¹Hematology, School of Clinical Sciences at Monash Health, Monash University, Clayton, Australia; ²Clinical, Peninsula Health, Frankston, Australia; ³Medicine, The University of Arizona Cancer Center, Tucson, United States; ⁴Hematology/Oncology, Henry Ford Hospital, Detroit, United States; ⁵Oncology, Wisconsin Institute of Medical Research, Madison, United States; ⁶Oncology, Roswell Park Comprehensive Cancer Center, Buffalo, United States; ⁷Hematology/Oncology, Tufts Medical Center, Boston, United States; ⁸Oncology, Penn State Hershey Cancer Institute, Hershey, United States; ⁹Oncology, Johns Hopkins Sidney Kimmel Cancer Center, Baltimore, United States; ¹⁰Oncology, Baylor University Medical Center, Dallas, United States; ¹¹Hematology, Swedish Cancer Institute, Seattle, United States; ¹²Hematology, Keck School of Medicine of USC, Los Angeles, United States; ¹³Biostatistics, Merck & Co., Inc., Kenilworth, NJ, United States; ¹⁴Clinical Research, Merck & Co., Inc., Kenilworth, United States; ¹⁵Hematology, University of Melbourne, St. Vincent's Hospital, Fitzroy, Australia

**Introduction:** KEYNOTE-155 (NCT02684617) evaluated the anti-tumor activity of pembrolizumab (pembro) plus dinaciclib in patients (pts) with relapsed/refractory (rr) CLL, MM, and DLBCL. Here we present data from an interim analysis of safety and efficacy in pts with rrDLBCL.

**Methods:** In this phase 1b, two-part, non-randomized, open-label study, pts with rrDLBCL who received ≥2 prior therapies were enrolled into a dose-evaluation phase (two 21-day [d] cycles of pembro 200 mg [d1] and dinaciclib 7 mg/m² [d1], 10 mg/m² [d8] in cycle 1 and 14 mg/m² [d1, d8] from cycle 2 onwards). The DLBCL expansion cohort was opened for the signal-detection phase (≥30 patients) if ≤4 pts had dose-limiting toxicities (DLTs) during dose-evaluation. Treatment continued up to 35 cycles or until progression (PD) or unacceptable toxicity. Response was assessed every 3 cycles for DLBCL (Revised Response Criteria for Malignant Lymphoma 2007). Pts who discontinued before PD were followed Q12W until PD, new anticancer treatment, or death. The primary endpoint was safety and tolerability. Secondary endpoints included ORR by investigator, DOR, PFS, and OS. Efficacy and safety were assessed in all pts who received ≥1 dose of pembrolizumab or dinaciclib.

**Results:** As of Apr 25, 2018, 38 pts with rrDLBCL were enrolled and treated. Median age was 64.5 years (range, 39-85), 22 (58%) pts had ECOG PS 1, and 22 (58%) had ≥3 prior therapies. After median follow-up of 2.79 mo (range, 1.5-6.8), 36 (95%) pts discontinued treatment: 21 (55%) PD, 12 (32%) by physician decision (10 [26%] clinical progression, 1[3%] logistics, 1 [3%] new therapy), 2 (5%) pt decision, and 1 (3%) AE. Median time on treatment was 51d (range, 1-471). Among 12 pts in all disease cohorts enrolled in the dose-evaluation phase, DLTs occurred in 1 of 6 (17%) evaluable pts (grade 4 thrombocytopenia) with rrDLBCL. Of 38 pts with rrDLBCL, 24 (63%) had a treatment-related AE. Common treatment-related AEs were decreased platelets (21%), lymphopenia (16%), nausea (13%), anemia (13%), diarrhea (13%), and pyrexia (13%). 12 (32%) pts had a grade 3-4 treatment-related AE. Common grade 3-4 treatment-related AEs were

lymphopenia (13%), decreased neutrophils (11%), decreased platelets (8%), leukopenia (8%), and laboratory TLS (5%). There was no clinical TLS and no treatment-related deaths. ORR was 18% (95% CI, 8-34) (3 CR and 4 PR) and median DOR was 4.9 mo (range, 2.1-13.8); 2 pts were on treatment at data cutoff. With 36 (95%) events, median PFS was 2.1 mo (95% CI, 2-3) with 6-mo and 12-mo PFS of 13% and 8%, respectively. 19 (50%) pts died, with median OS of 9.8 mo (95% CI, 3 to not reached), and 12-mo OS of 43%.

**Conclusion:** Despite limited overall response, the observed CRs and durability (up to 13 mo) provide proof of concept for activity of this combination in rrDLBCL and rationale for further studies to define optimal dosing of dinaciclib, given the brevity of CDK9 inhibition observed in preclinical models.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); PD-1L; Pembrolizumab.

