBV infection of the HRS cells as proven by the expression of viral latent membrane protein (LMP). 88,89 EBV is known to induce the surface expression of three latent antigens in EBV-infected HRS cells: LMP1, LMP2 and Epstein Barr nuclear antigen 1 (EBNA1). Immunotherapy targeting EBV related proteins in HRS cells is an area of active translational research. The generation and ex vivo expansion of cytotoxic T-cell lymphocytes (CTLs) specific for one or more EBV antigens had been studied in patients with hematological malignancies such as post-transplant lymphorpoliferative disorders and rel/ref HL. In general two EBV-related immunotherapy approached had been studied: 1) adoptive immunotherapy (administration of autologous or allogeneic EBV specific CTLs) and 2) vaccination of relevant epitopes from one of the EBV antigens to boost the patients' own immune response. 90,91 Lucas et al demonstrated the clinical efficacy of allogeneic EBV-specific CTLs in EBV-positive rel/ref HL who had previously failed HDC-ASCT. 92 Significant clinical activity was observed following allogenic CTLs infusion despite a lack to detect donor chimerism. In addition, in the limited number of patients evaluated the administration of fludarabine prior to CTLs infusion enhanced the clinical responses observed. While clinical effects had been observed in HL patients treated with EBV-specific CTLs, the anti-tumor effects are not as robust as what has been observed in patients with PTLDs. Several observations can account for such differences, EBV infected HRS usually express less immunogenic EBV proteins in contrast to PTLD patients (LMP2). In addition, HRS cell have mechanisms to evade immune response to EBV infection including down-regulation of the immunogenicity of latent EBV antigen (i.e. LMP2) and secretion of the immunosuppressive cytokines such as IL10, IL-13, TARC, TRAFs (tumor necrosis factor receptor-associated factors) and TGF which may suppress the efficacy of EBV specific CTL. [93][94][95] Using the strategies to enhance Th1 CTLs development and decrease Th2 cytokine production may overcome the defective immune recognition of HRS cells and improve the efficacy of the EBV specific CTL therapy in rel/ref HL (Table 5).  Table 5. Ongoing trials of EBV-Specific CTLs for patients with relapsed/refractory Hodgkin lymphoma.

Allogeneic bone marrow transplant (AlloSCT)
Approximately 50% of patients with relapsed Hodgkin lymphoma who undergo HDC-ASCT relapse as a consequence of refractory disease, usually within the first year posttransplant. 96,97 Relapsed HL patients after HDC-ASCT have a poor clinical outcome and represent a therapeutic challenge for the practicing oncologist. There are several proposed risk factors to identify patients at high risk to develop disease progression following HDC-ASCT such as chemotherapy resistant disease prior to HDC-ASCT, B symptoms at the time of relapse, residual disease at the time of transplantation by functional imaging, extra-nodal disease at the time of relapse, and bulky disease. 98,99 Patients with any of these high risk factors may be suited for alternative therapeutic strategies such as tandem transplant, allogeneic bone marrow transplant, and/or post-HDC-ASCT maintenance therapy in the context of a clinical trial.
A second HDC-ASCT could be considered for patients with a long period of remission following the initial transplant (>3 years) or those whom alloSCT is not feasible. 100 AlloSCT has been used in patients with rel/ref HL with controversial results. Often the high incidence of transplant related mortality (TRM) offsets the potential clinical benefit. 101 While the incorporation of reduced intensity conditioning regimens has been associated with lower TRM rates, the long-term PFS rates rarely exceed 20-25% questioning the validity of this approach. Patients who have chemotherapy sensitive disease, non-bulky disease, and have greater than 1 year of remission after the first HDC-ASCT seem to have most benefited with this approach. 102 Despite early good responses, the results of RIC-AlloSCT demonstrate a disappointing clinical outcome and lack of long term disease control with 2 year OS of 29-66% and 2 year PFS of 20-39% regardless of conditioning regimens or donor types. [103][104][105] Peggs et al reported durable response to donor lymphocyte infusion (DLI) in patients with relapsed HL post alloSCT that incorporated in vivo T-cell depletion. 106 DLI was administered to 46 patients for mixed chimerism (n=22) and relapsed disease post-alloSCT (n=24). Eighty-six percent of patients with mixed chimerism converted to full donor status and had a 4-year relapse incidence of 5%. More importantly, CR and PR were noted in 58.3% and 20.8% of 24 patients with relapsed disease respectively, suggesting the existence of graft vs. HL effects. Ongoing clinical studies are investigating the role of alloSCT in relapsed HL patients with poor-risk features who are at high risk to relapse after HDC-ASCT.
In summary, promising therapies are emerging for the treatment of rel/ref HL. Substantial and occasionally durable remissions have been observed with some therapeutic interventions, such as HDACi, drug immunoconjugates, and cellular therapy. Ongoing studies will hopefully guide the integration of these therapies in the current treatment of high-risk HL in an attempt to improve cure rates. In addition, ongoing scientific and translational research and the importance of the development of novel therapeutics for patients with ref/rel HL should not be underemphasized.