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WO2019195769A1 - Méthodes de diagnostic et de traitement de lymphomes à lymphocytes t cutanés agressifs - Google Patents

Méthodes de diagnostic et de traitement de lymphomes à lymphocytes t cutanés agressifs Download PDF

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WO2019195769A1
WO2019195769A1 PCT/US2019/026126 US2019026126W WO2019195769A1 WO 2019195769 A1 WO2019195769 A1 WO 2019195769A1 US 2019026126 W US2019026126 W US 2019026126W WO 2019195769 A1 WO2019195769 A1 WO 2019195769A1
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skin
cell
patients
tcf
subject
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John T. O'MALLEY
Thomas S. Kupper
Rachel A. CLARK
Adele De Masson D'AUTUME
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia

Definitions

  • the present disclosure generally relates to methods of identifying and treating subjects who are at risk of developing aggressive T-cell lymphomas, such as cutaneous T-cell lymphomas.
  • the methods include determining the tumor clone frequency.
  • Cutaneous T cell Lymphomas are uncommon non-Hodgkin lymphomas of mature skin- tropic memory T cells.
  • Mycosis Fungoides (MF) is the most common and prevalent CTCL, and typically presents as inflammatory patches and plaques on the skin. Diagnosis is often difficult, and has relied on a combination of clinical, histopathological, and molecular findings (1). The average time from appearance of lesions to definitive diagnosis has been estimated to be 3-6 years (2). Recently, the advent of next-generation high-throughput DNA sequencing has revolutionized the diagnosis of MF (3).
  • MF is nearly always a malignancy of CD4+ T cells with an ab T cell receptor, encoded by the TCRA and TCRB genes (3).
  • High-throughput sequencing of the TCRB gene can not only identify the unique T cell clone in MF, but can precisely determine the tumor clone frequency (TCF) in the entire T cell infiltrate (3, 4).
  • TCF tumor clone frequency
  • TCF tumor clone frequency
  • kits for treating a subject who has early-stage cutaneous T-cell lymphoma include obtaining a skin sample from the subject having CTCL; determining the tumor clone frequency (TCF) of said skin sample; and treating said subject with an aggressive treatment when the TCF is greater than a reference level.
  • CTCL early-stage cutaneous T-cell lymphoma
  • CTLC early-stage cutaneous T-cell lymphoma
  • the methods include obtaining a skin sample from a subject suspected of having CTCL; determining the tumor clone frequency (TCF) of said skin sample; and selecting a subject who has a TCF greater than a reference level for aggressive treatment.
  • TCF tumor clone frequency
  • Further provided are methods for predicting whether a subject with early-stage cutaneous T-cell lymphoma (CTLC) is likely to progress to aggressive disease.
  • the methods include obtaining a skin sample from the subject having CTCL; determining the tumor clone frequency (TCF) of said skin sample; and identifying a subject who has a TCF greater than a reference level as likely to progress to aggressive disease.
  • the CTCL is mycosis fungoides (MF), e.g., stage IA or IB mycosis fungoides.
  • MF mycosis fungoides
  • the subject has skin lesions on less than 10% of the body surface area (BSA) at time of diagnosis.
  • BSA body surface area
  • the subject has skin lesions on greater than 10% of the body surface area (BSA) at time of diagnosis.
  • BSA body surface area
  • the skin sample is from a biopsy from a skin lesion.
  • the analyzing step is performed by high-throughput DNA sequencing.
  • determining the tumor clone frequency (TCF) of said skin sample comprises analyzing T-cell receptor beta (TCR b) gene sequences in substantially every T cell in the sample, and determining the frequency of the most abundant single allele in the sample.
  • TCF tumor clone frequency
  • analyzing T-cell receptor beta (TCR b) gene sequences comprises:
  • TCRb T-cell receptor
  • CDR3 complementarity determining region-3
  • the methods include determining whether the T cell clone with the highest frequency of occurrence has a frequency of occurrence that is above or below a predetermined threshold.
  • a frequency of occurrence above the predetermined threshold indicates that the subject is likely to progress to aggressive disease.
  • the reference level is 25%
  • the aggressive treatment is allogeneic hematopoietic stem cell transplantation, skin-directed radiation, or chemotherapy.
  • the subject is in near complete or complete remission before administration of allogeneic hematopoietic stem cell transplantation.
  • the radiation therapy is total skin electron beam therapy (TSEB), surface brachytherapy, or other forms of ionizing radiation.
  • the chemotherapy comprises administration of etoposide, vincristine, doxorubicin,
  • cyclophosphamide and prednisone
  • EPOCH cyclophosphamide, and prednisone
  • cyclophosphamide vincristine, nr-16, adriamycin and prednisolone (COP, CHOP, CAVOP); CMED/ABV; pegylated liposomal doxorubicin; Pentostatin; Fludarabine plus IFN-a; Fludarabine plus cyclophosphamide;
  • FIGS. 1A-1E High throughput TCRB sequencing in 309 patients with cutaneous T cell lymphomas.
  • FIG. 1A depicts clinical diagnosis in 309 patients with cutaneous T cell lymphomas in the discovery and validation sets.
  • Pre-Sezary refers to the evidence of blood abnormalities (Bl ; elevated absolute CD4 T cell count or CD4/CD8 T cell ratio) that do not meet the criteria for stage B2 or Sezary syndrome (26).
  • FIG. IB depicts TCRBV gene family usage by the malignant clone in 309 cases of primary cutaneous T cell lymphomas.
  • FIG. 1A depicts clinical diagnosis in 309 patients with cutaneous T cell lymphomas in the discovery and validation sets.
  • Pre-Sezary refers to the evidence of
  • 1C depicts an example of the measurement of the malignant clone frequency in skin in two patients with stage IB mycosis fungoides.
  • FIG. IE depicts malignant clone frequency according to the extent of body surface area involved in patients with mycosis fungoides. Medians are indicated by horizontal bars and comparisons are carried out using Mann-Whitney U-test, *p ⁇ 0.05 considered significant.
  • FIGS. 2A-2E The tumor clone frequency in skin as predictor of progression-free and overall survival in patients with cutaneous T cell lymphomas.