**Disclosures:** Gregory, G: Consultant Advisory Role: Advisory board member at Gilead; Honoraria: Roche; Research Funding: Amgen; Other Remuneration: Travel expenses from Roche. Walker, P: Research Funding: Merck & Co., Inc. Chang, J: Research Funding: Genentech, Celgene. Hernandez-Ilizaliturri, F: Consultant Advisory Role: Advisory board member for Celgene, Novartis, Amgen, Pharmacyclics, Pfizer, and Seattle Genetics. Klein, A: Consultant Advisory Role: Consultant for Shire; Honoraria: Takeda; Other Remuneration: Travel expenses from Takeda. Rybka, W: Consultant Advisory Role: Consultant for Merck & Co., Inc.; Honoraria: Merck & Co., Inc.; Research Funding: Merck & Co., Inc.; Other Remuneration: Travel expenses from Merck & Co., Inc. Wagner-Johnston, N: Consultant Advisory Role: Advisory board: JUNO, ADC therapeutics, Janssen; Research Funding: Merck & Co., Inc., Novartis, Celgene, ASTEX. Escobar, C: Other Remuneration: Speakers' bureau: Kite; Gilead. Pagel, J: Consultant Advisory Role: Pharmacyclics; Gilead. Mohrbacher, A: Other Remuneration: Speakers' bureau: Genentech; Takeda. Opat, S: Consultant Advisory Role: Roche, Janssen, Celgene, Takeda, Abbvie, Gilead, BMS, Merck, Sanofi; Honoraria: Roche, Janssen, Celgene, Takeda, Abbvie, Gilead, Mundipharma; Research Funding: Roche, Janssen, Celgene, Takeda, Novartis, Abbvie, Gilead, Amgen; Other Remuneration: Trial Support: Roche, Janssen, Celgene, Takeda, Abbvie, Beigene, Merck, Epizyme. Ma, H: Employment Leadership Position: Merck & Co., Inc.; Stock Ownership: Merck & Co., Inc. Gwo, J: Employment Leadership Position: Merck & Co., Inc.; Stock Ownership: Merck & Co., Inc. Farooqui, M: Employment Leadership Position: Merck & Co., Inc.; Stock Ownership: Merck & Co., Inc.
P.F. Caimi¹ | D. Jagadeesh² | K. Boughan¹ |
R. Dean² | B. Cooper¹ | B. Pohlman² |
M. Gallogly¹ | S. Ondrejka¹ | E. Moore¹ |
K. Oduro¹ | E.D. Hsi³ | M. de Lima¹ | B.T. Hill²

¹Medicine - Hematology and Oncology, University Hospitals Seidman Cancer Center, Cleveland, United States; ²Hematology and Oncology, Cleveland Clinic, Cleveland, United States; ³Pathology, Cleveland Clinic, Cleveland, United States

**Introduction:** Diffuse large B cell lymphoma (DLBCL) patients (pts) with refractory or relapsed (r/r) disease after front line chemo-immunotherapy have poor survival. Overall response rates (ORR) to second line, platinum - containing regimens is approximately 50%, and complete response (CR) rates are 30 - 40%. Only 40% of patients proceed to an autologous stem cell transplant (ASCT). We conducted a phase I trial evaluating the combination of venetoclax (VEN) with RICE (rituximab, ifosfamide, carboplatin and etoposide) (VICER). We have reported the adverse event profile of this combination, with common but manageable hematologic toxicity and rare laboratory tumor lysis. The recommended phase 2 dose of VEN is 800mg/d. We present the final results of this phase I trial and outcomes of patients proceeding to ASCT.

**Methods:** Patients (≥18 years of age) with r/r DLBCL who failed one or two lines of therapy were enrolled. VEN was given orally on days 1 - 10 of each 21 - day cycle x 3 cycles. Dose escalation was conducted according to a 3+3 design, with 3 dose levels (400, 600 and 800mg). RICE was given at standard dose and schedule on days 1 -

<table><caption>TABLE 1 Baseline characteristics</caption><thead><tr><th>Patient Characteristic</th><th></th></tr></thead><tbody><tr><td>Gender, n (%)</td><td></td></tr><tr><td>Female</td><td>4 (22)</td></tr><tr><td>Male</td><td>14 (78)</td></tr><tr><td>Age, years, median (range)</td><td>55.5 (27 - 78)</td></tr><tr><td>Disease refractory to prior chemotherapy, n (%)</td><td>6 (46)</td></tr><tr><td>Bulky disease at enrollment</td><td>6 (33)</td></tr><tr><td>Involvement of > 1 extranodal site</td><td>2 (15)</td></tr><tr><td>Secondary IPI</td><td></td></tr><tr><td>Low (0 - 1)</td><td>12 (67)</td></tr><tr><td>High (≥ 2)</td><td>6 (33)</td></tr><tr><td>Cell of origin, n (%)</td><td></td></tr><tr><td>GCB</td><td>10 (42)</td></tr><tr><td>Non - GCB</td><td>8 (58)</td></tr><tr><td>Molecular abnormalities, n (%)</td><td></td></tr><tr><td>Double hit lymphoma</td><td>4 (22)</td></tr><tr><td>Double expressor lymphoma*</td><td>4 (22)</td></tr></tbody></table>

