REGULAR ARTICLE zary syndrome patient–derived models allow drug selection for Se personalized therapy Fernando Gallardo,1 Evelyn Andrades,1 Arnau Iglesias,2 Jessica Gonz alez,2 Laura Sol e,2 Yolanda Guill en,2 Gonzalo Blanco,3 Luis Colomo, Eva Gimeno, David Conde, Eva Rodriguez, Isabel Bielsa-Marso, Mar Iglesias, Beatriz Bellosillo,3 Ramon M. Pujol,1 3 4 5 6 7 3  R. Regueiro,8 Anna Bigas,2 and Lluıs Espinosa2 Jose 1 onoma de Barcelona, Barcelona, Spain; 2Cancer and Stem Cells Research Group, Institut Hospital del Mar Dermatology Department, Hospital del Mar, Universitat Aut Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 ediques (IMIM), and 3Pathology Department, Hospital del Mar, CIBERONC, Barcelona, Spain; 4Hematology Department, 5Pharmacy Department, and d’Investigacions M 6 Nephrology Department, Hospital del Mar, Barcelona, Spain; 7Dermatology Department, Hospital Germans Trias i Pujol, Barcelona, Spain; and 8Microbiology and Immunology Department, Universidad Complutense de Madrid, Madrid, Spain zary syndrome (SS) do not achieve a significant Current therapeutic approaches for Se Key Points improvement in long-term survival of patients, and they are mainly focused on reducing  Patient-derived SS blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic cells show highly and molecular heterogeneity. Determining effective and personalized treatments for SS is heterogeneous drug urgently needed. The present work compiles the current methods for SS patient–derived responses. xenograft (PDX) generation and management to provide new perspectives on treatment for  We have developed a patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh joint in vitro/in vivo peripheral blood of patients with SS (N 5 11). A selected panel of 26 compounds that are platform to predict SS inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, therapy response. histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient–derived Sezary cells xenotransplanted (PDX) into NOD-SCID-g mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner. Introduction Cutaneous T-cell lymphomas (CTCLs) are lymphoid malignant neoplasms that initially manifest in the skin. The most frequent CTCLs are mycosis fungoides (MF) and Se zary syndrome (SS). SS is clinically characterized by erythroderma associated with peripheral blood (PB) involvement manifested by circulat- ing malignant lymphoid cells with cerebriform nuclei (S ezary cells). SS is considered an aggressive CTCL with no curative alternative. Interferon, oral retinoids (bexarotene), and nonspecific histone deacety- lase (HDAC) inhibitors, alone or in combination with photopheresis, are currently prescribed as therapeu- tic options, but most cases achieve response rates of 30%. Multiple evidence shows that standard Submitted 17 December 2021; accepted 23 March 2022; prepublished online on Blood The full-text version of this article contains a data supplement. Advances First Edition 12 April 2022; final version published online 8 June 2022. DOI © 2022 by The American Society of Hematology. Licensed under Creative 10.1182/bloodadvances.2021006860. Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC- Requests for original images and data sharing may be made by contacting Lluıs ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other Espinosa (e-mail: lespinosa@imim.es). rights reserved. 3410 14 JUNE 2022 • VOLUME 6, NUMBER 11 polychemotherapy is not effective in SS, showing very short-lived enriched sample of mononuclear cells was obtained by Ficoll responses, associated in part with the high inter- and intra-tumoral separation. heterogeneity of the disease. Genetic characterization of SS cells zary cells Monitoring and quantification of aberrant malignant Se has been improved by next-generation sequencing studies.1-5 in vitro and in vivo were determined by FC for the standardized SS Because of the low incidence of SS,6 data from multiple small markers CD31/CD4low/CD26–/CD7–15-17 using fluorescein iso- cohorts have been combined for identifying rare driving mutations thiocyanate/phycoerythrin (FITC/PE)–conjugated monoclonal anti- and genomic copy number variations; this has led to multiple path- bodies (BD Biosciences, San Diego, CA). way alterations with impact on disease progression by modulating T-cell activation, inhibiting apoptosis, affecting chromatin remodeling In vitro drug screening platform and epigenetic regulators, DNA damage response, cell cycle, or Inhibitors used in the study were obtained from Selleck Chemicals immune surveillance impairment. Most relevant genomic alterations drug library (https://www.selleckchem.com/screening/fda-approved- found include genes such as TP53, PLCG1, CARD11, TNFRSF1B, drug-library.html) (Houston, TX). Selection of a single-agent multi- Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 STAT5B, DNMT3A, ARID1, FASN, or ZEB1.7,8 drug testing panel was based on