  • FIG. 2A depicts Kaplan-Meier estimates of progression-free (left panel) and overall survival (right) in 208 patients with cutaneous T cell lymphomas in the discovery set, according to the tumor clone frequency in skin ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIG. 2B depicts Kaplan-Meier estimates of progression-free survival in 101 patients with cutaneous T cell lymphomas in the validation set, according to the tumor clone frequency in skin ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIG. 2A depicts Kaplan-Meier estimates of progression-free (left panel) and overall survival (right) in 208 patients with cutaneous T cell lymphomas in the discovery set, according to the tumor clone frequency in skin ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIG. 2C depicts Kaplan-Meier estimates of progression-free (left panel) and overall survival (right) in 177 patients with mycosis fungoides in the discovery set, according to the tumor clone frequency in skin ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIG. 2D depicts Kaplan- Meier estimates of progression-free survival in 87 patients with mycosis fungoides in the validation set, according to the tumor clone frequency in skin ( ⁇ 25% versus >25% of the total T cells in skin)
  • p-values in FIGS. 2A-2D are estimated by Cox univariable analysis.
  • 2E depicts Kaplan-Meier estimates of progression- free (left panel) and overall survival (right) in 22 patients with Sezary syndrome in the discovery set, according to the tumor clone frequency in skin ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIGS. 3A-3F The tumor clone frequency in skin as predictor of progression-free and overall survival in patients with early-stage mycosis fungoides.
  • FIG. 3A depicts Kaplan- Meier estimates of progression-free (left) and overall survival (right) in 141 patients with early- stage (IA to IIA) mycosis fungoides in the discovery set, according to the tumor clone frequency ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIG. 3B depicts Kaplan-Meier estimates of progression-free survival in 69 patients with early-stage (IA to IIA) mycosis fungoides in the validation set, according to the tumor clone frequency ( ⁇ 25% versus >25% of the total T cells in skin).
  • FIG. 3A depicts Kaplan- Meier estimates of progression-free (left) and overall survival (right) in 141 patients with early- stage (IA to IIA) mycosis fungoides in the discovery set, according to the tumor clon
  • FIG. 3C depicts Kaplan-Meier estimates of progression-free (left) and overall survival (right) in 70 patients with stage IB mycosis fungoides in the discovery set, according to the tumor clone frequency ( ⁇ 25% versus >25% of the total T cells in skin, upper panels) or to the presence of plaques (lower panels).
  • FIG. 3D depicts Kaplan-Meier estimates of progression-free survival in 42 patients with stage IB mycosis fungoides in the validation set, according to the tumor clone frequency ( ⁇ 25% versus >25% of the total T cells in skin, upper panel) or to the presence of plaques (lower panel) p-values in FIG. 3A-3D are estimated by Cox univariable analysis.
  • FIG. 3C depicts Kaplan-Meier estimates of progression-free (left) and overall survival (right) in 70 patients with stage IB mycosis fungoides in the discovery set, according to the tumor clone frequency ( ⁇ 25% versus >25%
  • FIG. 3E depicts dot plot and linear regression of the time to progression/death according to the tumor clone frequency in skin in stage IB patients from the discovery and validation sets, who experienced disease progression during the follow-up. Pearson’s correlation coefficient and p- value are indicated.
  • FIG. 3F depicts receiver operating characteristic curve of the tumor clone frequency in skin (>25%) in patients with stage IB mycosis fungoides in the discovery and validation sets for 5-year progression or death.
  • Progressors are patients who progressed or died within 5 years after the test.
  • Nonprogressors are patients with at least 5 years of follow-up and no event of death or progression in 5 years.
  • the sensitivity is defined as the percentage of patients with a malignant clone >25% of T cells in skin among progressors.
  • FIGS. 4A-4C Samples with a high tumor clone frequency are not associated with a decreased anti-tumor immune response.
  • FIG. 4A depicts an example of CD8+ and granzyme immunostaining in lesional skin in 2 lesional CTCL skin biopsies.
  • FIG. 4B depicts percentage of CD8+ T cell % (left) and granzyme B positive cell % in lesional skin of CTCL patients with a low tumor clone frequency ( ⁇ 10% T cells) and high tumor clone frequency (>30% T cells).
  • FIG. 4C depicts reactive T cell clonality (left) and entropy (right) in lesional skin of CTCL patients with a low tumor clone frequency ( ⁇ 10% T cells) and high tumor clone frequency (>30% T cells). (Mann Whitney U-test, *p ⁇ 0.05).
  • FIGS. 5A-5F A high tumor clone frequency in skin is associated with a distinct gene expression profile and a higher number of somatic mutations.
  • FIG. 5A depicts unsupervised analysis by hierarchical clustering (complete linkage) according to the expression of 78 genes in 157 patients reveals 3 different clusters of patients. Intensity expression values in the heatmap are expressed as log2 fold changes compared to the average expression of each gene in the whole study group. The tumor clone frequency in each sample is represented by a colour scale at the bottom of the heatmap.
  • FIG. 5B depicts dot plots of the T cell percentages of nucleated cells) in patients in cluster 1, 2 and 3.
  • FIG. 5C depicts dot plots of the tumor clone frequency (TCF) in patients in cluster 1, 2 and 3. Means were compared by Mann-Whitney U-test with Bonferroni adjustment for multiple testing, * p ⁇ 0.05 and **p ⁇ 0.0l .
  • FIG. 5D depicts Kaplan-Meier estimates of progression-free survival in 157 patients with cutaneous T cell lymphomas in the training group, according to the gene expression clustering. Log-rank test with Bonferroni adjustment for multiple testing, * p ⁇ 0.05 and **p ⁇ 0.0l and ***p ⁇ 0.00l .
  • FIG. 5E depicts whole exome sequencing data of microdissected skin T cells in patients with mycosis fungoides. Number of somatic mutations according to the clinical stage. Mann-Whitney U-test, * p ⁇ 0.05, p ⁇ 0.05 considered significant.
  • FIG. 5F depicts whole exome sequencing data of microdissected skin T cells in patients with mycosis fungoides. Number of somatic mutations according to the malignant clone frequency in skin. Spearman correlation, p ⁇ 0.05 considered significant
  • FIGS. 6A-6C TCR nb high-throughput sequencing allows specific quantification of the frequency of the malignant T cell clone within a U b gene family.
  • FIG. 6A depicts TCR nb high throughput sequencing gives precise identification of V-J gene segment combinations including the variability of the malignant T cell clonal load within nb gene families.
  • Chord diagrams of Variable-Joining (V-J) segment combinations where each chord represents a set of clonotypes with a given V-J junction and is scaled according to the frequency of reads from TCR nb with such junction.
  • Each arc represents a V or J segment and is scaled to the relative number of reads containing the corresponding segment.