Abbreviations: GCB, germinal center B cell; IPI, International Prognostic Index.
*Does not include double hit subjects (all had positive IHC for MYC and BCL2)

3. All pts received tumor lysis prevention during cycle 1 and pegfilgrastim after every cycle.

**Results:** 18 pts enrolled and completed follow up (table 1). All pts received rituximab and anthracycline containing first line therapy, 4 patients had failed a second line of therapy and 6 had refractory disease (table 1). Fourteen pts (78%) achieved CR and 2 (11%) achieved a partial response (ORR = 89%). Two pts did not respond.

Successful stem cell collection was done in 14/16 responding pts. The median CD34 cell dose collected was 3.71 CD34+ cells/kg. Both pts with mobilization failure were older than 70 years and declined a second collection. All 14 subjects undergoing ASCT had hematopoietic engraftment. After a median follow up of 12 months, 5 pts have relapsed, including one subject who did not undergo ASCT. 1 - year PFS for intention to treat (ITT) cohort is 55%. 4 patients have died, including 2 pts without response to VICER and 2 patients relapsed after ASCT. One - year OS for the ITT cohort is 71%. Among patients undergoing ASCT, 1 - year PFS and OS were 62% and 77%, respectively.

**Conclusions:** This phase I trial of VICER has shown the combination is safe and active in r/r DLBCL, including high risk pts, achieving CR in 78%, with a high transplant rate (78%). The observed PFS and OS appear superior to those of comparable pts treated with R-ICE alone and those reported with other second line regimens. A phase II study of VICER will open to enrollment in mid-2019.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); venetoclax.

278

FEASIBILITY AND BENEFIT OF MOLECULARLY-INFORMED ENROLLMENT INTO EARLY PHASE CLINICAL TRIALS FOR PATIENTS WITH RELAPSED OR REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA

J. Michot¹ | V. Camara-Clayette¹ | A. Detolle¹ |
C. Chahine¹ | J. Lazarovici¹ | A. Danu¹ | J. Bosq¹ |
D. Ghez¹ | P. Romano-Martin¹ | P. Dartigues¹ |
J. Arfi-Rouche¹ | R. Bahleda¹ | W. Rahali¹ |
A. Varga² | C. Baldini² | L. Tselikas² | C. Paume² |
H. Lecourt² | S. Cotteret¹ | V. Vergé¹ | J. Soria² |
C. Massard² | V. Ribrag¹

¹Hematology, Gustave Roussy, Villejuif, France; ²Drug Development Department, Gustave Roussy, Villejuif, France

**Background:** Patients with relapsed or refractory diffuse large B cell lymphoma (r/r DLBCL) often do poorly with conventional salvage chemotherapy regimens. Next-generation sequencing (NGS) panels can identify recurrent molecular abnormalities, which can help to orient patient's treatment (pts) for potential molecularly oriented therapies or appropriated clinical trials with investigational targeted therapies. We aimed to evaluate whether orientating pts with r/r DLBCL through tumor genotyping is associated with a better outcome.
**Methods:** From 2013 to 2018, all pts with r/r DLBCL having molecular tumor portrait before enrollment in early clinical trials (eaCTs) for r/r DLBCL were analyzed for clinical and histomolecular characteristics, tumor response, progression-free survival (PFS) duration of response and overall survival. The main objective was to evaluate the feasibility and potential benefit of using tumor genotyping for orientating enrollment in eaCTs. Molecular Screening methods included immunohistochemistry and next-generation sequencing. All pts gave their written consent for the study.

**Results:** Sixty-two pts with r/r DLBCL had tumor molecular portraits. At the time of tumor molecular portrait, the median age was 69 years (range 26-77), median of previous line of therapies was 2 (range 1-9) and 14 pts (23%) had prior auto stem-cell transplant. Fifteen out of the 62 pts (24%) were molecularly oriented (MO) in eaCTs. Identification of potentially actionable targets was found in 30/62 (48%) of pts, of whom 15/30 (50%) received a molecularly-informed therapy. Beyond molecular portrait, fifty pts were enrolled in eaCTs (15 pts were MO and 35 were non-MO oriented) and 12 pts were not enrolled in eaCTs. The MO-oriented group of pts included the following therapeutic targets: CD79A/B or MYD88 (n = 10 pts), EZH2 or ARID1A (n = 3 pts), MYC (n = 1 pt) and BRAF (n = 1 pt). The overall responses rate was 60% (6 PR and 3 CR) in MO group versus 20% (5 PR and 2 CR) in non-MO group (p = 0.01). The median of PFS in MO and non-MO groups were 2.2 months and 1.9 months, respectively (p = 0.23; HR = 0.69 [CI95:0.38-1.26]). The median duration of response in MO and non-MO groups were 10.9 and 9.3 months, respectively (p = 0.78; HR = 0.76 [CI95: 0.26-2.18]). The mean PF2/PF1 ratio in MO and non-MO groups were 2.34 [CI95: 0.27-4.41] and 1.67 [CI:0.53-2.81], respectively (p = 0.093; HR = 0.61 [CI95: 0.33-1.14]). The median overall survival in MO and non-MO groups were 8.9 and 7.7 months, respectively (p = 0.34; HR = 0.69 [CI95: 0.33-1.47]).

