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Flipping the cyclin D1 switch in mantle cell lymphoma

Best Practice & Research Clinical Haematology, 2, 25, pages 143 - 152

Mantle cell lymphoma (MCL) is a rare, aggressive subtype of B cell NHL for which there is no standard of care. It is characterized by the t(11;14) translocation, implicating cyclin D1 (CCND1) in its pathogenesis. Cyclin D1 is one of a family of 3 unlinked D type cyclin genes, CCND1, 2, 3. CCND1 is not expressed in normal B cells. Deregulated expression occurs as a result of juxtaposition of cis IgH enhancer elements, Eμ and 3′ Cα, to the cyclin D1 gene. These enhancer elements and regions upstream of the CCND1 gene are hypomethylated on the translocated allele. Histones surrounding the translocation have shown hyperacetylation as well, a hallmark of transcriptionally active chromatin. The t(11;14) translocation is an epigenetic event, leading to cyclin D1 deregulated transcription. These findings provide the rationale for the use of epigenetic and targeted cyclin D1 therapies to overcome resistance and induce durable remissions in MCL.

Keywords: epigenetics, mantle cell lymphoma, MCL, cladribine, vorinostat, rituximab, cyclin D1, IgH, methylation, histone, CTCF, t(11;14), CD20, bortezomib, curcumin, PD0332991, nucleophosmin.

Introduction

Mantle cell lymphoma (MCL) is a distinct clinical and biological entity that comprises 6–8% of non-Hodgkin's lymphoma. Clinically it follows an aggressive course with a median survival of 3–5 years and has a poor response to traditional chemotherapeutic approaches. The pathogenesis of MCL can be linked to the t(11;14)(q13;32) translocation, which results in the deregulated expression of the cyclin D1 protein and proliferation of mature CD20+, CD5+ monoclonal B-lymphocytes [1], [2], and [3]. This review will focus on the regulation of cyclin D expression in normal and neoplastic B cells, particularly in MCL.

Regulation of cyclin D expression in normal B cells

The D type cyclins comprise a family of three unlinked proteins (cyclin D1, D2, and D3) that function mainly in the regulation of the G1 to S phase transition during the mammalian cell cycle. These proteins are not kinases but function in conjunction with cyclin dependent kinases such as cdk4 and cdk6. The cdk/cyclin D complex regulates the phosphorylation of the retinoblastoma protein (RB) which in turn regulates the E2F family of proteins that control entrance into the cell cycle [4] .

Cyclin D1 coordinates growth signals with cell cycle progression. When assembled into the cyclin D1/CDK4/6 complex, an active kinase that phosphorylates the retinoblastoma protein (RB) is formed. Phosphorylation of RB removes its repressive function on cell cycle progression [5] . Recently, cdk independent functions of the D type cyclins have been recognized. Cyclin D1 has been shown to be a DNA binding protein that functions through interaction with CCAAT enhancer binding protein alpha (CEBPα) in cancer cells [6] . Cyclin D1 also plays a role in neuronal development, and genomic analyses have defined multiple genes that are regulated by cyclin D1 in the nervous system [7] . The D type cyclins, D2 and D3, are expressed in both normal and neoplastic B and T lymphocytes. Cyclin D1 is not expressed in normal hematopoietic cells, and its expression in hematologic malignancies is predominantly in multiple myeloma (MM) and MCL. Both of these B cell malignancies contain the t(11;14) but with different breakpoints. A subset of CCND1(+) MM do not contain the t(11;14) but contain extra copies of chromosome 11 or a hyperdiploid phenotype that results in cyclin D1 overexpression. Cyclin D1 expression has also been reported in some cases of hairy cell leukemia (HCL) and MM that do not contain the t(11;14) translocation [8] .

This increased cyclin D1 expression is due to the juxtaposition of the BCL1 locus to an IGHJ segment of the IgH locus which gives control of cyclin D1 expression to IgH regulatory elements [3] and [9]. The IgH locus contains two classes of regulatory elements, an Eμ intronic enhancer and a 3′ Cα ( Fig. 1 ) [10] . For Burkitt's lymphoma (BL), patients are defined by breakpoints containing only the 3′ Cα region. This observation and published data from our lab suggests that the 3′ Cα region is necessary for IgH deregulated expression of oncogenes like c-myc, cyclin D1, and bcl-2 [11] .

gr1

Fig. 1 Map of CCND1 and IgH enhancer and promoter elements in the t(11;14)(q13;q32) translocation. Note that the IgH Locus contains two copies of the Cα enhancer and HS sites (second set not fully shown). Blue arrows denote epigenetically hypomethylated elements in MCL. (Adapted from Refs. [10] and [11]).