  • the black bar within TCRBV20 (left diagram) and TCRBV5-1 (right) in each diagram depicts the portion of the arc that makes up the malignant T cell clone in 2 different patients.
  • the green curved line represents the portion of the diagram representing the V gene families and the blue curved line represents the portion of the diagram making up the J gene families.
  • FIG. 6B depicts the frequency of the nb gene family expressed by the malignant T cell clone determined by immunostaining followed by cell counting shows variable frequencies but has a comparable overall frequency as to that determined by TCR nb high-throughput sequencing.
  • the upper panel depicts Patient 339. Co staining of nb2 and CD3 allows for determination of the % of CD3+ T cells that were also nb2 positive.
  • Representative 200x and 400x images from patient 339 are shown. Boxes represent similar tissue area at higher magnification from a serial section.
  • the lower panel depicts Patient 425. Co staining of nb5.1 and CD3 allows for determination of the % of CD3+ T cells that were also nb5.1+.
  • Representative 200x and 400x images taken from patient 425 are shown.
  • FIG. 6C depicts a summary of the high throughput sequencing data of the TCRb gene in lesional skin in patients 339 and 425.
  • FIGS. 7A-7B Continuous relationship between the tumor clone frequency and the hazard ratios for progression-free and overall survival.
  • FIG. 7A depicts hazard ratios and 95% confidence intervals (dotted lines) for progression- free survival in 177 patients with mycosis fungoides in the discovery set, according to tumor clone frequency (TCF) threshold. The vertical line indicates the 25% TCF threshold. Cox univariable analysis, p ⁇ 0.05 considered significant.
  • FIG. 7B depicts Hazard ratios and 95% confidence intervals (dotted lines) for overall survival in 177 patients with mycosis fungoides in the discovery set, according to tumor clone frequency (TCF) threshold. The vertical line indicates the 25% TCF threshold. Cox univariable analysis, p ⁇ 0.05 considered significant.
  • FIGS. 8A-8B Prognostic value of the Cutaneous Lymphoma International Prognostic Index (CLIPI) in early-stage mycosis fungoides.
  • FIG. 8A depicts Kaplan-Meier estimates of progression-free survival in 141 patients with early-stage mycosis fungoides in the discovery set, according to the CLIPI (Cutaneous Lymphomas International Prognostic Index, low versus intermediate versus high risk).
  • FIG. 8B depicts Kaplan-Meier estimates of progression-free survival in 69 patients with early-stage mycosis fungoides in the validation set, according to the CLIPI (Cutaneous Lymphomas International Prognostic Index, low versus intermediate versus high risk). Numbers at-risk are indicated at the bottom. Hazard ratios and p-values are estimated by Cox univariable analysis.
  • FIGS. 9A-9B Prognosis in early-stage patients according to body surface area involved and the presence of plaques.
  • FIG. 9A depicts Kaplan-Meier estimates of progression-free survival in patients with stage I mycosis fungoides in the training and validation sets, according to the ISCL/EORTC staging (body surface area involved and presence of plaques).
  • FIG. 9B depicts Kaplan-Meier estimates of overall survival in patients with stage I mycosis fungoides in the training and validation sets, according to the ISCL/EORTC staging (body surface area involved and presence of plaques).
  • FIGS. 10A-10B Prognosis in stage 1A patients.
  • FIG. 10A depicts Kaplan-Meier estimates of progression-free survival in patients with stage IA mycosis fungoides in the discovery set, according to the malignant clone frequency in skin.
  • FIG. 10B depicts B. Kaplan-Meier estimates of overall survival in patients with stage IA mycosis fungoides in the discovery set, according to the malignant clone frequency in skin.
  • FIGS. 11A-11C Reproducibility of the tumor clone frequency as measured by high throughput sequencing of the TCRP gene in different lesions in the same patient.
  • Clinical pictures and 3D histograms of the high throughput sequencing data of the TCRBV gene in lesional skin in 3 patients with cutaneous T cell lymphoma FIGS. 11A-11C.
  • the same type of skin lesion was biopsied at two different time points in each patient and sent for high throughput sequencing.
  • FIGS. 12A-12C Progression-free and overall survival in pre-treated and treatment-naive early-stage mycosis fungoides patients with a TCF>25%.
  • FIG. 12A depicts number of patients with early-stage mycosis fungoides having received previous treatments (upper left panel), systemic treatments (upper middle), UV therapy (upper right), oral bexarotene (lower left), interferon (lower middle) and methotrexate (lower right) in patients with a tumor clone frequency ⁇ 25% versus >25%. Fisher’s exact test, p ⁇ 0.05 considered significant.
  • FIG. 12A depicts number of patients with early-stage mycosis fungoides having received previous treatments (upper left panel), systemic treatments (upper middle), UV therapy (upper right), oral bexarotene (lower left), interferon (lower middle) and methotrexate (lower right) in patients with a tumor clone frequency ⁇ 25% versus >25%. Fisher’s exact test,
  • FIG. 12B depicts Kaplan-Meier estimates of progression-free survival in patients with early-stage mycosis fungoides and tumor clone frequency in skin >25%, treated prior to inclusion versus treatment- naive. Cox univariable analysis, p ⁇ 0.05 considered significant.
  • FIG. 12C depicts Kaplan-Meier estimates of overall survival in patients with early-stage mycosis fungoides and tumor clone frequency in skin >25%, treated prior to inclusion versus treatment-naive. Cox univariable analysis, p ⁇ 0.05 considered significant.
  • a major challenge in the management of CTCL and MF patients is the identification of early- stage patients who are at high risk for progression to advanced disease. More than 80% of early- stage patients will have an indolent life-long course free of disease progression, regardless of treatment modality (5). As a result, early-stage patients are treated and maintained with conservative skin-directed therapies unless their disease worsens (6). However, a subset of patients develops highly aggressive, treatment-resistant disease that can be fatal. Although greater percent skin surface area involvement is associated with a somewhat higher risk of progression, the majority of early-stage MF patients have indolent courses (5). In contrast, advanced- stage patients (stage IGB or higher) have dismal prognoses, with life expectancies ranging from 1.5 to 4 years (5). Recently, allogeneic hematopoietic stem cell transplantation has emerged as a potentially life-saving intervention in advanced-stage CTCL patients (7).
  • TCR genes TCRB, TCRG
  • CDR3 TCR complementarity-determining region 3
  • DNA sequencing allows the precise identification and absolute quantification of both malignant and benign T cell clones in CTCL (3, 4).