**Conclusions:** Molecularly oriented treatments of recurrent diffuse large B-cell lymphoma were associated with higher responses rates. A subset of patients with recurrent or refractory diffuse large B-cell lymphoma may benefit from incorporation of tumor genotyping to guide their enrollment in clinical trials. Accelerating the use of prospective genomics tumor molecular portraits may increase the chances for a precision medicine for recurrent diffuse large B-cell lymphoma.

**Keywords:** epigenetics; EZH2; high-grade B-cell lymphoma with or without rearrangement of MYC and BCL2 and/or BCL6.

279
SAFETY AND EARLY DATA FROM A PHASE
II TRIAL OF PEMBROLIZUMAB (PEM) AND
ENTINOSTAT (ENT) IN RELAPSED AND
REFRACTORY (R/R) HODGKIN LYMPHOMA
(HL) AND FOLLICULAR LYMPHOMA (FL)

D. Sermer¹ | S. Vardhana¹ | C. Batlevi¹ |

A. Hamilton¹ | A. Moskowitz¹ | P. Caron¹ |

C. Moskowitz¹ | M. Matasar¹ | E. Biggar¹ |

E. Leung¹ | J. Yahalom² | J. Rademaker³ |

A. Dogan⁴ | V. Seshan⁵ | A. Younes¹

¹Medicine, Memorial Sloan-Kettering Cancer Center, New York, United States; ²Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, United States; ³Radiology, Memorial Sloan-Kettering Cancer Center, New York, United States; ⁴Pathology, Memorial Sloan-Kettering Cancer Center, New York, United States; ⁵Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, United States

**Introduction:** Histone deacetylase (HDAC) inhibitors have single agent activity in HL and FL, and may enhance antigen-specific immune recognition in cancer in addition to modulating programmed cell death (PD)-1 expression. In preclinical studies, the combination of HDAC inhibitors and anti-PD-1 antibodies acts synergistically against various tumor models in mice. Accordingly, we investigated the safety and efficacy of the novel combination of the HDAC inhibitor ENT and the PD-1-blocking antibody PEM in patients with R/R FL or HL.

**Methods:** Patients with R/R HL or FL were eligible for this trial. Prior use of anti-PD-1 or HDAC inhibitor was allowed if there had been clinical benefit and this was not the most recent therapy. Patients received ENT 5-7 mg orally once weekly and PEM 200 mg intravenously once every three weeks. Tumor assessment was evaluated using the RECIL criteria. The primary objective is overall response rate (ORR) and 12-month progression-free survival (PFS).

**Results:** At data cutoff on 2/7/19, 12 patients (5 HL, 7 FL) have been enrolled. Median age was 60 (26-81). Median number of prior therapies was 3 (2-11) and 3 (2-4) in HL and FL, respectively.

With median duration of follow-up of 145 (33-288) days, 4 patients are currently receiving treatment on study, 3 patients have discontinued treatment due to toxicity, 3 due to disease progression, and 2 to proceed to consolidation with transplant or radiation. Out of

<figure><img src="image_171.png" /></figure>

Waterfall plot depicting best responses (% change) in 11 evaluable patients by RECIL criteria. Tumor size assessment was not available for the two patients with PD. The patient with the greatest tumor reduction in size did not have complete resolution of metabolic activity and thus was classified as a PR.
PD – Progressive disease, SD – Stable disease, MR – Minor response, PR – Partial response, CR – Complete response, Purple bars– FL, Blue bars – HL
11 evaluable patients, there was a 64% ORR across both disease types (100% and 33% ORR in the HL and FL groups, respectively). There was one complete response (CR) in each group, including one patient with FL who had relapsed after CAR T cell therapy. A patient with HL who had previously received both PEM and ENT as monotherapy achieved a partial response (PR). Median duration of response in both groups was 172 (69-209) days.

All patients experienced at least one adverse event (AE). 8/12 (67%) had grade 3 or higher adverse events (AE), which were mainly hematologic compared to non-hematologic (58% vs 8%), including neutropenia (50%), thrombocytopenia (25%), and anemia (17%). Two patients who experienced serious adverse events (SAEs) due to pericarditis and the hemophagocytic lymphohistiocytosis (HLH) syndrome and one patient with grade 3 bullous dermatitis were taken off study. ENT was dose-reduced in 5 patients and was held temporarily without dose-reduction in 4 patients. The median duration on treatment was 112 (27-288) days.

