Multidisciplinary Cutaneous Lymphoma Program
Identification and Functional Characterization of Novel Biomarkers and Therapeutic Targets in Cutaneous T-cell Lymphomas by Whole Transcriptome and Exome Sequencing
In collaboration with investigators of Program in Epithelial Biology
Paul Khavari, MD, PhD, Project Director
Lead investigators: Alexander Ungewickell, MD, PhD, Carolyn Lee, MD, PhD
Mycosis Fungoides (MF) and Sézary syndrome (SS) make up the majority of cutaneous T-cell lymphomas (CTCL). Most patients with MF have a favorable prognosis, however a significant subset of patients develop progressive disease despite treatment and have a similar prognosis to patients with SS, whose median survival is only 2-4 years. The genetic abnormalities of these CTCL subtypes are largely uncharacterized and a better understanding of the genetic aberrations present in these diseases may not only allow prognostic stratification of patients at diagnosis but also lead to novel therapeutic strategies. To address this issue, Dr. Youn Kim and her dermatology collaborators have conducted whole transcriptome sequencing (RNA-s eq) of Sézary cells (SC) and non-malignant CD4+ T cells to define a SS specific gene expression profile (Lee CS, Ungewickell A et al, Blood 2012;120:3288-97). Pathway analysis of differentially expressed genes identified mis-regulation of PI3K/Akt, TGF-beta, and NF-kB pathways as well as T-cell receptor signaling. Bioinformatic analysis did not support a viral etiology of SS or recurrently expressed gene fusions, but did identify 21 SC-associated annotated long noncoding RNAs (lncRNAs). Transcriptome assembly by multiple algorithms identified 13 differentially expressed unannotated transcripts termed Sezary cell-associated transcripts (SeCATs). High-throughput sequencing of archived tumors from MF patients confirmed the differential expression of lncRNAs and SeCATs in CTCL.
In a complementary approach, we are also performing whole exome-sequencing of CTCL samples and patient matched normal DNA to identify the spectrum of mutations acquired in the lymphoma cells. Further analysis and functional characterization of the genomic abnormalities in CTCL identified by RNA-seq and exome-sequencing are currently being investigated. We have recently performed whole-exome sequencing on 6 patients with MF and 6 patients with SS. For MF patients, either peripheral blood neutrophils or peripheral blood mononuclear cells were used to isolate control DNA, and the DNA from the malignant T-cells was isolated from fresh 5 mm punch biopsies that upon review with a dermatopathologist were deemed to have a high density of lymphoma cells. For SS patients, the CTCL cells were isolated from peripheral blood by staining for CD3, CD4 and TCR Vb to isolate the monoclonal T-cell lymphoma cells by FACS from the remaining white blood cells, which served as source of control DNA. The genes mutated in the discovery set of CTCLs will also be analyzed for recurrence within canonical signaling pathways using gene set enrichment analysis. Mutations of interest will be validated in a set of 60+ clinically annotated CTCL samples by targeted exon capture followed by high-throughput sequencing.
Clinical-Pathologic Collaborative Investigations to Improve Diagnosis of Rare Cutaneous Lymphomas
Stanford continue to lead or make practice-changing contributions in multicenter, national/international projects that enhance our ability to diagnose, prognosticate, and manage patients with rare cutaneous lymphomas. The very rare cutaneous lymphomas such as gamma-delta T cell lymphoma requires multicenter collaboration to produce papers that have utility (Guitart J et al, Am J Surg Pathol 2012;36:1656-1665). This is only possible because Stanford continues to be a lead referral center for patients with all cutaneous lymphomas including these rare subtypes.
Innovative Therapeutic Advancements in CTCL
1. Improved utility of Stanford's total skin electron beam therapy (TSEBT)
In collaboration with Radiation Oncology (Rich Hoppe and Lynn Million)
In collaboration with Radiation Oncology (Rich Hoppe and Lynn Million)
The "Stanford TSEBT technique" was originally established to deliver electrons to the entire skin surface in patients with extensive skin involvement with MF or SS. TSEBT has been widely accepted as standard therapy, however, it requires 9 weeks of intensive schedule and results in significant skin toxicities without a curative outcome. We have modified the Stanford technique by reducing the total dose by two-thirds (12 sessions in only 3 weeks); this low-dose regimen results in significantly less toxicity while preserving the efficient skin clearing activity of TSEBT (Harrison C et al, Int J Radiat Oncol Biol Phys 2011;81:e651-7). The lower-dose allows patients to receive repetitive courses of low-dose TSEBT downstream, and reduce exposure to toxicities of systemic therapies. We are further exploring strategies to enhance the clinical activity of TSEBT by combining with HDAC inhibitors that are known to enhance radiation effects or with immune-stimulatory agents, such as interferon. A multicenter investigator initiated, randomized control trial lead by Stanford investigators is ongoing comparing low-dose TSEBT vs. low-dose TSEBT combined with vorinostat, an HDAC-inhibitor.
2. Therapies targeting tumor-selective molecules/pathways or microenvironment factors
There has been an explosion of interest in targeted therapies that attack the tumor surface molecules, aberrant epigenetic regulation, cell signaling or survival pathways, or the microenvironment in lymphoma including CTCL.