  • Skin lesions of MF patients are infiltrated by large numbers of non-malignant memory T cells, and it is often impossible to distinguish the malignant T cell clone from activated benign infiltrating T cells in early-stage lesions by histopathology alone (16).
  • the high-throughput sequencing test greatly facilitates the diagnosis of early-stage disease (3), allows tracking of specific T cell clones over time and in different tissues (4, 17, 18) and detects residual disease after treatment with high sensitivity (19, 20).
  • the most commonly used diagnostic assay for clonality in CTCL patients employs polymerase chain reaction (PCR) amplification of a rearranged TCR gene, typically TCRG, followed by denaturing gradient gel electrophoresis and gel scanning or Biomed GeneScan analysis (21).
  • PCR polymerase chain reaction
  • TCF malignant T cell clone in skin
  • T2 body surface involvement
  • the present disclosure provides methods for diagnosing and treating subjects with a T-cell cancer, including non-Hodgkin lymphoma, peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma, lympoblastic lymphoma, precursor T-lymphoblastic lymphoma,
  • PTCL peripheral T-cell lymphoma
  • anaplastic large cell lymphoma lympoblastic lymphoma
  • precursor T-lymphoblastic lymphoma precursor T-lymphoblastic lymphoma
  • angioimmunoblastic T-cell lymphoma or Cutaneous T-Cell Lymphoma (CTCL), e.g., mycosis fungoides (MF), Sezary syndrome (SS), or CD30-positive lymphoproliferative disorder (see, e.g., Paulli and Berti, Haematologica January 2004 89: 1372-1388; Junkins-Hopkins, Seminars in Diagnostic Pathology 34(l):44-59, January 2017, and the like.
  • Cutaneous lymphomas can be diagnosed and classified using methods known in the art, e.g., WHO-EORTC classification for cutaneous lymphomas (see, e.g., Willemze et al, Blood.
  • T- cell lymphomas are lymphomas in which the T cells of the patient are determined to be cancerous.
  • T cell lymphomas encompass a variety of conditions including without limitation: (a) lymphoblastic lymphomas in which the malignancy occurs in primitive lymphoid progenitors from the thymus; (b) mature or peripheral T cell neoplasms, including T cell prolymphocytic leukemia, T- cell granular lymphocytic leukemia, NK-cell leukemia, cutaneous T cell lymphoma (Mycosis fungoides and Sezary syndrome), anaplastic large cell lymphoma, T cell type, enteropathy- type T cell lymphoma, Adult T-cell leukemia/lymphoma including those associated with HTLV-l, and angioimmunoblastic T cell lymphoma, and subcutaneous panniculitic T cell lymphoma; and (c) peripheral T cell lymphomas that initially involve a
  • the present disclosure provides methods for identifying subjects having a high likelihood of developing aggressive cancer, and optionally treating subjects identified using a method described herein.
  • this disclosure provides methods for diagnosing and treating subjects with aT-cell lymphoma, including CTCL, e.g., MF, SS.
  • the subjects have stage IA (Patchy or plaquelike skin disease involving less than 10% of skin surface area); IB (Patchy/plaquelike skin disease involving 10% or more of the skin surface area), or IIA (tumors present) CTCL (see, e.g., Olsen et al, Blood. 2007; 110(6): 1713-22; Al Hothali, Int J Health Sci (Qassim). 2013 Jun; 7(2): 220-239..
  • Risk stratification is one of the goals of precision oncology, and there is great interest in biomarkers that predict aggressive disease in malignancies in which a majority of patients have indolent disease, while a smaller subset develop aggressive disease. Identifying patients at risk for disease progression is particularly important in CTCL, a disease in which two patients with similar physical exams and histopathological morphology can have markedly different outcomes. High-throughput sequencing of the TCRB gene provides a precise and quantitative measurement of the malignant T cell clonal burden in CTCL lesions. Moreover, it is straightforward and readily accomplished using available platforms.
  • T-cell lymphoma e.g., a presently apparently indolent lymphoma
  • the methods include obtaining a sample from a subject, and evaluating the Tumor Clone Frequency (TCF) in the sample.
  • TCF Tumor Clone Frequency
  • the methods rely on detection and quantification of sequences of TCRB and/or TCRG in substantially every T cell in the sample to determine T cell clonality.
  • the sequence of the TCRB or TCRG CD3 region is determined.
  • the methods include summing the abundance of the most frequent single productive allele for TCRB and/or TCRG.
  • sample when referring to the material to be tested for the presence of a biological marker using the method of the invention, preferably includes a sample comprising lesional (affected) skin, e.g., obtained by a biopsy.
  • a sample comprising lesional (affected) skin, e.g., obtained by a biopsy.
  • An“isolated” or“purified” biological marker is substantially free of cellular material or other contaminants from the cell or tissue source from which the biological marker is derived i.e. partially or completely altered or removed from the natural state through human intervention.
  • nucleic acids contained in the sample are first isolated according to standard methods, for example using lytic enzymes, chemical solutions, or isolated by nucleic acid-binding resins following the manufacturer’s instructions.
  • TRB TRB
  • TCRG TRG nucleic acid sequence
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase polymerase chain reaction
  • quantitative or semi-quantitative real-time RT-PCR digital PCR i.e.
  • high throughput methods e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999, W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): l760-l763; Simpson, Proteins and
  • RNAs can be used to detect the presence and/or level of TCF.
  • Measurement of the level of a biomarker can be direct or indirect.
  • the abundance levels of each individual clone (sequence) of TCRB and/or TCRG can be directly quantitated and used to calculate TCF.
  • the amount of a biomarker can be determined indirectly by measuring abundance levels of cDNA, amplified RNAs or DNAs, or by measuring quantities or activities of RNAs, or other molecules that are indicative of the expression level of the biomarker.
  • a technique suitable for the detection of alterations in the structure or sequence of nucleic acids such as the presence of deletions, amplifications, or substitutions, can be used for the detection of biomarkers of this invention.
  • RT-PCR can be used to determine the expression profiles of biomarkers (U/S. Patent No.
  • RT-PCR The first step in expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction (Ausubel et al (1997) Current Protocols of Molecular Biology, John Wiley and Sons). To minimize errors and the effects of sample-to-sample variation, RT-PCR is usually performed using an internal standard, which is expressed at constant level among tissues, and is unaffected by the experimental treatment.