Results from previously performed targeted next-generation sequencing of lymph node biopsies were available for 8 patients. All (100%) had at least one mutation in epigenetic-modulating genes and 5/8 (63%) had at least one mutation in histone acetyltransferase-encoding genes. There was no association between mutation status and response rate.

**Conclusions:** Early results from this ongoing phase II clinical trial suggest that the combination of PEM and ENT is safe with encouraging responses in the study population, especially in those with HL.

**Keywords:** epigenetics; Hodgkin lymphoma (HL); PD-1.

**Disclosures:** Younes, A: Honoraria: Merck.

## 280
SAFETY AND EFFICACY OF ATEZOLIZUMAB, OBINUTUZUMAB AND VENETOCLAX COMBINATION FOR RELAPSED/REFRACTORY NON-HODGKIN LYMPHOMAS: RESULTS FROM THE SAFETY-RUN OF A LYSA STUDY

C. Herbaux<fnref n="1" /> | P. Feugier² | H. Tilly³ | G. Salles<fnref n="4" /> |

R. Gressin⁵ | S. Le Gouill⁶ | F. Morschhauser<fnref n="1" /> |

G. Cartron⁷

**Introduction:** Despite major advances introduced by immunochemotherapy, relapse and refractory B-lymphomas (R/R NHLs) treatment remains challenging. Atezolizumab and obinutuzumab are monoclonal antibodies acting respectively to inhibit T-lymphocyte exhaustion or by inducing lymphoma cells cytotoxicity,

whereas venetoclax is a small molecule inhibiting BCL-2. Combining tumor-targeted therapies with agents that enhance anti-tumor immunity represents an attractive treatment paradigm. The LYSA conducts a multicenter phase II trial (NCT03276468) evaluating the efficacy and safety of the chemotherapy-free combination of atezolizumab, obinutuzumab and venetoclax in R/R NHLs. We present safety and early response data of the patients included in the safety-run.

**Methods:** Patients ≥18 years with biopsy-confirmed R/R NHL (DLBCL or FL) who failed at least one line of therapy (rituximab and anthracycline containing regimen) were eligible. An initial safety-run was performed to assess this novel therapeutic combination. Obinutuzumab was given IV at the dose of 1000 g on day (D) 1, 8 and 15 of cycle (C) 1 and on D1 from C2 to C8 every 3 weeks. Atezolizumab was given IV, 1200 g every 3 weeks, started at D2 of C1, then administered at D2 of each cycle for 24 cycles. Venetoclax was given orally at 800 mg/D at full dose, started on D8C1 for 24 cycles. The primary endpoint of the safety-run was to evaluate the safety of this combination. Before entering in the main study, the safety-run had to include at least 12 patients treated at least 6 weeks (2 cycles).

**Results:** A total of 13 patients were enrolled in the safety-run between Feb and Apr 2018: 9 were DLBCL and 4 FL. Median age was 61.7 years [39-77]. With a median follow up of 4.4 months [3.5-5.1], and during the 4 first cycles of treatment, hematologic toxicity was the most common AE, with G≥3 neutropenia (7 [54%] pts) and G≥3 thrombocytopenia (4 [31%] pts). No patient experienced febrile neutropenia. The most common non-hematologic all-grade AE were infections (6 [46%] pts), back pain (3 [23%] pts), diarrhea (2 [15%] pts) and IRR (2 [15%] pts). All these non-hematologic AE were G1 or 2, except one G3 lung infection. No tumor lysis syndrome was observed. Four patients (3 DLBCL and 1 FL) have permanently discontinued study treatment, all of them due to progression. At intermediate analysis (after C4), 12 patients were evaluable for response: 3 (25%, 3 DLBCL) achieved complete metabolic response (CMR) and 5 (42%, 3 FL and 2 DLBCL) achieved partial metabolic response (PMR). The Overall Metabolic Response Rate (OMRR) was 67% (8 out of 12). A pseudo-progression was observed retrospectively in one FL patient. He was in tumoral metabolic progression at C4, and has showed a CMR at C8.

**Conclusion:** The atezolizumab, obinutuzumab and venetoclax combination appears to be well tolerated. The Data and Safety Monitoring Board decided to add primary prophylaxis of neutropenia. The OMRR of 67% is encouraging for a chemo-free regimen in R/R NHLs. Treatment and follow-up of these patients are currently ongoing and updated data will be presented at the congress.

