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Epigenetic therapy overcomes treatment resistance in T cell prolymphocytic leukemia

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Science Translational Medicine  24 Jun 2015:
Vol. 7, Issue 293, pp. 293ra102
DOI: 10.1126/scitranslmed.aaa5079
  • Fig. 1. One cycle of epigenetic therapy consisted of a three-drug combination over 28 days.

    Patients received cladribine at 5 mg/m2 intravenously (IV) each day on days 1 to 5. Those treated with vorinostat were given 400 mg orally (po) each day on days 1 to 5 as well. Patients received 30 mg of alemtuzumab intravenously or subcutaneously once a day on days 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26. One treatment cycle was 28 days. All patients were treated with alemtuzumab and cladribine. Those whose white blood cell counts did not respond well after relapse were also treated with vorinostat. Status of clinical and molecular remissions was evaluated after two to three cycles.

  • Fig. 2. Epigenetic therapy decreased leukemic white blood cell counts despite multiple relapses.

    Patient 2’s white blood cell (WBC) counts were monitored during treatment. Day 0 was the initial white blood cell count at presentation. The bars beneath the graph represent treatment received by the patient over the corresponding time periods. Graphs for all treated patients are available in fig. S3.

  • Fig. 3. Epigenetic therapy induced CD30 gene expression and chromatin reorganization.

    (A) Patient mRNA samples were assayed for CD30 gene expression using quantitative reverse transcription PCR (qRT-PCR) before and 5 days after treatment with epigenetic therapy. Fold change represents the fold increase or decrease of expression after treatment relative to before treatment. Numbers in parentheses indicate actual fold change. (B and C) Genomic DNA from patients 1 (B) and 5 (C) was assessed for changes in DNA and chromatin methylation as well as RNA Pol II binding before and after treatment using ChIP assays. For patient 5, samples were taken before and 5 days after treatment of relapse with romidepsin instead of vorinostat. For patient 1, samples were taken before and 5 days after initial epigenetic treatment. Fold change indicates fold increase or decrease in binding after treatment as measured by qRT-PCR. Three regions of the CD30 promoter were considered in this assay spanning roughly 400 bp on each side of the transcription start site. Prox refers to a 200-bp region around the transcription start site. *P < 0.05, **P < 0.005, ***P < 0.0005. Bars represent mean fold change ± SEM. n = 3.

  • Fig. 4. CD30 positivity correlates with response to brentuximab vedotin in patient 5.

    For time points at which blood samples of patient 5 were available, CD30 status was assessed by qRT-PCR. This is represented by the bar graph with days on the x axis. CD30 expression values are reported as fold change compared to initial treatment–naïve samples collected before study intervention. White blood cell counts (in red) from these same time points were also collected and graphed above their corresponding dates (black line graph). Antibody treatment received during each period is labeled above the graph. Switch from vorinostat to romidepsin occurred around day 200. The inset to the right shows flow cytometry data of CD30-negative (top) and CD52-positive (bottom) cell populations at the last time point at which CD30 expression was observed to disappear by qRT-PCR, after which treatment was switched back to alemtuzumab. ***P < 0.0005. Bars represent mean fold change ± SEM. n = 3.

  • Fig. 5. Brentuximab vedotin cleared CD30+ skin lesions in patient 5.

    (A) Immunohistochemistry (IHC) of biopsies of these lesions showed CD30+ infiltrate (left) in the dermis before treatment with brentuximab vedotin and absent CD30+ T-PLL cells after 2 months of treatment (right). Blue, nucleus; brown, CD30. Scale bars, 20 μm. (B) Images of the upper left shoulder (top) and lateral left chest wall (bottom) depict plaques infiltrated with CD30+ T-PLL cells before (left) and after (right) treatment with brentuximab vedotin.

  • Fig. 6. Epigenetic therapy induces HIN-200, CEBP, and globin gene expression.