  • Gene arrays are prepared by selecting probes which comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface.
  • the probes may comprise DNA sequences, RNA sequences, co-polymer sequences of DNA and RNA, DNA and/or RNA analogues, or combinations thereof.
  • the probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (e.g. by PCR), or non-enzymatically in vitro. High throughput sequencing can be used to determine the abundance of sequence in a sample (3, 31, 32, 33, and US 2015-0141261 Al).
  • the abundance of a particular CDR3 sequence of a TCRB or TCRG gene from a malignant T cell in a sample can be measured, e.g., using high throughput sequencing, Immunosequencing, or other methods. See, e.g., Kou et al., Clin Diagn Lab Immunol. 2000 Nov; 7(6): 953-959; Kirsch et al., Sci. Transl. Med. 7, 308ral58 (2015); Robins et al., Blood 114, 4099-4107 (2009); Keane et al., Clin Cancer Res. 2017 Apr l;23(7): 1820-1828.
  • a reference sequence of TCRB is at GenBank Ref. no. NG_00l333.2.
  • a reference sequence of TCRG is at GenBank Ref. no. NG 001336.2.
  • the presence and/or level of TCF is used to predict risk of an aggressive T-cell lymphoma, and the subject may have one or more symptoms associated with an T-cell lymphoma, then the subject is identified as having or at increased risk of developing an aggressive T-cell lymphoma.
  • the subject has no overt signs or symptoms of an aggressive T-cell lymphoma, but the presence and/or level of TCF evaluated is comparable to the presence and/or level of the TCF in the disease reference, then the subject is identified as having an increased risk of developing an aggressive T-cell lymphoma.
  • a treatment e.g., as known in the art or as described herein, can be administered.
  • a subject who has an increased risk is a subject who has a level of TCF > 25%, compared to a subject in a reference cohort who has a TCF ⁇ 25%, and therefore does not have a significant risk of developing aggressive disease, e.g., within one, two, five, or ten years.
  • Suitable reference values can be determined using methods known in the art, e.g., using standard clinical trial methodology and statistical analysis.
  • the reference values can have any relevant form.
  • the reference comprises a predetermined value for a meaningful level of TCF, e.g., a control reference level that represents a normal level of TCF, e.g., a level in a subject who is not at risk (or who has a normal risk) of developing aggressive disease described herein, and/or a disease reference that represents a level of the TCF associated with increased risk of developing an aggressive form of T-cell lymphoma, e.g., a level in a subject having CTCL (e.g., MF).
  • CTCL e.g., MF
  • the predetermined level can be a single cut-off (threshold) value, such as a median or mean, or a level that defines the boundaries of an upper or lower quartile, tertile, or other segment of a clinical trial population that is determined to be statistically different from the other segments. It can be a range of cut-off (or threshold) values, such as a confidence interval. It can be established based upon comparative groups, such as where association with risk of developing disease or presence of disease in one defined group is a fold higher, or lower, (e.g.,
  • n- quantiles i.e., n regularly spaced intervals
  • the predetermined level is a level or occurrence in the same subject, e.g., at a different time point, e.g., an earlier time point.
  • the predetermined reference level is 25% TCF.
  • a control reference subject has T cell lymphoma, e.g., indolent lymphoma, but does not have an increased risk of developing an aggressive T-cell lymphoma.
  • a disease reference subject is one who has an increased risk of developing an aggressive T-cell lymphoma.
  • An increased risk is defined as a risk above the risk of subjects in the general population.
  • the level of TCF in a subject being less than or equal to a reference level of TCF is indicative of a clinical status (e.g., indicative of a disorder as described herein, e.g., risk of developing an aggressive T-cell lymphoma.
  • the level of TCF in a subject being greater than or equal to the reference level of TCF is indicative of the absence of disease or normal risk of the disease.
  • the amount by which the level in the subject is the less than the reference level is sufficient to distinguish a subject from a control subject, and optionally is a statistically significantly less than the level in a control subject.
  • the“being equal” refers to being approximately equal (e.g., not statistically different).
  • the predetermined value can depend upon the particular population of subjects (e.g., human subjects) selected. Accordingly, the predetermined values selected may take into account the category (e.g., sex, age, health, risk, presence of other diseases) in which a subject (e.g., human subject) falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
  • a TCF greater than about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% is used to identify as subject as at risk of developing an aggressive T-cell lymphoma .
  • a TCF greater than or equal to about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% is used to identify as subject as at risk of developing an aggressive T-cell lymphoma.
  • a TCF of about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% is used to identify as subject as at risk of developing an aggressive T-cell lymphoma (.
  • the methods described herein include methods for the treatment of subjects identified as at risk of developing an agressive T-cell lymphoma.
  • the subject has CTCL, e.g., MF or SS.
  • the methods include administering an aggressive treatment, e.g., allogenic hematopoietic stem cell transplantation, as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment.
  • to“treat” means to ameliorate at least one symptom of the T cell lymphoma and/or reduce risk of developing an aggressive T-cell lymphoma.
  • CTCL results in dermatitis, pruritic plaques, lymphadenopathy, and tumors, e.g., ulcerating tumors; thus, a treatment can result in a reduction in dermatitis, pruritic plaques, lymphadenopathy, and tumors, e.g., ulcerating tumors and a return or approach to normal skin appearance and feeling.
  • the TCF is used to recommend administering a method of treatment for an aggressive T-cell lymphoma to a subject who does not have aggressive disease, but who has been identified using a method described herein as being at risk of developing aggressive disease.
  • the method of treatment includes allogenic hematopoietic stem cell transplantation; total skin electron beam therapy (TSEB), e.g., at a low dose (12 Gy) or high dose (36 Gy); unrelated cord blood transplantation; and/or Multiagent chemotherapy.
  • TSEB total skin electron beam therapy
  • the method of treatment includes one or more of (i) allogenic hematopoietic stem cell or cord blood transplantation, (ii) administering a steroid, a P-glycoprotein multiple drug resistance (MDR) antagonist, a retinoid and/or an active agent targeted to a T-cell receptor, (iii) the use of chemotherapy, and (iv) the use of radiation therapy or any combination thereof.
  • administering a steroid, a P-glycoprotein multiple drug resistance (MDR) antagonist, a retinoid and/or an active agent targeted to a T-cell receptor precedes allogenic hematopoietic stem cell or cord blood transplantation.
  • chemotherapy precedes allogenic hematopoietic stem cell or cord blood transplantation.
