Integrating novel agents into the treatment of advanced mycosis fungoides and Sézary syndrome.

Agents targeting the unique biology of mycosis fungoides and Sézary syndrome are quickly being incorporated into clinical management. With these new therapies, we are now capable of inducing more durable responses and even complete remissions in advanced disease, outcomes which were exceedingly rare with prior therapies. Yet, even this new generation of therapies typically produce objective responses in only a minority of patients. As our therapeutic options increase, we are now challenged with selecting treatments from a growing list of options. To gain the full benefit of these novel agents, we must develop strategies to match treatments for the patients most likely to benefit from them. Here, we consider both the current approaches to treatment selection based on clinical features and the future of molecular biomarker-guided therapy for patients with this heterogeneous disease.

Resistance to mogamulizumab is associated with loss of CCR4 in cutaneous T-cell lymphoma.

Mogamulizumab is a humanized anti-CC chemokine receptor 4 (CCR4) antibody approved for the treatment of mycosis fungoides and Sézary syndrome. Despite almost universal expression of CCR4 in these diseases, most patients eventually develop resistance to mogamulizumab. We tested whether resistance to mogamulizumab is associated with loss of CCR4 expression. We identified 17 patients with mycosis fungoides or Sézary syndrome who either were intrinsically resistant or acquired resistance to mogamulizumab. Low expression of CCR4 by immunohistochemistry or flow cytometry was found in 65% of patients. Novel emergent CCR4 mutations targeting the N-terminal and transmembrane domains were found in 3 patients after disease progression. Emerging CCR4 copy number loss was detected in 2 patients with CCR4 mutations. Acquisition of CCR4 genomic alterations corresponded with loss of CCR4 antigen expression. We also report on outcomes of 3 cutaneous T-cell lymphoma (CTCL) patients with gain-of-function CCR4 mutations treated with mogamulizumab. Our study indicates that resistance to mogamulizumab in CTCL frequently involves loss of CCR4 expression and emergence of CCR4 genomic alterations. This finding has implications for management and monitoring of CTCL patients on mogamulizumab and development of future CCR4-directed therapies.

Radiation Therapy for Primary Cutaneous Gamma Delta Lymphoma Prior to Stem Cell Transplantation.

We present a patient with widespread PCGD-TCL of the bilateral arms and legs, who underwent radiotherapy with 34 Gy in 17 fractions using circumferential VMAT and 3-D printed bolus to the four extremities prior to planned stem cell transplant, who was then found to have progression in the liver, lung, and skin, followed by drastic regression of all in and out-of-field lesions on imaging 1.5 months later. The cause of regression may be related to a radiation-induced abscopal effect from the immunomodulatory effects of radiation, or related to immune reactivation in the setting of cessation of systemic immunosuppressive agents.

Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens.

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

IPH4102, a first-in-class anti-KIR3DL2 monoclonal antibody, in patients with relapsed or refractory cutaneous T-cell lymphoma: an international, first-in-human, open-label, phase 1 trial.

BACKGROUND: IPH4102 is a first-in-class monoclonal antibody targeting KIR3DL2, a cell surface protein that is expressed in cutaneous T-cell lymphoma, and predominantly in its leukaemic form, Sézary syndrome. We aimed to assess the safety and activity of IPH4102 in cutaneous T-cell lymphoma. METHODS: We did an international, first-in-human, open-label, phase 1 clinical trial with dose-escalation and cohort-expansion parts in five academic hospitals in the USA, France, the UK, and the Netherlands. Eligible patients had histologically confirmed relapsed or refractory primary cutaneous T-cell lymphoma, an Eastern Cooperative Oncology group performance score of 2 or less, were aged 18 years or older, and had received at least two previous systemic therapies. Ten dose levels of IPH4102, administered as an intravenous infusion, ranging from 0·0001 mg/kg to 10 mg/kg, were assessed using an accelerated 3 + 3 design. The primary endpoint was the occurrence of dose-limiting toxicities during the first 2 weeks of treatment, defined as toxicity grade 3 or worse lasting for 8 or more days, except for lymphopenia. Global overall response by cutaneous T-cell lymphoma subtype was a secondary endpoint. Safety and activity analyses were done in the per-protocol population. The study is ongoing and recruitment is complete. This trial is registered with ClinicalTrials.gov, number NCT02593045. FINDINGS: Between Nov 4, 2015, and Nov 20, 2017, 44 patients were enrolled. 35 (80%) patients had Sézary syndrome, eight (18%) had mycosis fungoides, and one (2%) had primary cutaneous T-cell lymphoma, not otherwise specified. In the dose-escalation part, no dose limiting toxicity was reported and the trial's safety committee recommended a flat dose of 750 mg for the cohort-expansion, corresponding to the maximum administered dose. The most common adverse events were peripheral oedema (12 [27%] of 44 patients) and fatigue (nine [20%]), all of which were grade 1-2. Lymphopenia was the most common grade 3 or worse adverse event (three [7%]). One patient developed possibly treatment-related fulminant hepatitis 6 weeks after IPH4102 discontinuation and subsequently died. However, the patient had evidence of human herpes virus-6B infection. Median follow-up was 14·1 months (IQR 11·3-20·5). A confirmed global overall response was achieved in 16 (36·4% [95% CI 23·8-51·1]) of 44 patients, and of those, 15 responses were observed in 35 patients with Sézary syndrome (43% [28·0-59·1]). INTERPRETATION: IPH4102 is safe and shows encouraging clinical activity in patients with relapsed or refractory cutaneous T-cell lymphoma, particularly those with Sézary syndrome. If confirmed in future trials, IPH4102 could become a novel treatment option for these patients. A multi-cohort, phase 2 trial (TELLOMAK) is underway to confirm the activity in patients with Sézary syndrome and explore the role of IPH4102 in other subtypes of T-cell lymphomas that express KIR3DL2. FUNDING: Innate Pharma.