    Changes in expression of AIM2, MNDA, IFI16, PYHIN, CEBPA, CEBPB, CEBPD, HBA, and HBB were assessed by qRT-PCR before and 5 days after treatment of six patients with epigenetic therapy. Sufficient samples for mRNA analysis were only available for patients 1 to 6. Only three CEBP family members were tested. Values are shown as fold changes as compared to treatment-naïve expression in each patient. Numbers in parentheses indicate actual fold change. *P < 0.05, **P < 0.005, ***P < 0.0005. Bars represent mean fold change ± SEM. n = 3.

  • Fig. 7. Epigenetic therapy induces TRIB1 expression and increases active chromatin marks.

    (A) Changes in expression of TRIB1 were assessed by qRT-PCR before and after treatment of six patients with epigenetic therapy. Values are shown as fold changes as compared to treatment-naïve expression in each patient. Numbers in parentheses indicate actual fold change. (B) Genomic DNA from patients 1 (gray) and 5 (black) was assessed with ChIP to detect changes in DNA and chromatin methylation as well as RNA Pol II binding before and 5 days after treatment, the same time points as in Fig. 3 (B and C). Fold change indicates fold increase or decrease in binding after treatment as measured by qRT-PCR. The region represented is 400 bp around the transcriptional start of the TRIB1 gene. Acytl, pan–histone acetylation. *P < 0.05, **P < 0.005, ***P < 0.0005. Bars represent mean fold change ± SEM. n = 3.

  • Table 1. Summary of patient treatment response.

    Disease status, previous treatments, treatment administered, extent of clinical and molecular remission, duration of response, overall survival, and expected survival were tabulated. Survival times are calculated from first treatment with alemtuzumab. Mean time between relapses was 6 months. CR, complete remission; PR, partial response (decrease in white blood cell counts); CNS, central nervous system; Allo, allogeneic; N/A, not applicable.

    PatientDisease status
    before epigenetic
    therapy
    Previous
    treatment
    Treatment
    administered
    Clinical
    remission
    Molecular
    remission
    Duration of
    response
    Overall
    survival time
    Expected
    survival*
    1First relapseCVP (cyclophosphamide,
    vincristine, prednisone)
    Alemtuzumab,
    cladribine
    CRYes16 months34.3 months4 months
    Second relapseAlemtuzumab,
    cladribine
    Alemtuzumab,
    cladribine
    PRN/A11.6 months
    Third relapseAlemtuzumab,
    cladribine
    Alemtuzumab,
    brentuximab vedotin
    PRN/A6.7 months to death,
    persistent disease
    2First relapseAlemtuzumab
    (6 months CR)
    Alemtuzumab,
    cladribine, vorinostat
    CRYes5.3 months17 months7.5 –
    10 months
    Second relapseAlemtuzumab,
    cladribine, vorinostat
    Alemtuzumab,
    cladribine, vorinostat
    PRN/A2 months
    Third relapseAlemtuzumab,
    cladribine, vorinostat
    Alemtuzumab,
    cladribine, vorinostat
    PRN/A3.7 months to death,
    persistent disease
    3Initial presentationN/AAlemtuzumab,
    cladribine
    PRN/A5.7 months5.7 months20 months
    4Initial presentationN/AAlemtuzumab,
    cladribine
    CRYes13.3 months14.8 months20 months
    First relapseAlemtuzumab,
    cladribine
    Alemtuzumab,
    cladribine
    PRN/A1.5 months to death,
    persistent disease,
    CNS hemorrhage
    5Initial presentationN/AAlemtuzumab,
    cladribine, vorinostat
    CRYes6.3 months15.3 months20 months
    First relapse with
    skin involvement
    Alemtuzumab,
    cladribine, vorinostat
    Alemtuzumab,
    cladribine, romidepsin
    PRN/A4 months—blood
    Second relapse with
    skin involvement
    Alemtuzumab,
    cladribine, romidepsin
    Brentuximab vedotinPRN/A5 months to Allo
    transplant, death,
    persistent disease
    6Initial presentationN/AAlemtuzumab,
    cladribine
    CRYes0.8 months to
    Allo transplant
    0.8+ monthsN/A
    7First relapseAlemtuzumab
    (0.3 months)
    Alemtuzumab,
    cladribine, valproic acid
    CRYes12 months to Allo
    transplant referral
    12+ months4 months
    8First relapseAlemtuzumab
    (16 months)
    Alemtuzumab,
    cladribine
    CRYes6.7 months23.7+ months7.5 –
    10 months
    Second relapseAlemtuzumab,
    cladribine
    Alemtuzumab,
    cladribine
    CRYes1+ month