  • radiation therapy precedes allogenic hematopoietic stem cell or cord blood transplantation.
  • both administering a steroid, a P-glycoprotein multiple drug resistance (MDR) antagonist, a retinoid and/or an active agent targeted to a T-cell receptor and the use of radiation therapy precedes allogenic or cord blood hematopoietic stem cell transplantation.
  • radiation therapy precedes allogenic hematopoietic stem cell or cord blood transplantation.
  • administering precedes allogenic hematopoietic stem cell or cord blood transplantation.
  • MDR P-glycoprotein multiple drug resistance
  • Radiation therapy includes electron beam radiation therapy, surface brachytherapy, UVB,
  • the radiation therapy is total skin therapy. In some embodiments, the radiation therapy is local skin therapy.
  • steroids examples include, but are not limited to, glucocorticoids.
  • a steroid is administered topically.
  • P-glycoprotein antagonists are also known in the art and include, but are not limited to, cyclosporin A, verapamil, quinidine, dihydro-pyridines, calcium channel blockers, cyclosporin analogues (e.g., PSC833 (Novartis, East Hanover, NJ)), phenothiazines,
  • a P- glycoprotein antagonist is administered topically or systemically.
  • Retinoids include agents that bind to the retinoic acid receptor, such as 9-cis- retinoic acid, 4- hydroxy-retinoic acid, all traro-retinoic acid, (E)-4-[2-(5,6,7,8- tetrahydro-2-naphthylenyl)-l - propenyl] -benzoic acid, 3-methyl-(E)-4-[2-(5,6,7,8- tetrahydro-2-naphthylenyl)-l-propenyl]- benzoic acid), and the like as known in the art.
  • a retinoid is administered topically or systemically.
  • Retinoid related drugs bind to the RXR receptor and can also be used therapeutically in CTCL.
  • An active agent that is targeted to a T-cell receptor can be any suitable agent that is targeted to a T-cell receptor, such as the IL-2 receptor, and has an effect, which is an anti-cancer effect.
  • the active agent can be an antibody (or an antigenically reactive fragment thereof) to a T-cell receptor, such as the IL-2 receptor.
  • a commercially available antibody to a T-cell receptor is Zenapax, which is available from Hoffinan-LaRoche, Inc., Nutley, NJ. The antibody is preferably administered systemically.
  • the active agent can be a fusion protein or a conjugate of a means of targeting a T-cell receptor, such as an antibody (or an antigenically reactive fragment thereof) to a T-cell receptor or a ligand to a T-cell receptor, and an active agent, such as a drug (or a prodrug or derivative or pharmaceutically acceptable salt thereof) or a toxin as are known in the art.
  • a drug or a prodrug or derivative or pharmaceutically acceptable salt thereof
  • the drug is an anti-cancer drug and the toxin is an anti cancer toxin.
  • An example of such an agent is an anti-IL- 2 antibody fused to a toxin, such as the agent known as OntakTM (Ligand Pharmaceuticals, San Diego, CA).
  • Chemotherapy can include administration of one or more antimetabolites, alkylating agents, topoisomerase II inhibitors, anthracyclines, and/or purine analogues.
  • methotrexate, chlorambucil, vorinostat, or etoposide is used.
  • an HD AC inhibitor is used, e.g. romidepsin.
  • the treatment includes etoposide, vincristine, doxorubicin, cyclophosphamide, and prednisone (EPOCH); cyclophosphamide, vincristine, nr-16, adriamycin and prednisolone (COP, CHOP, CAVOP); CMED/ABV;
  • CCR4-directed monoclonal antibody e.g., Mogamulizumab
  • HD AC inhibitors e.g., fusion toxins (e.g., DAB- interleukin 2 (DAB-IL2)); Adcetris (brentuximab vedotin), an antibody-drug conjugate focused on targeting CD30; bexarotene; Denileukin diftitox; Alemtuzumab; IFN-alpha; .
  • the primary discovery cohort comprised 208 patients with CTCL seen at the Dana-Farber Cancer Institute’s Cutaneous Lymphoma Clinic from 2002 to 2016 (Table A). This discovery set included 177 patients with MF with a median follow-up of 8 years. Samples were typically collected at the time of diagnosis or at the time of referral to the DFCI Cutaneous Lymphoma Clinic for management of established disease.
  • UVB therapy 46 (26)
  • Mogamulizumab 2 (1) Abbreviations: ISCL, International Society for Cutaneous Lymphomas; EORTC, European Organization for Research and Treatment of Cancer; LDH, Lactate Dehydrogenases; UVB, ultraviolet B; PUVA, psoralen and ultraviolet A; *Pre-Sezary refers to the evidence of blood abnormalities (elevated absolute CD4 T cell count or CD4/CD8 T cell ratio) that do not meet the criteria for stage B2 or Sezary syndrome.** available data in 203 patients ***treatments received, alone or in combination, between the inclusion and first evidence of progression, death or censoring.
  • the independent validation set included 101 distinct CTCL patients recently included in the same study, including 87 patients with MF (Table B). The data were collected through December 23, 2016.
  • ISCL International Society for Cutaneous Lymphomas
  • EORTC European Organization for Research and Treatment of Cancer
  • LDH Lactate Dehydrogenases
  • UVB ultraviolet B
  • PUVA psoralen and ultraviolet A.
  • pre-Sezary refers to the evidence of blood abnormalities (elevated absolute CD4 T cell count or CD4/CD8 T cell ratio) that do not meet the criteria for stage B2 or Sezary syndrome. * treatments received, alone or in combination, between the inclusion and first evidence of progression, death or censoring.
  • DNA and RNA were extracted from four 20 pm -thick formalin-fixed, paraffin-embedded tissue scrolls from a lesional skin biopsy using the Allprep DNA/RNA FFPE isolation kit (Qiagen) as per the manufacturer’s instructions. DNA and RNA amounts were measured using a BioDrop spectrophotometer (Denville Scientific Inc.). For fresh frozen samples from the validation set, DNA was isolated from thirty cryosections of 10 pm thickness. DNA extraction was carried out using the QIAamp DNA Mini Kit (Qiagen) kit as per manufacturer’s instructions with overnight tissue digestion. High throughput sequencing of the TCRB gene
  • ImmunoSEQ Adaptive Biotechnologies
  • the ImmunoSeq platform is available as a kit or service (adaptivebiotech.com/immunoseq). All TCRB characterization was performed by Adaptive Biotechnologies using the ImmunoSeq TCRB 'survey level' human assay (4, 34) which has previously described in detail (3).