The mechanism of cyclin D1 deregulated expression by translocation of IgH regulatory elements remains obscure. RNA polymerase II (pol II) binds the cyclin D1 promoter and IgH regulatory elements, similar to published data for the β−globin Locus Control Region (LCR). However, the two model systems differ in other ways. The Groudine laboratory demonstrated that the β−globin LCR functions to regulate transcriptional elongation of the β−globin like genes [12] . We did not find any influence of the IgH elements on transcriptional elongation in the cyclin D1 locus [13] . We and others have investigated the chromatin structure of the cyclin D1 locus in normal B cells, MCL and MM cell lines with the t(11;14) and primary MCL cells [13] . The chromatin structure of the CCND1 locus is open with a hypomethylated promoter and upstream region that also contains acetylated histones. Surprisingly, the CCND1 chromatin domain is open in normal B cells, and the translocation of IgH regulatory elements serves not to open the locus but to recruit RNA pol II and associated cofactors to the IgH LCR and the CCND1 promoter. This work has been independently reproduced in different MM cell lines and patient samples [11] and [13].

The translocation of IgH elements also created a role for the master epigenetic regulatory protein CTCF (CCC containing binding protein) in the deregulated expression of cyclin D1. CTCF binds to the CCND1 locus and IgH regulatory elements in MCL cells. Our laboratory has found that CTCF's binding partner, nucleophosmin, is also cobound with CTCF at the CCND1 and IgH loci and together these proteins tether the translocated and nontranslocated CCND1 loci to the nucleolus in MCL cell lines and primary patient samples ( Fig. 2 ). Both CCND1 loci are linked epigenetically through a transallelic interaction termed transvection. Thus, the translocated CCND1 allele exerts an epigenetic effect on the nontranslocated CCND1 locus as assayed by changes in DNA methylation. This observation represents the first description of transvection in human cancer [11] .

gr2

Fig. 2 ImmunoFISH of colocalization of cyclin D1 and nucleolus. Cyclin D1 (yellow), nucleophosmin/nucleolus (pink), DAPI (blue). (Adapted from Ref.  [11] ).

The epigenetic control of cyclin D1 expression in B cell malignancies

DNA methylation and histone acetylation, mainly on histones H3 and H4, are known to be important in controlling gene expression in eukaryotes [14] . DNA hypomethylation and histone hyperacetylation have been shown to correlate with active transcription, and agents that prevent deacetylation and block methylation activate transcription of silenced genes [15] . Methylation usually occurs in regions of DNA that contain long segments of repeating CpG dinucleotides (CpG islands). Located within genes, in promoters and even in enhancer elements, these islands give rise to an epigenetic control of gene expression through inhibition of transcription when the islands are methylated. Additionally, methylated DNA can affect the expression of distant genes by changing the structure of chromatin domains and vice versa [16] and [17]. To make matters more complicated, histone acetylation is but one of many different kinds of epigenetic changes to silence or activate genes through histone modification; phosphorylation, sumoylation, ubiquitination, and methylation being some others [18] .

The CCND1 promoter has a CpG island, so it could theoretically be regulated by methylation [19] , and in cell lines, methylation of the cyclin D1 promoter can be found. For many years after the discovery of the t(11;14) translocation, it was believed that the IgH regulatory elements caused the cyclin D1 gene to be expressed by opening up its chromatin domain [20] . However, although hypomethylated DNA and hypoacetylated histones can be found near the translocated CCND1 gene in cell lines, an identical pattern can be found in normal B cells [21] . This means that there are more factors than just the aforementioned at play when the translocation is present.

Methylation of the cyclin D1 locus

The CCND1 locus is critical to the pathogenesis of MCL [22] . Though the t(11;14) translocation remains the most noteworthy hallmark of the disease, it is by no means the only alteration in the expression of cyclin D and its role in tumorigenesis. Epigenetics has been found to play an important role as well through modulation of DNA methylation and histone acetylation [21] . In order to appreciate this role, the molecular anatomy of the CCND1 locus and the various transcripts of cyclin D1 must be understood.