standard treatments used in the Some of the described molecular alterations are currently being clinic (ie, bexarotene, steroids, methotrexate, HDAC inhibitors) and investigated in clinical trials for their therapeutic use in MF or SS. the signaling pathways previously identified as relevant for SS pro- The effectiveness of using the phosphatidylinositol 3-kinase (PI3K) gression7 (ie, JAK/STAT, PI3K/protein kinase B/mammalian target of (eg, duvelisib) or NF-kB (eg, bortezomib) inhibitors has been dis- rapamycin, MAPK, or TCR/NF-kB pathway inhibitors). Compounds cussed in translational research and tested in few patients in clinical (supplemental Table 2) were prepared as dimethyl sulfoxide (DMSO) stocks and then diluted as indicated. Then, 4 3 105 cells trials, and both have shown good tolerance and durable responses. per well were seeded in 96-well plates and treated with the thera- The HDAC inhibitors vorinostat and romidepsin are approved in the peutic agents for 72 hours. Additional controls were included that United States for use in MF, and resminostat is in a phase 3 trial in were treated with the minimum and maximum doses of dimethyl sulf- the European Union. Currently, alisertib, an oral inhibitor of the oxide used as vehicle (0.1% and 20%). aurora A kinase, and cobomarsen, an oligonucleotide inhibitor of miR-155, are under investigation, among other interesting targeted Quantification of the response to pharmacologic drugs. Finally, JAK/STAT inhibitors are increasingly being used in dif- inhibition in the in vitro assay ferent hematologic and skin conditions and represent promising drugs for further clinical development in CTCL. However, advances The effect of pharmacologic inhibition on cell viability after 72 hours of incubation was determined by using CellTiter-Glo_3D Cell Viabil- in the genetic knowledge of CTCL tumor cells have not yet been ity Assay (Promega, Madison, WI). We initially tested all compounds translated into the implementation of targeted therapies with curative at 20, 4, and 0.8 mM. Compounds showing a minimum of a 50% response rates. Immunotherapy-related treatments such as moga- decrease in cell viability at a concentration of 1 mM were further mulizumab have changed the management of patients with SS, and tested in dose–response assays. brentuximab, as well as activators of the antitumor immune response (anti–programmed death-1 [PD1]/programmed cell death ligand 1), In vivo murine model seem to be well positioned as alternative therapeutics.9-11 Transplantation of Sezary cells was performed by intradermal injec- In this context, new pathway-based personalized treatments are tion of 2 million mononuclear cells in 20 mL of phosphate-buffered clearly needed to combat SS. Currently, ex vivo culture of patient- saline (obtained from apheresis) in the ear of NOD-SCID-g (NSG) derived cancer cells and xenograft (PDX) models represent a tool mice (The Jackson Laboratory, Bar Harbor, ME) at 8 to 12 weeks of for expanding human tumors, which can then be banked and used age. PDX assays were performed in accordance with the regula- as in vivo models for translational research. These models have tions of Parc de Salut Mar-Parc de Biome dica de Barcelona- recurrently been shown to closely resemble patient tumor features Universitat Autonoma de Barcelona (TG-09-1223P3-ABS) and the and retain molecular and histologic characteristics. Recently, a few current regulations on Protection of Personal Data and Guarantees PDX models of primary SS cells have been established,12 but most of Digital Rights (General Data Protection Regulation of the EU, in vivo investigations have been performed by using SS cell Regulation EU-2016/679 and Spanish Organic Law 3/2018), and lines.13,14 The predictive value of in vitro and in silico data are ulti- the Biomedical Research Law 14/2007 and Royal Decree mately limited by the complexity of the whole-organism systems. RD1716/2011. Thus, combining in vitro and in vivo models will allow a better trans- Mice treatment was initiated when a minimum of 1% human lation of in vitro drug testing to patient treatment. CD451 cells were detected in circulation. Vehicle alone was used in the untreated controls at the same doses and protocol of admin- Methods istration as the therapeutic agents. Specific drug schedule treatment was determined according to pharmacokinetic, 50% inhibitory con- Patients and study samples centration dosage (obtained in the in vitro analysis), and/or maximum Eight patients with SS were included in the study. Clinical charac- reported doses for the specific drug in the in vivo mouse models. teristics, peripheral blood flow cytometry (FC) parameters, patient Quantification of the response to pharmacologic treatments, and clinical status are presented in supplemental Table 1. Primary Se zary cells for in vitro drug screening and in vivo gener- inhibition in the in vivo model ation of PDX were obtained during the apheresis procedure cur- PB samples from mice were obtained at different time points after rently performed as adjuvant treatment for patients with SS. An transplantation and treatment, and at the experimental end point. 