  • the putative malignant clone was defined by sequence abundance.
  • a clone can have either one or two rearranged TCR alleles. For most of the clones, both TCRG alleles are rearranged, and for TCRB, a minority have both alleles rearranged.
  • a clone’s abundance was defined by summing the abundance of the most frequent single productive allele for TCRB.
  • the putative malignant clone was defined by relative abundance of its unique CDR3 sequence (3). The percent of T cells consisting of the malignant clone was determined by dividing the abundance of the malignant clone (number of reads) by the total number of T cells (number of total reads).
  • the diversity of the reactive T cell clones was studied using the Shannon’s index. Shannon’s entropy quantifies the uncertainty in predicting the sequence identity of a random sequence from a dataset.
  • the Shannon’s index of the reactive clones (H) was calculated according to the following formula:
  • entropy was normalized by division of log 2 of the number of unique productive sequences.
  • CTCL skin samples were co-immunostained for anti-VP2 (Beckman-Coulter, clone: MPB2D5) or anti-VP5. 1 (Beckman-Coulter, clone: IMMU 157) conjugated to R-phycoerythrin with anti- CD3 conjugated to Alexa Fluor 647 (Biolegend, clone: UCHT1) with 3 five-minute wash steps in TBS-saponin before mounting. Single color controls confirmed specificity of staining and no bleed through into the other channel.
  • the samples were analyzed using an Olympus BX43 microscope with the objective lens of l0x/0.40, 20x/0.75 and 40x/0.95 Olympus UPlanFL (Olympus). Images were acquired with the Mantra Quantitative Pathology Imaging System, and analyzed using inForm software (Perkin-Elmer) and the manual counting feature from Adobe Photoshop CS5 (Adobe). We analyzed lOx images of non-overlapping fields.
  • NanoString gene expression profiling using a custom codeset including 78 probes directed against potential biomarkers identified in previous gene expression studies by our group (13-15) or in exome sequencing studies by others (23, 28-30), and 3 housekeeping genes.
  • the Nanostring technology uses molecular barcode and single molecule imaging for the direct hybridization and detection of hundreds of unique transcripts in a single reaction. Each color-coded barcode is attached to a single target-specific probe corresponding to an analyte of interest. Combined together with invariant controls, the probes form a multiplexed CodeSet. The samples are run on the nCounter platform. Gene expression data were background subtracted and normalized to positive controls and
  • Nanostring nSolver software (nanostring.com/products/analysis- software/nsolver). Gene expression values were expressed as log2 fold changes (FC) of the average gene expression of the considered gene in the whole study group. Gene expression assays were performed and analyzed blinded to the patient’s outcome.
  • the flashlamp is an intense pulsed light (IPL) that emits a bright range of wavelengths from ultraviolet to visible light and infrared, but ultraviolet light is filtered out and does not reach the tissue.
  • IPL intense pulsed light
  • the light excites and heats the stained cells that transfer to the membrane.
  • the membrane was then placed in lysis buffer and DNA extracted using a QIAmp DNA microkit (Qiagen). The DNA quantity and integrity were measured by using a Bioanalyzer. A matched blood sample from the same patient, without blood involvement as confirmed by high-throughput sequencing of the TCR)3 gene, was used as a germline control.
  • SNV single nucleotide variants
  • VEP Variant Effect Predictor
  • PFS Progression-free survival
  • OS overall survival
  • CLIPI Cutaneous Lymphoma International Prognostic Index
  • TCF tumor clone frequency
  • TCF histopathological analyses demonstrated that a high TCF was not associated with higher absolute numbers of mononuclear cells in the skin infiltrate (FIG. 1D).
  • Tl skin surface area involved with patches and plaques
  • T2 >10% body surface area involved with patches and plaques
  • T4 erythroderma
  • Counting T cells by immunostaining with antibodies to nb gene products has been used to identify clonal populations in skin, since all malignant clonal cells express the same Ub gene product. Therefore, we asked whether immunostaining could substitute for high-throughput sequencing of the TCRB.
  • antibodies are available for only about 50% of nb families.
  • immunostaining for nb is inherently imprecise in the identification and quantification of a specific T cell clone. In part, this is because a given TCRVB exon can rearrange and pair with one of 13 TCRJB exons during intrathymic T cell maturation.
  • 28.4% of skin T cells were TCRVB20+, but only 39.8% of these TCRVB20 T cells shared the specific CDR3 sequence of the malignant clone
  • FIGS. 6A-6C Here, antibody staining more accurately estimated the malignant clone, but was still variable from histological section to section. These approaches appear to be fundamentally inferior at quantifying the malignant clone when compared to the highly quantitative metric of TCF.
  • TCF was still significantly associated with PFS (pO.OOl) and OS (pO.OOl).
  • PFS pO.OOl
  • OS pO.OOl
  • a TCF of 25% was found to be the best cutoff as determined by the concordance index (25) in univariable analysis on PFS and OS.
  • stage T2/IB patients with a malignant clone ⁇ 25% of the skin T cells were alive without disease progression 4 years later, vs. 30% (95% Cl, 7-58) of stage T2/IB patients with a malignant clone>25% (FIG. 3C).
  • stage T2/IB patients with a malignant clone ⁇ 25% were alive and progression-free 4 years later, vs.
  • FIG. 3D 19% (95% Cl, 5-40%) of patients with a malignant clone>25%.
  • the TCF in skin was also significantly associated with OS (pO.Ol) (FIG. 3C).
  • 7A-7B compares PFS and OS of Tla, Tlb, T2a, and T2b patients, and confirms that both skin stage (T2/IB versus Tl/IA) and the presence of plaques are associated with decreased PFS (p ⁇ 0.0l and p ⁇ 0.05 for skin stage and plaques, respectively) and that the skin stage is associated with decreased OS (p ⁇ 0.0l) in Cox univariable analysis.
  • PFS and OS in Stage IB/T2 patients were assessed according to the presence or absence of plaques (IB/T2a vs. IB/T2b) or the TCF>25% (FIG. 3C-D), the latter was far more predictive.
  • TCF>25% was highly predictive of PFS and OS, and was far more predictive than the presence of plaques vs patches.
  • stage IB patients who experienced progression or death during the follow-up there was an inverse correlation between the TCF and the time to progression or death (rho -0.6, pO.OOl) (FIG. 3E).