The CCND1 promoter is about 1.8–3.0 kb long and contains a CRE binding site as well as two SP-1 sites through which the majority of cyclin D1 transcription occurs [23] and [24]. The rat CCND1 promoter contains CpG islands in the regions of its SP-1 binding sites, and in the absence of CCND1 mutations or gene amplifications, methylation content inversely correlates with cyclin D1 expression [19] . In addition to these three factor binding sites near the CCND1 gene, the P519 and MTC regions that both also contain CpG islands are located 90–120 kb upstream of the CCND1 gene ( Fig. 1 ) [13] . Bisulfite sequencing was carried out on MCL cell lines (Granta) and MM cell lines (U266). The promoter of the CCND1 gene as well as the upstream MTC and P519 regions were found to be hypomethylated. To determine if hypomethylation of the upstream MTC and P519 regions always accompanied that of the cyclin D1 promoter, we analyzed two cyclin D1 overexpressing breast cancer cell lines that did not have IgH translocations with bisulfite sequencing. In these breast cancer lines, it was found that without the IgH translocation, the MTC and P519 sites remained methylated though the promoter was hypomethylated suggesting that MTC and P519 hypomethylation are unique to B cell disorders expressing the IgH-cyclin D1 translocation [13] .

In addition to methylation changes along the CCND1 promoter and gene, the IgH translocation can lead to the formation of a truncated cyclin D1 protein [25] . The shorter RNA transcript from which it is made leads to a worse prognosis, because it no longer contains the mir-16-1 binding motif, a regulatory domain for microRNAs. Additionally, the truncated RNA is more stable than the normal one [26] . It is important in future studies to determine if the truncation results from aberrant or alternate splicing or a post-transcriptional modification. Splicing variants are a possibility, because the CCND1 locus does have a few different splice variants that can be generated from its 5 exons, cyclin D1a and cyclin D1b being the most prominent. In MCL and B cell disorders carrying cyclin D1 overexpression, the D1a variant is dominantly translated even though both have comparable RNA expression levels [25] .

The aforementioned epigenetic controls of gene expression are all mediated through cis-acting mechanisms, i.e. promoter methylation, and have been extensively explored [14] . Less explored but just as important are trans-acting mechanisms. Coined paramutation, studies in maize and mice have shown that alleles can control expression of each other in a heritable fashion [27], [28], and [29] and can do so through cytosine methylation [30] . Furthermore, it has been shown that recombination events can lead to transfer of methylation from one allele to another indicating that homologous chromosomal recombination events may be a prominent mechanism of co-allelic induced expression [31] . The cyclin D1 locus can be regulated in a similar fashion in the presence of the t(11;14) translocation, an aberrant form of recombination. Two MCL cell lines, Granta 519 and NCEB-1 and one multiple myeloma (MM) cell line, U266, were analyzed for cyclin D1 expression before and after the loss of the t(11;14) translocation. As expected, high levels of cyclin D1 expression were noted in the presence of the translocation; however, when the translocation was lost, as shown by FISH and Southern blot, cyclin D1 expression was undetectable, consistent with the idea that the translocation exerts a trans-activating effect on the normal CCND1 gene. To provide more insight into the mechanism of trans-activation, DNA methylation was assessed from the promoter all the way up to the P519 site 90 kb upstream by Southern blot. In line with the translocation, the CCND1 regions assessed showed very low methylation consistent with high transcription. In the cells lacking the translocation, the same regions were found to be hypermethylated with resulting low cyclin D1 expression levels [11] . The t(11;14) chromosome was reintroduced into the D1 (−) cell line variants by cell fusion. Southern blot and bisulfite sequencing showed hypomethylation of a region 3 kb upstream of the promoter after reintroduction of a translocated chromosome, supporting the cause-effect relationship between the t(11;14) translocation and methylation of the CCND1 locus [11] .