**Keywords:** B-cell lymphoma; immune system; venetoclax.

## 281
PHASE 1B STUDY OF ViPOR (VENETOCLAX, IBRUTINIB, PREDNISONE, OBINUTUZUMAB, LENALIDOMIDE) IN RELAPSED/REFRACTORY B-CELL LYMPHOMA: SAFETY, EFFICACY AND MOLECULAR ANALYSIS

<footnote n="1">¹Hématologie, CHRU de Lille, Lille, France; ²Hematology, CHU Nancy, Vandoeuvre-Lès-Nancy, France; ³Hematology, CHU de, Rouen, France;</footnote>

<footnote n="4">⁴Hematology, Hôpital Lyon Sud, Pierre-Bénite, France; ⁵Hematology, CHU de Grenoble, Grenoble Cedex 9, France; ⁶Hematology, CHU de Nantes, Nantes Cedex 1, France; ⁷Hematology, CHU Montpellier, Montpellier Cedex 5, France</footnote>
<caption>Figure 1</caption>

A. Change in Tumor Burden

<figure><img src="image_172.png" /></figure>

B. Response Characteristics

<figure><img src="image_173.png" /></figure>

C.J. Melani¹

R. Lakhotia¹

S. Pittaluga²

M.D. Miljkovic¹

C.A. Portell³

R.J. Farah⁴

A.M. Juanitez¹

L.L. Chou¹

S.M. Steinberg⁵

E.S. Jaffe²

L.M. Staudt¹

M.J. Roschewski¹

W.H. Wilson¹

¹Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States;

²Laboratory of Pathology, Clinical Center, National Cancer Institute, National Institutes of Health, Bethesda, United States; ³Emily Couric Clinical Cancer Center, University of Virginia, Charlottesville, United States; ⁴Mario Lemieux Center for Blood Cancers, University of Pittsburgh School of Medicine, Pittsburgh, United States; ⁵Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States

**Introduction:** Aggressive B-cell lymphomas can be cured with chemoimmunotherapy; however, those who fail primary therapy and indolent lymphomas are rarely curable. Novel targeted therapies can disrupt key survival pathways in lymphoma such as BCL2 (venetoclax), BTK (ibrutinib), and NFκB (lenalidomide). These agents are active as monotherapy but do not induce deep responses and require continuous therapy. Also, genetically defined subtypes of lymphoma that best respond to these targeted therapies are undefined. We hypothesized that combining synergistic agents that target unique survival pathways will leverage efficacy and time-limited, cyclic dosing will limit toxicities.

**Methods:** Relapsed/refractory B-cell lymphoma pts, excluding MCL and CLL/SLL, with adequate organ function were eligible. A 3+3 design was used to determine the MTD of 4 DLs of venetoclax (200mg, 400mg, 600mg, 800mg) PO D2-14 (starts cycle 2 for DL1) in combination with fixed-dose ibrutinib 560mg PO D1-14, prednisone 100mg PO D1-7, obinutuzumab 1000mg IV D1-2, and lenalidomide (Revlimid®) 15mg PO D1-14. Aggressive and indolent expansion cohorts were included at the MTD. Max. 6 cycles of ViPOR q21 days was given unless PD or unacceptable AE. TLS prophylaxis was given to all pts and VTE prophylaxis and G-CSF use was per investigator

discretion. Pre-treatment biopsies were obtained and analyzed for WES, RNA-sequencing, and CNAs. Baseline CT, PET and BM was performed with CT scans after cycles 1, 2, 4, and 6 and PET after cycle 6 or at time of suspected CR.

**Results:** 17 pts were enrolled in the dose-escalation cohort (DL1-8, DL2-3, DL3-3, DL4-3). Subtypes included FL (7), DLBCL (6), HGBCL (2), and MZL (2). Median (range) of prior therapies was 2 (1-6) and 10 (59%) pts were refractory to last therapy. A single DLT of G3 intracranial hemorrhage (ICH) occurred at DL1 with concomitant enoxaparin. The MTD was not identified and a dose of 800mg for venetoclax was used in expansion. G3/4 heme AEs (%pts/%cycles) included neutropenia (56%/27%), thrombocytopenia (25%/17%), and anemia (19%/4%). G3/4 non-heme AEs (%pts) included diarrhea (13%), edema, a.fib, ICH, and hypophosphatemia (6% each). SAEs (%pts) included fever (25%), thromboembolic event (13%), and ICH (6%). Of 14 pts off-therapy (10 completed, 1 PD, 3 AE), 13 are evaluable for response with an ORR of 77% (10/13) and 46% (6/13) CR. In 16 evaluable pts overall, 81% (13/16) had tumor reduction (Fig.1A). All responses are on-going with a median DOR of 4.8m (Fig.1B). One DLBCL pt with PR received XRT and 1 FL pt with PR had transformation and is proceeding to CAR-T.

**Conclusions:** ViPOR demonstrates rapid responses including CR in refractory lymphoma pts. No TLS or unexpected toxicities were observed and hematologic AEs, mainly neutropenia, were most common. Updated response durability and molecular correlates of response will be presented at the meeting.