  • TCF in skin >25% was associated with a positive predictive value of 92% for 5-year disease progression or death, and a negative predictive value of 83% (FIG. 3F).
  • stage IA/T1 patients who have limited skin involvement with ⁇ 10% of the body surface area involved
  • had an excellent prognosis regardless of the TCF (FIG.10A-10B).
  • TCF malignant T cell clone in skin
  • Elevated LDH levels 1 .2 0.4 - 3.1 .8 1.0 .3 - 3.4 1
  • Patches, papules, or plaques covering > 10% of the skin surface may further stratify into T2 T2a (patch only) v T2b (plaque +/- patch)
  • T3 One or more tumors (> 1 cm diameter)
  • High blood tumor burden > 1 ,000/pL Sezary cells with positive clone that matches the skin B2 clone; one of the following can be substituted for Sezary cells: CD4/CD8 > 10, CD4+ CD7- cells > 40% or CD4+ CD26- cells > 30%
  • ISCL International Society for Cutaneous Lymphomas
  • EORTC European Organisation for Research and Treatment of Cancer
  • MF mycosis fungoides
  • SS Sezary syndrome
  • NCI National Cancer Institute.
  • *Patch any size lesion without induration or significant elevation above the surrounding uninvolved skin.
  • Plaque any size lesion that is elevated or indurated.
  • the standard staging classification system for MF and SS is the TNMB system, which is based on an evaluation of the skin (T), lymph nodes (N), visceral involvement (M), and blood (B).
  • IVB 1-4 0-3, X 1 0-2 Abbreviations: ISCL, International Society for Cutaneous Lymphomas; EORTC, European Organisation for Research and Treatment of Cancer; MF, mycosis fungoides; SS, Sezary syndrome; X, clinically abnormal lymph nodes without histologic confirmation or inability to fully characterize histologic subcategories
  • Example 5 Tumor Microenvironment / High TCF Not Associated with Lower Numbers of Reactive CD8 T-Cells or a Less Clonal Reactive T-Cell Environment
  • T cell specific genes such as cell surface markers ( CD4 , CCR4, CCR7, CD28, CD52, PDCD1 ), genes in the IL-21/JAK/STAT pathway (IL21, IL2RG, JAK3 ), and genes in the TCR signaling pathway (I ⁇ K, LCK, PRKCQ, SH2D1A, FYB, LAT, PTPRCAP, RAC2, GIMAP4, T3JAM, CARD 11, SIT1, PIK3CD, VAV1, LEF1 ).
  • cell surface markers CD4 , CCR4, CCR7, CD28, CD52, PDCD1
  • genes in the IL-21/JAK/STAT pathway IL21, IL2RG, JAK3
  • genes in the TCR signaling pathway I ⁇ K, LCK, PRKCQ, SH2D1A, FYB, LAT, PTPRCAP, RAC2, GIMAP4, T3JAM, CARD 11, SIT1, PIK3CD, VAV1, LEF1 .
  • cluster 1 a cluster of patients with a distinct gene expression profile, high tumor clone frequency in skin, and poor prognosis.
  • the overexpression of genes in the JAK- STAT and TCR signaling pathways in patients with a high TCF are consistent with the role of these pathways in T cell proliferation and survival.
  • Whole exome sequencing of tumors has yielded valuable data in a variety of cancers, but has been difficult to perform in patches or plaques of MF because of the paucity of tumor cells relative to total nucleated cells.
  • the mean target coverage was 70x in tumor samples and l03x in peripheral blood mononuclear cells.
  • ISCL/EORTC Revisions to the staging and classification of mycosis fungoides and Sezary syndrome: a proposal of the International Society for Cutaneous Lymphomas (ISCL) and the cutaneous lymphoma task force of the European Organization of ISCL/EORTC
  • T cells in cutaneous T-cell lymphoma expression of cytotoxic proteins, Fas Ligand, and killing inhibitory receptors and their relationship with clinical behavior, J. Clin. Oncol.

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Abstract

L'invention concerne des procédés d'identification et des méthodes de traitement de sujets présentant un risque de développer des lymphomes à lymphocytes T agressifs, tels que des lymphomes à lymphocytes T cutanés. Les procédés consistent à déterminer la fréquence de clonage de la tumeur.
PCT/US2019/026126 2018-04-06 2019-04-05 Méthodes de diagnostic et de traitement de lymphomes à lymphocytes t cutanés agressifs Ceased WO2019195769A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130251711A1 (en) * 2010-11-22 2013-09-26 Innate Pharma Sa Nk cell modulating treatments and methods for treatment of hematological malignancies
US20150218656A1 (en) * 2014-02-03 2015-08-06 Adaptive Biotechnologies Corp. Methods for detection and diagnosis of a lymphoid malignancy using high throughput sequencing
WO2016109452A1 (fr) * 2014-12-31 2016-07-07 Guardant Health , Inc. Détection et traitement d'une maladie faisant preuve d'hétérogénéité des cellules malades et systèmes et procédés de communication des résultats de test
US9824179B2 (en) * 2011-12-09 2017-11-21 Adaptive Biotechnologies Corp. Diagnosis of lymphoid malignancies and minimal residual disease detection
US20180208984A1 (en) * 2017-01-17 2018-07-26 Life Technologies Corporation Compositions and methods for immune repertoire sequencing
WO2019067092A1 (fr) * 2017-08-07 2019-04-04 The Johns Hopkins University Méthodes et substances pour l'évaluation et le traitement du cancer

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130251711A1 (en) * 2010-11-22 2013-09-26 Innate Pharma Sa Nk cell modulating treatments and methods for treatment of hematological malignancies
US9824179B2 (en) * 2011-12-09 2017-11-21 Adaptive Biotechnologies Corp. Diagnosis of lymphoid malignancies and minimal residual disease detection
US20150218656A1 (en) * 2014-02-03 2015-08-06 Adaptive Biotechnologies Corp. Methods for detection and diagnosis of a lymphoid malignancy using high throughput sequencing
WO2016109452A1 (fr) * 2014-12-31 2016-07-07 Guardant Health , Inc. Détection et traitement d'une maladie faisant preuve d'hétérogénéité des cellules malades et systèmes et procédés de communication des résultats de test
US20180208984A1 (en) * 2017-01-17 2018-07-26 Life Technologies Corporation Compositions and methods for immune repertoire sequencing
WO2019067092A1 (fr) * 2017-08-07 2019-04-04 The Johns Hopkins University Méthodes et substances pour l'évaluation et le traitement du cancer

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