    *Expected survival without transplant (5).

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/293/293ra102/DC1

      Supplemental patient case information

      Fig. S1. Cladribine inhibits DNA methylation in vivo and histone methylation in vitro.

      Fig. S2. Epigenetic therapy does not induce apoptosis in T-PLL.

      Fig. S3. Epigenetic therapy lowered leukemic white blood cell counts despite multiple relapses.

      Fig. S4. CD52 expression was unchanged by epigenetic therapy.

      Fig. S5. CD30 induction was not observed in circulating MCL cells.

      Fig. S6. Skin biopsy from patient 5 shows CD52-positive dermis-infiltrating lymphocytes.

      Fig. S7. Epigenetic treatment of T-PLL causes mRNA expression changes in the HIN-200, CEBP, and tribbles families.

      Fig. S8. All tested patients showed CDKN1B haploinsufficiency, and four of six were TCL1-positive.

      Fig. S9. Forty percent of patients were positive for the N642H STAT5B mutation.

      Table S1. Patient presentation at diagnosis.

      Table S2. Patient characteristics and adverse events.

      Table S3. Primer sequences.

      Table S4. Significant P values for all statistically significant comparisons in figures.

      References (61, 62)

    • Supplementary Material for:

      Epigenetic therapy overcomes treatment resistance in T cell prolymphocytic leukemia

      Zainul S. Hasanali, Bikramajit Singh Saroya, August Stuart, Sara Shimko, Juanita Evans, Mithun Vinod Shah, Kamal Sharma, Violetta V. Leshchenko, Samir Parekh, Thomas P. Loughran Jr.,* Elliot M. Epner*

      *Corresponding author. E-mail: tploughran{at}virginia.edu (T.P.L.); epner5{at}msn.com (E.E.)

      Published 24 June 2015, Sci. Transl. Med. 7, 293ra102 (2015)
      DOI: 10.1126/scitranslmed.aaa5079

      This PDF file includes:

      • Supplemental patient case information
      • Fig. S1. Cladribine inhibits DNA methylation in vivo and histone methylation in vitro.
      • Fig. S2. Epigenetic therapy does not induce apoptosis in T-PLL.
      • Fig. S3. Epigenetic therapy lowered leukemic white blood cell counts despite multiple relapses.
      • Fig. S4. CD52 expression was unchanged by epigenetic therapy.
      • Fig. S5. CD30 induction was not observed in circulating MCL cells.
      • Fig. S6. Skin biopsy from patient 5 shows CD52-positive dermis-infiltrating lymphocytes.
      • Fig. S7. Epigenetic treatment of T-PLL causes mRNA expression changes in the HIN-200, CEBP, and tribbles families.
      • Fig. S8. All tested patients showed CDKN1B haploinsufficiency, and four of six were TCL1-positive.
      • Fig. S9. Forty percent of patients were positive for the N642H STAT5B mutation.
      • Table S1. Patient presentation at diagnosis.
      • Table S2. Patient characteristics and adverse events.
      • Table S3. Primer sequences.
      • Table S4. Significant P values for all statistically significant comparisons in figures.
      • References (61, 62)

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