Histone modifications

Histone modifications, mainly methylation and acetylation, are other important epigenetic marks that contribute to control of gene expression [14] . The previously mentioned cell lines (Granta, NCEB-1 and U266) were analyzed for histone modifications around the CCND1 locus by chromatin immunoprecipitation (ChIP) and were found to have hyperacetylated H3 histones 3 kb and 10 kb upstream of the promoter and at the MTC region. Hematopoietic cell lines showed similar acetylation patterns with the exception of the hyperacetylated MTC region, which appears to be unique to MCL. Other cancer lines were also assessed for CCND1 histone acetylation. MCF-7 breast cancer cells were found to have hypoacetylated regions around cyclin D1, consistent with low cyclin D1 transcriptional activity through a mechanism not involving chromosomal translocation [13] .

The translocated chromosomal aberration plays an important role in histone acetylation as well. In the same models that were used to determine the allele specific methylation effect of the t(11;14) translocation, the effect upon histone acetylation was also surveyed through the use of ChIP assays. In the parental cell lines that contained the translocation, the histones 3 kb and 10 kb upstream of the promoter as well as those around the MTC were found to be hyperacetylated. In the mutant cell lines that had lost the translocation, the 3 kb upstream region was found to be hypoacetylated. The other two regions remained hyperacetylated, similar to the methylation patterns observed. Though there is a role for histones in the epigenetic regulation of cyclin D1 and MCL, ChIP assays done in allele differentiable U266 cells to determine if the observed histone patterns were on both alleles or just the translocated one found only H3 hyperacetylation on the translocated allele, suggesting that transallelic epigenetic affects with histone modifications like acetylation and methylation, (unpublished) may not accompany DNA methylation [11] .

Is cyclin D1 necessary and sufficient to cause MCL?

This question is seemingly straightforward, but the data and interpretation remain controversial. Transgenic mice injected with the cyclin D1 gene driven by the IgH Eμ enhancer element did not develop lymphoma, while mice with similar c-myc or bcl-2 gene constructs did develop lymphoma [5] . These data were interpreted as suggesting additional genetic abnormalities are required for the development of MCL. Numerous genetic abnormalities including p53, ATM, p16, and more recently NOTCH1 have been described [32] .

However, additional data suggests that intranuclear localization of cyclin D1 is important for cell transformation and lymphomagenesis. Nuclear cyclin D1 staining is found in the majority of human MCLs, and cyclin D1 mutations that impair its degradation by the proteasome and nuclear export are associated with tumorigenesis. Expression of a constitutive nuclear cyclin D1 in transgenic mice was sufficient to cause B cell lymphomas, although pathologically the lymphomas were not identical to MCL [33] . Nuclear cyclin D1 retention during the S phase allows DNA re-replication, activation of DNA damage checkpoints and leads to genomic instability. The mechanism is thought to involve stabilization of the replication factor CDT1. The E3 ubiquitin ligase/SCF Fbx4 has been shown to regulate cyclin D1 accumulation and serves as a tumor suppressor gene that prevents neoplastic transformation by cyclin D1 [33] .

Are MCL cells addicted to cyclin D1?

Cyclin D1 knockdown studies in MCL and MM cells have shown limited effects on stopping cell growth, and upregulation of cyclin D2 has been demonstrated [34] . Experiments were carried out in our laboratory using a gene targeting strategy to ablate cyclin D1 message and protein in MCL and MM cells. These cyclin D1 (−) cells did not die or show reduced growth. Cyclin D3 was upregulated transcriptionally, and cyclin D3 protein was markedly stabilized [35] . The cyclin D1 (−) cells grew faster than their controls in vitro and in vivo in immunodeficient mice. When cyclin D3 was knocked down, apoptosis and growth arrest were observed. Thus, MCL cells appear not to be exclusively addicted to cyclin D1 but rather to cyclin D1 and D3 (submitted). Therefore, therapeutic agents which target cyclin D1 alone may not be effective. Agents or combinations that target both cyclin D1 and D3, like bortezomib combined with curcumin, are worthy of further study (see section on Turning Off Cyclin D1).

Targeting the epigenome

Because MCL is a disease primarily of the elderly, their baseline health status and co-morbidities largely contribute to the choice of therapy they will receive. In MCL, R–CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), a widely accepted regimen, is often utilized. Clinical trials established the efficacy of CHOP vs. R–CHOP in this disease, and it has been shown that the addition of rituximab increases response rates and remissions but not necessarily long term outcomes [36] . In addition to the minimal benefit gained by the addition of rituximab, R–CHOP therapy has many accompanying adverse effects, like bone marrow suppression and hair loss [37] and [38]. Epigenetic therapies utilize drugs that target DNA and histone modifications and alter DNA structure, offering a way to induce remission with fewer side effects, leading to a better quality of life during and after treatment, especially in an elderly population.