14 JUNE 2022 • VOLUME 6, NUMBER 11 NEW SS MODEL FOR PATHWAY-BASED MEDICINE 3411 Patient #23 (sample from photopheresis) 105 105 CD3+ CD45+ 88.0 PerCP-Cy5-5-A hCD3 4 APC-Cy7-A hCD45 10 99.3 104 103 103 102 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 0 0 –102 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K FSC-A FSC-A 104 104 CD4+ 100 103 PE-Cy7-A hCD4 103 PE-A hCD26 102 CD26– 10 2 91.5 0 0 –102 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K FSC-A FSC-A CD26– CD7+ CD26+ CD7+ 104 104 10.6 6.24 103 103 FITC-A hCD7 FITC-A hCD7 102 CD7– 102 83.6 0 0 CD26– CD7– CD26+ CD7– –102 –102 81.3 1.85 0 50K 100K 150K 200K 250K –103 0 103 104 105 FSC-A PE-A hCD26 Figure 1. High efficiency purification of SS cells from patient photopheresis. Representative FC analysis of a blood sample obtained from the photopheresis of the indicated SS patient. Noteworthy is the high prevalence of malignant CD41, CD26–, and CD7– lymphocytes. They were further analyzed by FC with antibodies for Se zary cells. quantify the aberrant (and normal) human T-cell lymphoid popula- First bleeding was performed at 4 weeks after transplantation and tions by FC. Compounds used for in vivo testing were selected subsequently repeated every 2 weeks. We established detection of based on their therapeutic efficacy in the in vitro assay and the pos- 1% of human CD451 cells in circulation as the criterion to start the sibility of a rapid translation to the clinics (ie, we favored compounds in vivo treatment. After starting treatment, mice were bled weekly to already used in the clinical practice or in clinical trials). At 3412 GALLARDO et al 14 JUNE 2022 • VOLUME 6, NUMBER 11 A Measuring Plating cells cell viability Leukocytes ! 72h treatment B Example: patient #30 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 287 322 302 322 291 322 322 275 Relative cell growth Relative cell growth Relative cell growth Relative cell growth Relative cell growth 226 210 258 163 181 167 142 129 118 80 106 96 [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. Bardoxolone methyl Dasatinib Sorafenib Temsirolimus Bortezomib 247 287 318 313 324 301 283 283 287 265 Relative cell growth Relative cell growth Relative cell growth Relative cell growth Relative cell growth 200 212 198 198 164 176 144 156 135 121 [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. [ ] PM 20 4 0.8 Ct. Panobinostat Belinostat Tacrolimus Methotrexate Doxorubicin C 125 Bortezomib 125 Bortezomib 125 Vorinostat Relative cell growth Relative cell growth Relative cell growth Panobinostat Panobinostat Lenalidomide 100 Romidepsin 100 100 Romidepsin Temsirolimus Belinostat Belinostat Ruxolitiniub 75 Sorafenib 75 75 Sorafenib Bortezomib Bexarotene Temsirolimus 50 50 50 25 25 25 Patient #23 Patient #24 Patient #25 0 0 0 –7 –6 –5 –4 –3 –2 –1 –7 –6 –5 –4 –3 –2 –1 –7 –6 –5 –4 –3 –2 –1 Log [ ] PM Log [ ] PM Log [ ] PM 125 Bortezomib 125 Dasatinib 125 Dasatinib Relative cell growth Relative cell growth Relative cell growth Panobinostat Panobinostat Panobinostat 100 Romidepsin 100 Bortezomib 100 Bortezomib Belinostat 75 Sorafenib 75 75 Temsirolimus 50 50 50 25 25 25 Patient #26 Patient #30 Patient #31 0 0 0 –7 –6 –5 –4 –3 –2 –1 –7 –6 –5 –4 –3 –2 –1 –7 –6 –5 –4 –3 –2 –1 Log [ ] PM Log [ ] PM Log [ ] PM Figure 2. Variable drug sensitivity in leukemic cells from individual patients with SS. (A) Schematic representation of the protocol used for the in vitro drug screening. (B) Representative results obtained from patient #30. (C) Graphs representing the dose–response analysis of different SS patients treated as indicated. (D) Bar plots showing the high variation in the response of individual patients to specific compounds. (E) FC analysis of cells from patient #30 treated as indicated. 14 JUNE 2022 • VOLUME 6, NUMBER 11 NEW SS MODEL FOR PATHWAY-BASED MEDICINE 3413 D E CD26– CD7+ CD26+ CD7+ 105 104 104 11.1 7.19 Vehicle (control) FITC-A hCD7 FITC-A hCD7 PE-A hCD26 104 103 103 1e–03 103 CD26– CD7– 83.8 102 82.9 102 0 0 0 CD26– CD7– CD26+ CD7– –102 –102 75.4 6.29 –103 Patient 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K –10 3 0 10 3 104 105 1e–05 #23 FSC-A FSC-A PE-A hCD26 #24 #25 10 5 4 4 CD26– CD7+ CD26+ CD7+ 10 10 #26 Vorinostat [1PM] 14.2 36.7 #30 FITC-A hCD7 FITC-A hCD7 PE-A hCD26 104 103 103 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 1e–07 #31 103 CD26– CD7– 43.2 102 49.7 102 0 0 0 CD26– CD7– CD26+ CD7– –102 –102 29.6 19.5 –103 0 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K –10 3 0 103 104 105 ib b So sin sir nib us FSC-A FSC-A PE-A hCD26 rte at m at no tini m Bo ost Ro ost im ep Te rafe zo Pa sa ol id lin n a bi D Be 5 CD26– CD7+ CD26+ CD7+ m 4 4 10 10 10 Vorinostat [10PM] 9.76 80.5 FITC-A hCD7 FITC-A hCD7 PE-A hCD26 4 10 103 103 CD26– 103 12.2 CD7– 102 12.7 102 0 0 0 CD26– CD7– CD26+ CD7– –102 –102 2.44 7.32 –103 3 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K –10 0 103 104 105 FSC-A FSC-A PE-A hCD26 Figure 2. (continued) euthanasia, either at the end point of the study or when mice that were selected based on their target selectivity and clinical appli- showed symptoms of suffering, we analyzed tumor burden in PB by cability (supplemental Table 2). We detected a high heterogeneous FC, and we also measured visceral infiltration by hematoxylin and response of individual primary SS samples against the different eosin and immunohistochemical (IHC) analysis of paraffin- compounds tested (Figure 2B-C; supplemental Table 3). Likewise, embedded tissue sections. single compounds produced very distinctive effects on the various patient samples, with the proteasome inhibitor bortezomib, bardoxo- Histopathologic examination lone methyl, and the HDAC inhibitor panobinostat showing the high- Paraffin blocks of all tumor tissues in mice were fixed in 10% forma- est activity in most patient-derived samples (supplemental Figure lin solution, processed by standard methods, embedded in paraffin, 1A; supplemental Table 3). sectioned at 2 mm, and stained with hematoxylin and eosin and with We next performed a more accurate dose–response assay for com- an IHC lymphoid panel. pounds that produced a minimum of a 50% decrease in cell viability (on specific SS samples) at concentrations of 1 mM (Figure 2C-D). Results Our results confirmed the high heterogeneous response of individ- ual patient-derived SS cells against specific compounds, with some SS patients and sample characterization patients displaying high sensitivity against most drugs tested (ie, We collected clinical samples from 8 patients with SS, newly diag- patient #24), whereas others were mostly insensitive to all treat- nosed and under first-line treatments. Se zary cell count in PB ments (ie, patient #26). Importantly, FC analysis of patient ranged from 5.4% to 59% of total leukocytes, and up to 95% of #25–derived SS cells with the HDAC inhibitor vorinostat confirmed the T lymphocytes (supplemental Table 1) measured by FC analysis selective killing of the malignant CD31CD26– population after 72 (Figure 1). Of the 8 patients with SS analyzed, only 1 displayed hours of treatment at 1 mM and 10 mM (Figure 2E). In contrast, peripheral lymph node involvement (stage IVA2) as determined by treatment with bortezomib imposed a nonspecific removal of the histopathologic examination of the biopsy specimens. whole CD3 population (supplemental Figure 1B). Primary Se  zary cells display a highly heterogeneous Generation of a robust model for primary SS PDX sensitivity to specific pharmacologic inhibition in mice We first performed a single-agent titration (at 20, 4, and 0.8 mM) in To establish an in vivo model for studying SS, we injected various primary patient-derived SS samples (schematic representation of numbers of fresh mononuclear cells (0.5, 1, and 2 3 106 cells) iso- the assay in Figure 2A) using 26 different therapeutic compounds lated from patient samples during apheresis procedures into the ear 3414 GALLARDO et al 14 JUNE 2022 • VOLUME 6, NUMBER 11 A Sample preparation Intra-ear inoculation B 8 weeks post-injection 2×105 1×106 2×106 hCD4— hCD45+ hCD4+ hCD45+ hCD4— hCD45+ hCD4+ hCD45+ hCD4— hCD45+ hCD4+ hCD45+ 0.070 0.00 0.092 0.037 0.61 4.85 APC-Cy7-A hCD45 APC-Cy7-A hCD45 APC-Cy7-A hCD45 103 103 103 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 102 102 102 hCD4— hCD45— hCD4— hCD45— hCD4+ hCD45— hCD4— hCD45— hCD4+ hCD45— 101 99.9 101 99.9 0.016 101 94.5 0.016 0 103 104 105 0 103 104 105 0 103 104 105 PE-A hCD4 PE-A hCD4 PE-A hCD4 C PerCP-Cy5-5-A hCD3 APC-Cy7-A hCD45 103 CD45+ 103 103 PE-Cy7-A hCD4 8.83 CD3+ 95.8 CD4+ 102 102 102 94.1 101 101 101 0 0 0 0 200 400 600 800 1.0K 0 200 400 600 800 1.0K 0 200 400 600 800 1.0K FSC-A FSC-A FSC-A CD26—CD7+ CD26+CD7+ 4.69 6.25 103 103 103 FITC-A hCD7 FITC-A hCD7 PE-A hCD26 102 102 102 CD26— CD7— CD26—CD7— CD26+CD7— 101 10.9 101 89.1 101 6.25 82.8 0 0 0 0 200 400 600 800 1.0K 0 200 400 600 800 1.0K 100 101 102 103 FSC-A FSC-A PE-A hCD26 D PerCP-Cy5-5-A hCD3 APC-Cy7-A hCD45 103 CD45+ 103 103 PE-Cy7-A hCD4 5.87 CD3+ 99.6 CD4+ 102 102 102 62.7 101 101 101 0 0 0 0 200 400 600 800 1.0K 0 200 400 600 800 1.0K 0 200 400 600 800 1.0K FSC-A FSC-A FSC-A CD26—CD7+ CD26+CD7+ 1.63 64.1 103 103 103 FITC-A hCD7 FITC-A hCD7 PE-A hCD26 102 102 102 CD26— CD7— CD26—CD7— CD26+CD7— 101 2.29 101 34.0 101 0.98 33.3 0 0 0 0 200 400 600 800 1.0K 0 200 400 600 800 1.0K 100 101 102 103 FSC-A FSC-A PE-A hCD26 Figure 3. 