The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity.

Heterochromatin integrity is crucial for genome stability and regulation of gene expression, but the factors involved in mammalian heterochromatin biology are only incompletely understood. Here we identify the oncoprotein DEK, an abundant nuclear protein with a previously enigmatic in vivo function, as a Suppressor of Variegation [Su(var)] that is crucial to global heterochromatin integrity. We show that DEK interacts directly with Heterochromatin Protein 1 α (HP1α) and markedly enhances its binding to trimethylated H3K9 (H3K9me3), which is key for maintaining heterochromatic regions. Loss of Dek in Drosophila leads to a Su(var) phenotype and global reduction in heterochromatin. Thus, these findings show that DEK is a key factor in maintaining the balance between heterochromatin and euchromatin in vivo.

T-Cell Immunopeptidomes Reveal Cell Subtype Surface Markers Derived From Intracellular Proteins.

Immunopeptidomes promise novel surface markers as ideal immunotherapy targets, but their characterization by mass spectrometry (MS) remains challenging. Until recently, cell numbers exceeding 109 were needed to survey thousands of HLA ligands. Such limited analytical sensitivity has historically constrained the types of clinical specimens that can be evaluated to cell cultures or bulk tissues. Measuring immunopeptidomes from purified cell subpopulations would be preferable for many applications, particularly those evaluating rare, primary hematopoietic cell lineages. Here, we test the feasibility of immunopeptidome profiling from limited numbers of primary purified human regulatory T cells (TReg ), conventional T cells (Tconv ), and activated T cells. The combined T cell immunopeptide dataset reported here contains 13 804 unique HLA ligands derived from 5049 proteins. Of these, more than 700 HLA ligands were derived from 82 proteins that we exclusively identified from TReg -enriched cells. This study 1) demonstrates that primary, lineage-enriched T cell subpopulations recovered from single donors are compatible with immunopeptidome analysis; 2) presents new TReg -biased ligand candidates; and 3) supports immunopeptidome surveys' value for revealing T cell biology that may not be apparent from expression data alone. Taken together, these findings open up new avenues for targeting TReg and abrogating their suppressive functions to treat cancer.

Multi-omic profiling reveals the endogenous and neoplastic responses to immunotherapies in cutaneous T cell lymphoma.

Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA+CD39+ T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA+CD39+ T cells in non-responders and activated cytotoxic CLA+CD39+ T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials.

A human lymphoma organoid model for evaluating and targeting the follicular lymphoma tumor immune microenvironment.

Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3 weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and T cell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.

A clinical trial of therapeutic vaccination in lymphoma with serial tumor sampling and single-cell analysis.