The purine analog cladribine has a number of known mechanisms of action. The classic one is incorporation into elongating DNA strands leading to inhibition of ribonucleotide reductase and certain DNA polymerase isoforms [39] , but there is also evidence that cladribine inhibits the enzymes S-adenosylhomocysteine (SAH) hydrolase and DNA methyl transferases through covalent binding, effectively depleting the methyl donor pool [40] . This second mechanism of action leads to global hypomethylation and the reactivation of potentially silenced regulatory genes making it a previously unrecognized epigenetic drug as well [41] and [42].

Rituximab, a monoclonal antibody directed against the CD20 antigen of B cells, is desirable as additive therapy for MCL, a B cell lymphoma due to its exquisite B cell specificity. The CD20 antigen that it is specific for is preferentially expressed on active B cells and overexpressed in MCL, side stepping both memory and plasma cells as well as the entire thymus lineage [43] . Rituximab's cytotoxic effects result from a number of different mechanisms, including complement activation, ceramide generation, and induction of caspases and cytochrome c [44] . Single agent treatment with rituximab in MCL has shown response rates of about 27% and is accompanied by relapses, and thus, single agent rituximab is rarely indicated [45] . However, when combined with epigenetic drugs, rituximab becomes more potent, providing evidence that epigenetic changes play an important role in resistance to monoclonal antibodies [46] . This provides support for the use of combination therapies to attack differing proposed mechanisms of resistance in MCL and that MCL is an epigenetic disease.

Vorinostat is a histone deacetylase inhibitor (HDACi) currently approved for treatment of cutaneous manifestations in cutaneous T-cell lymphoma (CTCL). In MCL, vorinostat induces apoptosis and caspase dependent cell death through the hyperacetylation of promoters of the BH3 proapoptotic family members [47] . It also has the ability to sensitize cells to death by opening chromatin domains that turn on genes silenced by cancer. A study by Xargay-Torrent et al. to assess the efficacy of this drug in MCL found that the HDACi properties of vorinostat are not dependent upon HDAC concentration or basal HDAC activity level. These authors also demonstrated by ChIP assay that the chromatin domains of proapoptotic proteins like BIM and BAF and those of tumor suppressor and regulator genes like p21 and p27 were opened after treatment. Protein levels were also increased and led to apoptotic cell death, even at low concentrations of the drug. Additionally, vorinostat preferentially targets cancer cells in vitro with little effect on normal tissues, further strengthening the rationale for its use [47] . The known in vitro synergy between HDACi and DNA hypomethylating agents increases the scientific rationale for utilizing both drugs in combination [48] .

Turning off cyclin D1

Cyclin D1's critical role in the pathogenesis of MCL makes it a logical target for small molecule inhibitors. However, the expression of the cyclin D1 gene in multiple nonhematopoietic tissues may make it a less specific target. Expression of other D type cyclins may compensate and may serve as additional targets, albeit with additive toxicity.

Bortezomib (Velcade) is the only FDA approved agent for MCL in the setting of relapsed disease. Data from several labs have reported that bortezomib downregulates cyclin D1 protein but not mRNA levels [35] . Since cyclin D1 is degraded by the proteasome, inhibition of the proteasome should increase cyclin D1 protein levels. However, as shown by our lab and others, cyclin D1 levels decline. These observations point to additional nonproteasomal mechanisms of action of bortezomib on cyclin D1 protein levels such as the endoplasmic reticulum stress or the unfolded response pathways [49] .

Curcumin, a plant flavonoid that is available naturally in turmeric and as an herbal supplement, has been shown in vitro to downregulate cyclins D1 and D3 at both the transcriptional and post-transcriptional levels in MCL and MM cell lines [50] . Curcumin is known to inhibit the COP9 signalosome, a multiprotein complex similar to the proteasome [51], [52], and [53]. Our laboratory has demonstrated that curcumin and bortezomib synergize in downregulating protein levels of cyclins D1 and D3 in MM and MCL cells [35] . Clinical trials of this agent in combination in lymphoid malignancies alone or in combination with bortezomib are ongoing.