14 JUNE 2022 • VOLUME 6, NUMBER 11 NEW SS MODEL FOR PATHWAY-BASED MEDICINE 3415 of NSG mice (n 5 5 patients, 2 mice per condition). Schematic rep- response to the drug). We performed a first experiment with 10 resentation of the procedure used is shown in Figure 3A. Human mice (5 in the control group and 5 on treatment) and a second CD451 cells were consistently found in the circulation in the major- experiment with 12 mice (7 controls and 5 treated) (2 3 106 SS ity of recipient mice transplanted with 2 3 106 cells (Figure 3B), cells per mice transplanted). No significant differences were indicative of successful engraftment. In addition, a variable degree detected between the control and treatment groups in terms of sur- of human T lymphocytes was detected in the blood of mice that vival or selective reduction of the pathologic cell population in the exhibited an aberrant phenotype (CD31, CD41, CD7–, and/or successive bleedings (data not shown) at the bortezomib doses CD26–) consistent with Se zary cells (Figure 3C). As a control, reported in the literature.19 These results were in agreement with NSG mice transplanted with mononuclear cells from healthy donors the nonselective activity of bortezomib observed in vitro (supplemen- showed a consistent expansion of human T cells that were mainly tal Figure 1B). CD7 and CD26 double positive (Figure 3D). In a third experiment, we transplanted 12 NSG mice with 2 3 106 SS PDX cells reproduce the main characteristics of SS cells (per mice) from patient #30. Here, the HDAC inhibitor pan- Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 the original patient tumor obinostat was used as the therapeutic agent based on the high sen- sitivity of these cells in vitro (Figure 2C). Four weeks after We next investigated whether this PDX model reproduced the clini- transplantation, the presence of human SS cells was confirmed in copathologic features of the original patient SS tumor. After 4 weeks the NSG mice circulation by FC. Because slight differences in the of transplantation, mice injected with human SS cells rapidly devel- number of SS cells were detected in each recipient mouse, animals oped symptoms of the disease; this included erythematous and scaly were homogeneously ascribed to the different experimental groups skin lesions and alopecia (Figures 4A) associated with the presence (control or panobinostat, 10 mg/kg oral administration for 5 consec- of a robust CD7– T-cell population in PB (supplemental Figure 2A-B) that was positive for the immune checkpoint PD1 (not depicted), as utive days). After a first round of treatment (15 days), animals were reported previously.18 However, we detected no obvious signs of SS left untreated for 15 days before starting a second round of treat- infiltrates in the skin of mice (Figure 4B). From week 4 to week 6, the ment (15 days) to mimic the procedure used in patients (Figure 5A). percentages of CD7– T cells were primarily maintained, but a rapid FC analysis was performed every 2 weeks to monitor the presence expansion of the human CD26– population was detected in most of of human T cells in the PB of mice. A significant increase was the transplanted animals (supplemental Figure 3). Analysis of trans- detected in the number of human CD451CD31 cells after the first planted mice at euthanasia revealed the presence of human CD41 round of treatment in the PB of control mice, which was not cells in bone marrow, PB, spleen, lymph nodes, and liver (Figure 4C). observed in 5 of 6 animals in the panobinostat-treated group IHC analysis of the spleens (Figure 4D) and livers (not depicted) (Figure 5B). The average percentage of CD451CD31 cells in the showed extensive lymphomatous infiltrates that were CD31, CD41, PB of control mice was 22.4 6 20.8 compared with 2.0 6 2.4 in and CD7– or low, further indicating that they correspond to malignant the treated group (P 5 .06). Sezary cells. Unexpectedly, by comparative analysis of the TCR reper- Further indicating the efficacy of panobinostat in vivo, we detected toire in patient blood and PDX samples, we did not detect a predomi- a transient increase in the human CD451CD31 population in both nant clonal amplification of the main patient-derived SS population in groups after concluding the first round of treatment, which was the transplanted mice but a heterogeneous expansion of different pri- again reduced in the panobinostat group after restarting a second mary SS cell clones (Figure 4E). round of treatment. IHC analysis of the livers at euthanasia revealed Further supporting the concordance between patient samples and the presence of a robust CD31, CD41 infiltrate, as previously their corresponding PDX models, several of the mice transplanted found, in the vehicle-treated group that was absent in all but one of with cells from SS patient #23 died as a result of massive B-cell the animals treated with panobinostat (Figure 5C). These findings lymphomatous infiltrates in the spleen and liver (Figure 4F); these indicate that SS PDX retains the drug sensitivity observed in vitro were associated with the