ABSTRACT: In situ vaccination (ISV) triggers an immune response to tumor-associated antigens at 1 tumor site, which can then tackle the disease throughout the body. Here, we report clinical and biological results of a phase 1/2 ISV trial in patients with low-grade lymphoma, combining an intratumoral toll-like receptor 9 (TLR9) agonist with local low-dose radiation and ibrutinib (an inhibitor of B- and T-cell kinases). Adverse events were predominately low grade. The overall response rate was 50%, including 1 complete response. All patients experienced tumor reduction at distant sites. Single-cell analyses of serial fine needle aspirates from injected and uninjected tumors revealed correlates of clinical response, such as lower CD47 and higher major histocompatibility complex class II expression on tumor cells, enhanced T-cell and natural killer cell effector function, and reduced immune suppression from transforming growth factor ß and inhibitory T regulatory 1 cells. Although changes at the local injected site were more pronounced, changes at distant uninjected sites were more often associated with clinical responses. Functional immune response assays and tracking of T-cell receptor sequences provided evidence of treatment-induced tumor-specific T-cell responses. Induction of immune effectors and reversal of negative regulators were both important in producing clinically meaningful tumor responses. The trial was registered at www.clinicaltrials.gov as #NCT02927964.

Minimal/Measurable Residual Disease Monitoring in Patients with Lymphoid Neoplasms by High-Throughput Sequencing of the T-Cell Receptor.

High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies. A custom bioinformatics pipeline and database was also developed to facilitate minimal/measurable residual disease analysis and clinical reporting. This assay demonstrated excellent test performance characteristics, achieving a sensitivity of 1 of 100,000 T-cell equivalents for the DNA inputs evaluated and high concordance with orthogonal testing methods. This assay was further utilized to correlate disease burden in several patients, demonstrating its potential utility for monitoring patients with T-cell malignancies.

Two cases of mycosis fungoides with large cell transformation with KMT2A rearrangements.

Cutaneous T-cell lymphomas (CTCL) are a clinically and molecularly heterogeneous class of lymphomas of the skin-homing T cell, and their genetic profiles are not fully characterized. Previously, rearrangements of the Lysine Methyltransferase 2A (KMT2A) gene have been identified as driver mutations only in acute leukemias. KMT2A plays a role in epigenetic regulation, and cancers with such rearrangements are responsive to epigenetic therapy including hypomethylating agents. Here, we report two cases of CTCL with novel genetic profiles. KMT2A rearrangements were identified in two aggressive cases of mycosis fungoides with large cell transformation. A KMT2A::DSCAML1 gene rearrangement was seen in Case 1, while a KMT2A::MAPRE1 fusion was identified in Case 2. These cases demonstrate that KMT2A rearrangements can be found in primary CTCLs rather than solely acute leukemias, illustrating the importance of correlating molecular findings with clinical and histologic features in diagnosis. Additionally, this finding suggests that the subset of CTCLs driven by aberrancy of the KMT2A pathway may be responsive to therapy with hypomethylating agents or menin inhibitors, as seen in acute leukemias.

Tracing Founder Mutations in Circulating and Tissue-Resident Follicular Lymphoma Precursors.

Follicular lymphomas (FL) are characterized by BCL2 translocations, often detectable in blood years before FL diagnosis, but also observed in aging healthy individuals, suggesting additional lesions are required for lymphomagenesis. We directly characterized early cooperating mutations by ultradeep sequencing of prediagnostic blood and tissue specimens from 48 subjects who ultimately developed FL. Strikingly, CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, and largely distinguished patients developing FL (14/48, 29%) from healthy adults with or without detected BCL2 rearrangements (0/13, P = 0.03 and 0/20, P = 0.007, respectively). CREBBP variants were detectable a median of 5.8 years before FL diagnosis, were clonally selected in FL tumors, and appeared restricted to the committed B-cell lineage. These results suggest that mutations affecting the CREBBP KAT domain are common lesions in FL cancer precursor cells (CPC), with the potential for discriminating subjects at risk of developing FL or monitoring residual disease. SIGNIFICANCE: Our study provides direct evidence for recurrent genetic aberrations preceding FL diagnosis, revealing the combination of BCL2 translocation with CREBBP KAT domain mutations as characteristic committed lesions of FL CPCs. Such prediagnostic mutations are detectable years before clinical diagnosis and may help discriminate individuals at risk for lymphoma development. This article is highlighted in the In This Issue feature, p. 1275.

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

Single-cell RNA-sequencing reveals predictive features of response to pembrolizumab in Sézary syndrome.