PD0332991 is a small molecule inhibitor of cdk4/cdk6. In vitro, it can cause cell growth arrest but not apoptosis. In one study, 8 MCL patient samples were found to only express the cyclin D1a protein despite expressing detectable cyclin D1b mRNA [25] . Cell lines and tissue samples displayed constitutive activation of the cyclin D1 signaling cascade, as evidenced by strong expression of CDK4, Rb phosphorylation, and cyclin D1/CDK4 coassociation. All MCL cell lines and tissues examined displayed nondetectable to diminished expression of the cyclin D1 inhibitor p16. At low nanomolar concentrations, the novel small molecule CDK4/CDK6 inhibitor PD0332991 profoundly suppressed Rb phosphorylation, proliferation, and cell cycle progression at the G0/G1 phase of MCL cells. Cdk4/6 inhibitor PD0332991 was shown to inhibit the cell cycle via FLT-PET imaging and tissue analysis in patients with recurrent MCL. Stable disease was observed in single agent trials [54] . This agent has been studied in MCL and a phase I/II trial using combination therapy with bortezomib is in progress [55] and [56].

Vorinostat (SAHA) and other HDAC inhibitors have also been shown to downregulate cyclin D1 protein. The precise mechanism of action has not been elucidated, though effects on translation through inhibition of the phosphatidylinositol 3-kinase (PI3 K)/Akt/mTOR/eIF4E-BP pathway, probably by PI3 K inhibition, have been demonstrated [57] and [58].

The mainstays of treatment for MCL include R–CHOP, R-HyperCvad or R-bendamustine regimens [59] ; however, recent evidence from our lab and others has been emerging for the use of epigenetic drugs in combination with monoclonal antibodies in order to induce remission and overcome resistance. Cladribine has recently shown improved survival in MCL when given in combination with rituximab [60] . If used alone, resistance to cladribine develops quickly, but if used in combination with rituximab, it causes a sharp decline in circulating white cell counts, leading to remission in about 60% of MCL patients [60] . The combination of these epigenetic agents and cyclin D targeted drugs with rituximab may result in durable remissions comparable to and perhaps better than most widely accepted regimens for this disease. Clinical trials are currently underway to assess the efficacy of these new therapies on newly diagnosed MCL patients with promising preliminary data.

Summary

Epigenetics play an important and indispensable role in the pathogenesis of MCL. Hypomethylation of enhancer and promoter elements, hyperacetylated histones, and binding of CTCF, Pol II, and nucleophosmin has been shown to be associated with the aberrant expression of cyclin D1 in malignant B cells with the t(11;14) translocation [61] . These epigenetic modifications imply that MCL can epigenetically silence and activate genes that support its survival. This provides a rationale for the use of epigenetic and cyclin D1 specific drugs, like cladribine, vorinostat, curcumin and bortezomib, in addition to monoclonal antibodies such as rituximab to treat this disease. By preventing MCL cells from silencing genes, it may be possible to increase susceptibility and prevent resistance to chemoimmunotherapy and induce durable remissions. Ongoing research is focused on finding these specific genes and the pathways that they modulate.

 

  • MCL is an epigenetic disease.
  • Preclinical and clinical studies of epigenetic therapies are warranted.
  • Drugs that downregulate cyclin D1 in vitro and decrease epigenetic silencing in combination with monoclonal antibodies have shown efficacy in MCL.

Practice points

 

  • Further development of cyclin D1 targeted therapies.
  • Further development of epigenetic agents and monoclonal antibodies for use in MCL.

Research agenda

Conflict of interest

Epner: speakers bureau and research funding from Merck and Millenium.

Other authors have no conflicts.

Funding

Work in the authors' laboratory has been supported by the NIH, LRF, and IMF.

Acknowledgments

We thank August Stuart and Sara Shimko for assistance with manuscript preparation.

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Footnotes

a Penn State Hershey Cancer Institute, Experimental Therapeutics A – CH74, Room T3319, 500 University Drive, Hershey, PA 17033-0850, USA

b Penn State Hershey Cancer Institute, Division of Hematology/Oncology, 500 University Dr., P.O. Box 850, Hershey, PA 17033-0850, USA

Corresponding author. Tel.: +1 717 531 0003x281238.

1 Tel.: +1 717 531 0003x281239.

2 Tel.: +1 717 531 7782.