presence of an aberrant CD1911, and may represent a suitable preclinical model for human SS, as a CD2011. k11 B-cell population (20% of lymphocytes) in the PB previous step for personalized medicine. of this patient (supplemental Table 1). Finally, we determined the effectiveness and specificity of panobino- These results indicate that the SS PDX model phenocopies the stat treatment by measuring the levels of acetylated histones in original SS patient populations. blood cells obtained at experimental end point. We performed west- ern blot analysis of PB cell lysates with specific antibodies against SS PDX models provide a platform to screen acetylated histones H3 and H4. Increased histone acetylation targeted drug treatments was detected in samples from panobinostat-treated mice, indicative We investigated whether our in vitro drug screening was capable of of effective HDAC inhibition even after long-term treatments predicting the therapeutic response of primary SS cells in the PDX (Figure 5D). In contrast, we did not detect off-target effects on model. Based on in vitro data, we selected bortezomib for in vivo IkBa, the classical readout of NF-kB activity, or p-ERK1/2, the treatment of samples from patient #23 (showing a medium downstream effector of the MAPK pathway. Figure 3 (continued) Robust in vivo xenograft model of patient-derived SS. (A) Schematic representation of the procedure used to generate the SS xenograft. (B-C) FC analysis of PB from mice at 8 weeks after transplantation with different numbers of SS cells from patient #23. (C) FC analysis with the indicated marker to confirm the outgrowth of malignant CD41; CD7– SS cells in the recipient mice. (D) Same analysis as in panel C from the bone marrow (BM) of a mouse transplanted with mononucleated cells from a healthy donor. 3416 GALLARDO et al 14 JUNE 2022 • VOLUME 6, NUMBER 11 A C Bone marrow Lymph nodes Liver 4 hCD4— hCD45+ hCD4+ hCD45+ 4 hCD4— hCD45+ hCD4+ hCD45+ 4 hCD4— hCD45+ hCD4+ hCD45+ 10 0.049 1.92 10 0.16 9.63 10 0.74 86.7 APC-Cy7-A hCD45 103 103 103 hCD4— hCD45— hCD4+ hCD45— hCD4— hCD45— hCD4+ hCD45— hCD4— hCD45— hCD4+ hCD45— 102 94.3 3.70 102 89.6 0.69 102 10.8 1.76 –102 –102 –102 0 103 104 105 0 103 104 105 0 103 104 105 PE-A hCD4 PE-A hCD4 PE-A hCD4 Spleen Peripheral blood B Skin section 104 hCD4— hCD45+ 0.33 hCD4+ hCD45+ 81.4 104 hCD4— hCD45+ 0.18 hCD4+ hCD45+ 62.2 103 103 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 H&E hCD4— hCD45— hCD4+ hCD45— hCD4— hCD45— hCD4+ hCD45— 102 16.5 1.69 102 36.4 1.25 –102 –102 50 PM 0 103 104 105 0 103 104 105 PE-A hCD4 PE-A hCD4 D H&E hCD3 hCD7 hCD4 E 135 165 195 12000 8000 4000 sz 172.03 Patient sample sz 178.00 PDX#2 PDX#3 sz 185.06 PDX#4 F Liver Spleen IHC: hCD4 IHC: hCD3 IHC: hCD4 IHC: hCD3 IHC: hCD5 IHC: hCD20 IHC: hCD5 IHC: hCD20 Figure 4. 14 JUNE 2022 • VOLUME 6, NUMBER 11 NEW SS MODEL FOR PATHWAY-BASED MEDICINE 3417 Taken together, our data indicate that the SS PDX mouse model CTCLs,14 which functionally links NF-kB and b-catenin with CTCL identified an efficacious therapy for patients with SS, strongly sug- initiation and/or progression. Recently, it was shown that transgenic gesting that the SS PDX model is a valid experimental platform that mice with aberrant STAT3 activation in CD4 T cells develop clinical can guide clinical decision-making regarding therapeutic agents. features of MF and SS, being microbiota required for acquisition of the complete clinical phenotype. This mouse model, which develops Discussion progressive disease, provides a new and useful tool for studying the cutaneous biology of T-cell lymphoma.22,23 Similarly, transgenic CTCLs represent heterogeneous non-Hodgkin lymphomas, with SS mice with constitutive overexpression of interleukin-15 develop an being one of the most aggressive subtypes manifested by pruritic infiltration of the skin by mature T cells immunophenotypically mim- erythroderma, lymphadenopathy, and atypical circulating lympho- icking the human disease.24 These models are an alternative tool for cytes (S ezary cells). SS is a devastating disease with a high impact CTCL investigation. on the quality of life of patients. The pathogenic mechanisms are poorly understood, being molecularly complex and heterogeneous. PDX models currently represent a promising tool for translational Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 Consequently, it is essential to obtain molecular-based personalized research, as they closely resemble individual patient tumor features therapies (precision medicine). and retain molecular and histologic characteristics. PDX models have already been used in several solid tumors and validated as a There is enough evidence to state that standard polychemotherapy robust model to evaluate drug response and therapeutic efficacy. regimens are not effective in SS, showing very short-lived responses However, their use in CTCL is still preliminary and