**Keywords:** ibrutinib; lenalidomide; venetoclax.

**Disclosures:** Farah, R: Consultant Advisory Role: Celgene advisory board; Other Remuneration: Celgene speaker bureau.

282
RESULTS FROM THE SAFETY RUN-IN
PERIOD OF THE SYMPATICO STUDY
EVALUATING IBRUTINIB IN COMBINATION
WITH VENETOCLAX IN PATIENTS WITH
RELAPSED/REFRACTORY MANTLE CELL
LYMPHOMA
M. Wang¹ | R. Ramchandren² | R. Chen³ |

L. Karlin⁴ | G. Chong⁵ | W. Jurczak⁶ | K. Wu⁷ |

M. Bishton⁸ | G. Collins⁹ | P. Eliadis¹⁰ |

F. Peyrade¹¹ | K. Freise¹² | J. Sukbuntherng¹³ |

Y. Lee¹⁴ | E. Dobkowska¹⁵ | V. Fedorov¹⁵ |

J. Neuenburg¹⁵ | C. Tam¹⁶

¹Department of Lymphoma/Myeloma, University of Texas MD Anderson Cancer Center, Houston, United States; ²Department of Oncology, Karmanos Cancer Institute, Detroit, United States; ³Department of Hematology & Hematopoietic Cell Transplantation, City of Hope, Duarte, United States; ⁴Department of Hematology, Centre Hospitalier Lyon Sud, Pierre-Bénite, France; ⁵Department of Medical Oncology and Clinical Haematology, Olivia Newton-John Cancer Centre, and University of Melbourne, Melbourne, Australia; ⁶Department of Hematology, Jagiellonian University, Kraków, Poland; ⁷Department of Hematology, Ziekenhuis Netwerk Antwerpen, Antwerp, Belgium; ⁸Department of Clinical Haematology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; ⁹Department of Hematology, Oxford University Hospitals, NHS Foundation Trust, Oxford, United Kingdom; ¹⁰Department of Oncology, ICON Cancer Care, South Brisbane, Australia; ¹¹Department of Oncology, Centre Antoine Lacassagne, Nice, France; ¹²Department of Clinical Pharmacology and Pharmacometrics, AbbVie Inc, North Chicago, United States; ¹³Department of Drug Metabolism & Pharmacokinetics, Pharmacyclics LLC, an AbbVie Company, Sunnyvale, United States; ¹⁴Department of Biostatistics, Pharmacyclics LLC, an AbbVie Company, Sunnyvale, United States; ¹⁵Department of Clinical Science, Pharmacyclics LLC, an AbbVie Company, Sunnyvale, United States; ¹⁶Department of Haematology, Peter MacCallum Cancer Centre & St. Vincent's Hospital and the University of Melbourne, Melbourne, Australia

**Introduction:** Ibrutinib (ibr), a once-daily Bruton tyrosine kinase inhibitor, is indicated in the US and EU for treatment of relapsed/refractory (R/R) mantle cell lymphoma (MCL). Combining ibr with venetoclax (ven; B-cell lymphoma-2 inhibitor) has shown synergistic activity in preclinical and early phase clinical studies (Portell Blood 2014; Tam NEJM 2018). SYMPATICO (PCYC-1143; NCT03112174), an ongoing phase 3 study, is evaluating the efficacy and safety of concurrent ibr + ven (I+V) in patients (pts) with R/R MCL. A safety run-in (SRI) period is included per protocol to determine whether concurrent I+V is tolerable or if a 1-mo ibr lead-in is needed.

**Methods:** SYMPATICO has an open-label SRI and double-blind randomized period. In the SRI, pts received ibr 560 mg QD plus ven starting at 20, then 50, 100, 200, 400 mg QD in weekly dose ramp-up over 5 wks to a final dose of 400 mg QD. After SRI, pts were randomized 1:1 to I+V or ibr + placebo (pbo); ven or pbo is to be discontinued after 24 mo and ibr continued until progressive disease (PD) or unacceptable toxicity. Primary endpoints for SRI are frequency of tumor lysis syndrome (TLS) and dose-limiting toxicities (DLTs; grade [G] ≥3 non-TLS adverse events [AEs] possibly related to ibr and/or ven observed during the ≥5-wk DLT assessment period). Pts at high

baseline TLS risk had ≥1 lesion >10 cm or ≥1 lesion >5 cm with circulating lymphocytes >25000 cells/mm³ and/or creatinine clearance (CrCl) <60 mL/min. Pts not meeting high risk criteria were classified as low risk for TLS.

**Results:** 21 pts with R/R MCL were enrolled in SRI (low risk, n = 6; high risk, n = 15). Median age was 67 yrs (range, 53-84) and median prior therapies was 2 (range, 1-4); 8 pts had ≥1 lesion >10 cm, no pt had ≥1 lesion >5 cm with circulating lymphocytes >25000 cells/mm³, and 9 pts had CrCl <60 mL/min at baseline. One high-risk pt with a Wk 1, D 1 WBC of 111.89 x 10⁹/L had laboratory TLS for 5 d; no clinical TLS events occurred. In total, 3 pts had DLTs (high risk, n = 3; low risk, n = 0): G4 neutropenia, G4 infection; and G3 atrial fibrillation and G3 hypotension. Treatment-emergent AEs occurred in all pts, with G3/4 events in 12 pts (high risk, n = 10; low risk, n = 2). The most common AEs were diarrhea (n = 15; high risk, n = 10; low risk, n = 5) and fatigue (n = 11; high risk, n = 6; low risk, n = 5); 1 death occurred ≤30 d after last dose (12 d) due to retroperitoneal hemorrhage and was unrelated to ibr or ven in a low risk pt with PD and MCL in the spleen. Two deaths occurred in high risk pts >30 d after the last dose due to MCL.