The PD-1 inhibitor pembrolizumab is effective in treating Sézary syndrome, a leukemic variant of cutaneous T-cell lymphoma. Our purpose was to investigate the effects of pembrolizumab on healthy and malignant T cells in Sézary syndrome and to discover characteristics that predict pembrolizumab response. Samples were analyzed before and after 3 weeks of pembrolizumab treatment using single-cell RNA-sequencing of 118,961 peripheral blood T cells isolated from six Sézary syndrome patients. T-cell receptor clonotyping, bulk RNA-seq signatures, and whole-exome data were integrated to classify malignant T-cells and their underlying subclonal heterogeneity. We found that responses to pembrolizumab were associated with lower KIR3DL2 expression within Sézary T cells. Pembrolizumab modulated Sézary cell gene expression of T-cell activation associated genes. The CD8 effector populations included clonally expanded populations with a strong cytotoxic profile. Expansions of CD8 terminal effector and CD8 effector memory T-cell populations were observed in responding patients after treatment. We observed intrapatient Sézary cell heterogeneity including subclonal segregation of a coding mutation and copy number variation. Our study reveals differential effects of pembrolizumab in both malignant and healthy T cells. These data support further study of KIR3DL2 expression and CD8 immune populations as predictive biomarkers of pembrolizumab response in Sézary syndrome.

CD20-Targeted Therapy Ablates De Novo Antibody Response to Vaccination but Spares Preestablished Immunity.

To obtain a deeper understanding of poor responses to COVID-19 vaccination in patients with lymphoma, we assessed blocking antibodies, total anti-spike IgG, and spike-specific memory B cells in the peripheral blood of 126 patients with lymphoma and 20 age-matched healthy controls 1 and 4 months after COVID-19 vaccination. Fifty-five percent of patients developed blocking antibodies postvaccination, compared with 100% of controls. When evaluating patients last treated from days to nearly 18 years prior to vaccination, time since last anti-CD20 was a significant independent predictor of vaccine response. None of 31 patients who had received anti-CD20 treatment within 6 months prior to vaccination developed blocking antibodies. In contrast, patients who initiated anti-CD20 treatment shortly after achieving a vaccine-induced antibody response tended to retain that response during treatment, suggesting a policy of immunizing prior to treatment whenever possible. SIGNIFICANCE: In a large cohort of patients with B-cell lymphoma, time since anti-CD20 treatment was an independent predictor of neutralizing antibody response to COVID-19 vaccination. Comparing patients who received anti-CD20 treatment before or after vaccination, we demonstrate that vaccinating first can generate an antibody response that endures through anti-CD20-containing treatment. This article is highlighted in the In This Issue feature, p. 85.

Low-Dose Total Skin Electron Beam Therapy Combined With Mogamulizumab for Refractory Mycosis Fungoides and Sézary Syndrome.

PURPOSE: Management of patients with refractory mycosis fungoides and Sézary syndrome (SS) is often challenging, as available therapies lack durable response and consistent activity across disease compartments. Combining low-dose total skin electron beam therapy (LD-TSEBT) upfront with mogamulizumab could optimize the clinical outcome of these patients. LD-TSEBT is effective in clearing skin disease, and mogamulizumab is an antitumor immunotherapy with long-term tolerability, suggesting its potential as a maintenance therapy after maximal response. We examine the combination regimen in patients with SS who were previously treated. METHODS AND MATERIALS: Two patients with SS were treated with combination LD-TSEBT and mogamulizumab. Both patients received mogamulizumab 1 mg/kg weekly × 4 and then bi-weekly; LD-TSEBT (12 Gy) was initiated within 2 days of starting mogamulizumab and given over 2-3 weeks. Safety and clinical response were evaluated. RESULTS: Total skin electron beam therapy plus mogamulizumab (TSE-Moga) was well-tolerated without any unanticipated adverse events. Patient 1 (T4N2bM0B2) was a 63-year-old woman with 4 prior systemic therapies; time to global response with TSE-Moga was 9 weeks. Patient 2 (T4NxM0B2) was a 75-year-old man with 5 prior systemic therapies; time to global response was 4 weeks. Both patients lacked global response to their prior therapies but achieved global complete response (blood and skin) with TSE-Moga. After a follow-up of 72 weeks and 43 weeks, respectively, global complete response continued. CONCLUSIONS: TSE-Moga demonstrated excellent tolerability and promising clinical activity with ongoing global complete responses in 2 patients with refractory SS. This encouraging experience supports our ongoing clinical trial evaluating the efficacy and safety of TSE-Moga in mycosis fungoides and SS.

Low-dose total skin electron beam therapy for refractory cutaneous CD30 positive lymphoproliferative disorders.

We describe a case of a 48-year-old woman with a refractory cutaneous CD30 positive lymphoproliferative disorder treated successfully with total skin electron beam radiotherapy (TSEBT).