scarce.21,25 The and short survivals. Thus, there is an urgent need for new effective European consortium EuroPDX (www.europdx.eu) experience pro- treatments that have a safe profile, are well tolerated, and maintain posed PDX and orthotopic PDX as realistic and promising upcom- long-term activity because standard therapeutic approaches in SS ing technologies to deep into: (1) the mechanisms of resistance to do not lead to a significant improvement in long-term survival. In standard chemotherapy treatments; (2) the development of new recent years, knowledge regarding specific therapeutic targets has allowed the design of immunotherapies (both cytotoxic and immuno- therapeutic strategies; (3) the contribution of tumor microenviron- regulatory) such as brentuximab, mogamulizumab, or check-point ment to cancer progression and its role in the development of resis- inhibitors (PD1/programmed cell death ligand 1 inhibitors), which tance to targeted therapies; and (4) the improvement of models to have modified the disease-free survival curves of CTCL patients in evaluate personalized therapies in real time integrating the tumor the short term. In younger patients, the therapeutic option of alloge- genetics with the response to drugs.26,27 neic hematopoietic stem cell transplantation can be proposed. We have now established a robust PDX model for SS and showed Integrated genomic data sets identified key elements and pathways its usefulness in predicting therapeutic response of tumor cells. that could be used to stratify patients with SS who would potentially Unexpectedly, we found that the main clone present in the patient benefit from future targeted therapies. Therefore, given the genetic was not predominant in the recipient mice, but several clones and molecular heterogeneity of CTCL and particularly SS, it is already present in the patient were randomly selected. Almost identi- essential to develop realistic and practical in vitro and in vivo models cal subclonal heterogeneity of the SS PDX system has been to test personalized targeted therapies in individual cases. recently described by Poglio et al.12 Subclonal selection is likely associated with immunophenotypic plasticity and limited genomic Many of the in vivo models use CTCL cell lines that do not repre- evolution in response to environmental conditions. Unfortunately, we sent an appropriate tool for personalized medicine. Among cell were not able to reproducibly expand human SS cells in secondary line–derived xenograft models, one the most notable was generated NSG recipients, suggesting that a low number of tumor-initiating by injecting CTCL cells into mice in the context of an inflammatory cells recovered from the primary transplantation. environment, a scenario similar to the real pathologic situation.20,21 Also, intrahepatic injection of CTCL cell lines in NOD/SCID/IL2Rg The present work compiles current methods for an SS PDX genera- mice leads to successful engraftment and has been used to evalu- tion and its utility, providing new perspectives for the rapid transla- ate cell line tumorigenicity and therapeutic responses in preclinical tion of results to clinical practice. Our ongoing work is focused on studies.13 Using in vitro and in vivo models of human SS cell lines, studying the likely correlation between drug responses and the we previously showed that canonical NF-kB is constitutively acti- mutational landscape of individual SS patients to uncover bio- vated in CTCL cells, downstream of the TAK1 kinase. Inhibition of markers that could eventually inform future personalized therapies. TAK1 prevented signaling through NF-kB and b-catenin, thus induc- This is particularly important in highly heterogeneous diseases such ing apoptosis of CTCL cell lines and primary SS cells both in vitro as SS in which specific therapies will fail in the absence of patient and in vivo. In addition, the presence of activated TAK1, as deter- selection criteria. For example, we have now reported the preclinical mined by specific antibodies, correlated with activation of NF-kB efficacy of panobinostat on patient-derived SS cells previously char- and b-catenin, being indicative of CTCL, compared with different T acterized as sensitive in vitro; however, the same compound lymphocyte–mediated inflammatory dermatoses. Thus, activated showed a modest but demonstrated efficacy in a phase 2 clinical TAK1 was identified as a novel biomarker and therapeutic target for trial but still higher in SS than in MF.28 In addition, we found that Figure 4 (continued) Transplanted SS cells reproduce the human disease in mice. (A) Representative photograph of a mouse at 8 weeks after being transplanted with 2 million SS cells. (B) Hematoxylin and eosin (H&E) staining of a skin section of the same animal obtained at euthanasia. (C) FC analysis of the different organoids indicated the presence of SS infiltrates. (D) IHC analysis of liver sections to confirm FC data. (E) Clonality analysis by PCR analysis of SS from patient #23 and blood from 2 different mice transplanted with SS cells from the same patient. (F) IHC of the liver and spleen of a representative mouse transplanted with cells from patient #23. Note the high amounts of aberrant CD201 B cells, already present in the patient, that infiltrate the organs in addition to the CD31;CD41;CD51 SS population. 