CD30 and CCR4 has been a particular interest in CTCL as malignant T cells preferentially express CCR4 and/or CD30 molecules over normal/resting T cells. Unlike Hodgkin's lymphoma and systemic ALCL, malignant cells in MF and SS have variable expression of CD30. Dr. Kim and colleagues are exploring the efficacy and safety of an anti-CD30 mab-drug-conjugate, brentuximab vedotin (BV), in the MF/SS populations in their ongoing investigator-initiated study. This study also explores the effect on local tissue microenvironment with BV therapy. Furthermore, methods to improve the detection of CD30 molecule and to better delineate what cells express CD30 (malignant CD4 cells, Tregs, TILs, macrophages) are being explored.
Stanford CL program continues to have impactful contribution towards the optimal design and conduct of phase 3 multicenter trials that lead to FDA-approval of new systemic agents in CTCL. This includes the two HDAC inhibitors that are already approved and the two phase 3 trials currently ongoing with newer targeted agents, mogamulizumab and brentuximab vedotin. Mogamulizmab (anti-CCR4 defucosylated, humanized mAb) has significant activity in SS with minimal toxicity. In addition to targeting tumor cells expressing CCR4, it has potential additional immune-modulatory effects as Th2 and Tregs are also known to have CCR4 expression.
Utilizing our results of the next generation sequencing data, we are preparing to explore targeted agents against the PI3K/Akt/mTOR pathways in CTCL. The study designs are underway in collaboration with industry partners. Molecular correlative science is planned for these studies.
3. Clinical development of new skin-directed therapies
We continue to partner with industry in the clinical development of newer skin-directed therapies including a topical HDAC inhibitor (novel reverse pro-drug), photodynamic therapy, and topical chemotherapies (Lessin SR et al, Arch Dermatol 2012: Epub ahead of print PMID 23069814).
In Situ Vaccination Against Mycosis Fungoides by Intratumoral Injection of a TLR9 Agonist Combined with Radiation
As part of Dr. Levy's investigation of a novel in situ vaccination strategy (Brody JD et al, J Clin Oncol 2010:28:4324-4332), our cutaneous lymphoma group collaborated in the therapeutic trial exploring the feasibility, efficacy, and safety of this unique immunotherapy in our patients with MF. This in situ maneuver of combining intratumoral CpG and local radiation, was designed to circumvent the need to produce labor-intensive and time-consuming ex vivo vaccine product. The in situ strategy was used to induce distant tumor regression supporting a systemic antitumor immune response Kim YH et al, Blood 2012; 119:355-363). This strategy of stimulating tumor-specific immune response is now being applied to augment donor cell antitumor response in patients who have disease progression after allogeneic HSCT.
Novel Allogeneic HSCT Regimen with Successful Long-Term GVL with Absent Non-Relapse Mortality in MF and SS: Demonstration of Molecular Remission by High-Throughput Sequencing of T Cell ReceptorIn collaboration with BMT (Wen-Kai Weng)
We have demonstrated curative or long-term clinical benefit with a novel non-myeloablative allogeneic HSCT regimen utilizing a preparatory regimen of total skin electron beam therapy (TSEBT), total lymphoid irradiation (TLI), and antithymocyte globulin (ATG). Stanford investigators have shown that TLI/ATG conditioning protects the host against GVHD by altering the host immune profile to favor regulatory NK/T cells that suppress GVHD by polarizing donor T cells toward secretion of non-inflammatory cytokines (IL4) and by promoting expansion of donor CD4+CD25+FoxP3+ Treg cells. These immune effects reduces GVHD complications however, does not affect donor CD8+ T-cell cytolytic function and graft antitumor activity. The TSEBT was added to consolidate the clearing of malignant T cells in the skin compartment and to possibly enhance donor cell trafficking to the skin. We have now successfully transplanted 21 patients with advanced stage MF or SS using our novel regimen. In the 17 evaluable patients 14 have achieved CR and 2 had PR (ORR 94%). Neither median OS nor PFS was reached with a median f/u of 14 months. Patients with SS have superior OS and PFS when compared with those outcomes in advanced stage MF. Patients tolerated transplant extremely well with 0% one-year non-relapse mortality. Three patients had grade II acute GVHD and one developed chronic GVHD.
The current monitoring method using flow cytometry, review of skin samples, and TCR PCR lacks sensitivity or specificity in detecting minimal residual disease (MRD). We applied high-throughput sequencing of TCR CDR3 region to monitor MRD. This method was able to detect Sezary cells in whole blood as low as 1 in 50,000 PBMCs. The percent of malignant clones decreased in all cases immediately post transplant. Four patients eventually achieved molecular remission by our high-throughput sequencing method. Furthermore, we were also able to demonstrate successful reconstitution of TCR repertoire in these patients with molecular remission.
We are currently continuing to enroll patients in this protocol and plan to perform final clinical and correlative analyses after 36 patients.
Future PlansWe have ongoing activity in all topics detailed above and to further consolidate and expand collaborations to produce meaningful basic and correlative/translational science.
As part of this endeavor, we are further developing our collaborations with Drs. Kohrt and Levy to explore and enhance our understanding of the role of immunologic strategies in attaining sustained responses in patients with CTCL. This includes involvement in trials and correlative science with agents that either co-stimulate immune cells or block immune checkpoints (anti-PD-L1 mAb; MPDL3280A, H Kohrt Protocol Director).