3418 GALLARDO et al 14 JUNE 2022 • VOLUME 6, NUMBER 11 A Xenograft confirmation hCD3 Analysis hCD4 Panobinostat 10mg/Kg Panobinostat 10mg/Kg (daily I.P. injection) (daily I.P. injection) 8—10 weeks-old mice Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 First cycle Rest Second cycle 4 weeks 2 weeks 1 week 2 weeks End point B D 85 Vehicle Panobinostat 75 % Human CD45+/CD3+ cells #1 #2 #3 #1 #2 #3 65 WB: 15 kDa 55 Acetyl H4 45 35 Acetyl H3 15 kDa 25 15 kDa 15 Histone H4 5 0 Chromatin extracts First round Break Second round Panobinostat 37 kDa Vehicle p-INBD INBD 37 kDa C Vehicle Panobinostat 50 kDa `p-ERK1/2 IHC:hCD3 50 kDa ERK1/2 Tubulin 50 kDa 100 Pm 100 Pm Soluble extracts IHC:hCD4 100 Pm 100 Pm Liver infiltrates Figure 5. In vivo treatment reproduces the SS drug sensitivity determined in vitro. (A) Schematic representation of the treatment protocol in mice. (B) Representation of disease progression in the different groups of treatment as determined by FC analysis of PB. (C) Representative IHC analysis of the liver showing the presence of malignant SS cells specifically in vehicle-treated animals. (D) Western blot analysis of the indicated proteins in blood samples from 3 different vehicle-treated or panobinostat-treated mice. Note the increased levels of acetylated histone in the panobinostat-treated blood samples consistent with the HDAC inhibitory activity of this drug. 14 JUNE 2022 • VOLUME 6, NUMBER 11 NEW SS MODEL FOR PATHWAY-BASED MEDICINE 3419 SS cells from patient #23 exhibited the highest response to inhibi- Authorship tors of the NF-kB pathway such as bardoxolone methyl and bortezo- Contribution: F.G., R.M.P., A.B., and L.E. designed the study, ana- mib in the in vitro assays (supplemental Table 3). Although we did lyzed data, and wrote the manuscript; E.A., A.I., J.G., and L.S. per- not perform any in vivo experiment with these cells, patient #23 is formed experiments and analyzed data; Y.G., G.B., L.C., E.G., D.C., now on mogamulizumab treatment, a blocking antibody against E.R., and I.B.-M. analyzed data; and M.I., B.B., and J.R.R. performed CCR4 that was found to induce NF-kB and PI3K pathways, and zary cell the phenotypic characterization of SS cells. has achieved complete remission of both the circulating Se population and the aberrant B-cell population. Conflict-of-interest disclosure: The authors declare no competing In case of treatments that are indicated for other tumor types, the financial interests. possibility of recommending their use for treatments in selected ORCID profiles: A.I., 0000-0002-7195-7868; L.S., 0000-0002- patients could be considered. Our goal is to develop a realistic and 3195-0565; Y.G., 0000-0001-8673-6470; G.B., 0000-0001-6557- practical preclinical strategy to identify new biomarkers of prognos- 3764; E.G., 0000-0002-3534-6189; D.C., 0000-0002-9414-9409; Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3410/1900456/advancesadv2021006860.pdf by guest on 13 April 2023 tic and pathway-based therapeutic strategies. B.B., 0000-0002-5335-2726; J.R.R., 0000-0001-8442-7762; A.B., 0000-0003-4801-6899; L.E., 0000-0002-2897-4099. Acknowledgments Correspondence: Fernando Gallardo, Dermatology Department, The authors thank all members of the Gallardo, Espinosa, and Hospital del Mar, Passeig Marıtim 25-29, 08003 Barcelona, Spain; Bigas laboratories for their support and helpful comments and e-mail: fgallardo@parcdeslautmar.cat; Anna Bigas, Cancer and suggestions. They also thank Patricia Charoenrook for helping Stem Cells Research Group, Institut Hospital del Mar with English editing. d'Investigacions Mediques, CIBERONC, Dr Aiguader 88, 08003 This work has been supported by grants PI19/00013, PI18/ Barcelona, Spain; e-mail: abigas@imim.es; and Lluıs Espinosa, 00021, and PI21/0390 from Instituto de Salud Carlos III/FEDER Cancer and Stem Cells Research Group, Institut Hospital del Mar and grant 2017-SGR 135 from AGAUR from the Government of d'Investigacions Mediques, CIBERONC, Dr Aiguader 88, 08003 Catalonia and CIBERONC. Barcelona, Spain; e-mail: lespinosa@imim.es. References 1. da Silva Almeida AC, Abate F, Khiabanian H, et al. The mutational landscape of cutaneous T cell lymphoma and S ezary syndrome. Nat Genet. 2015;47(12):1465-1470. 2. Prasad A, Rabionet R, Espinet B, et al. Identification of gene mutations and fusion genes in patients with S ezary syndrome. J Invest Dermatol. 2016;136(7):1490-1499. 3. 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