**Conclusions:** In the SRI of SYMPATICO that evaluated concurrent I+V, 1 laboratory TLS event occurred in a pt at high risk for TLS. No clinical TLS events occurred. DLTs were infrequent and observed in 3/21 pts. Based on these data, concurrent I+V was recommended per protocol for pts at low and high risk for TLS. The ongoing phase 3 randomized period is evaluating the efficacy (primary endpoint: PFS) and safety of I+V vs ibr + pbo in pts with R/R MCL.

**Keywords:** ibrutinib; mantle cell lymphoma (MCL); venetoclax.

**Disclosures:** Wang, M: Consultant Advisory Role: Pharmacyclics LLC, an Abbvie Company, Celgene, Janssen; Stock Ownership: MoreHealth; Honoraria: Pharmacyclics LLC, an Abbvie Company; Research Funding: Pharmacyclics LLC, an Abbvie Company, Janssen, AstraZeneca, BeiGene, Novartis, Kite Pharma, Juno, Celgene; Other Remuneration: Expert Testimony: AstraZeneca; Travel, Accomodations, Expenses: Pharmacyclics LLC, an Abbvie Company, Janssen. Ramchandren, R: Consultant Advisory Role: Pharmacyclics LLC, an Abbvie Company; Research Funding: Pharmacyclics LLC, an Abbvie Company, Janssen. Chen, R: Consultant Advisory Role: Pharmacyclics LLC, an Abbvie Company. Karlin, L: Employment Leadership Position: AGUETTANT; Consultant Advisory Role: Celgene, Amgen, Janssen; Honoraria: CELGENE, AMGEN, JANSSEN, GILEAD; Other Remuneration: Travel, Accomodations, Expenses: Amgen, Janssen, Sanofi. Chong, G: Research Funding: Pharmacyclics LLC, an Abbvie Company, Hutchison, Beigene, Novartis, Merck KGaA, Bristol Myers Squibb. Jurczak, W: Consultant Advisory Role: Sandoz Novartis, Roche, Janssen, Gilead, Acerta; Research Funding: Celgene, Abbvie, Gilead, TG Therapeutics, Janssen, Acerta, Merck, Beigene, Pharmacyclics, Pfizer, Roche, Sandoz-Novartis. Wu, K: Consultant Advisory Role: Celgene, Janssen; Other Remuneration: Travel, Accomodations, Expenses: Celgene. Bishton, M: Consultant Advisory Role: Janssen; Research Funding: Abbvie, Roche, Gilead; Other Remuneration: Travel, Accomodations, Expenses: Takeda, Roche. Collins, G: Consultant Advisory Role: Roche, Takeda, Gilead, MSD, Pfizer, Celleron; Honoraria: Roche, Takeda, BMS, MSD, Gilead, Pfizer,
Celleron; Research Funding: Amgen, Celgene, Pfizer, BMS; Other Remuneration: Speaker's Bureau: Roche, Takeda, Gilead; Travel, Accommodations, Expenses: Roche, Takeda. Peyrade, F: Consultant Advisory Role: Merck; Honoraria: MSD, BMS, Merck; Other Remuneration: Travel, Accommodations, Expenses: Merck. Freise, K: Employment Leadership Position: Abbvie Inc.; Stock Ownership: Abbvie Inc.. Sukbuntherng, J: Employment Leadership Position: Pharmacyclics LLC, an Abbvie Company (self), Global Blood Therapeutics (spouse); Stock Ownership:ABBV, GBT, PTLA. Lee, Y: Employment Leadership Position: Pharmacyclics LLC, an Abbvie Company; Stock Ownership: Pharmacyclics LLC, an Abbvie Company; Other Remuneration: Travel, Accommodations, Expenses: Pharmacyclics LLC, an Abbvie Company. Dobkowska, E: Employment Leadership Position: Pharmacyclics LLC, an Abbvie Company; Stock Ownership: Abbvie. Fedorov, V: Employment Leadership Position: Pharmacyclics LLC, an Abbvie Company; Stock Ownership: Abbvie. Neuenburg, J: Employment Leadership Position: Pharmacyclics LLC, an Abbvie Company; Stock Ownership: Abbvie. Tam, C: Honoraria: Janssen and Pharmacyclics; Research Funding: Janssen.

## 283
### PHASE I STUDY OF ROMIDEPSIN AND LIPOSOMAL DOXORUBICIN IN RELAPSED OR REFRACTORY T-CELL LYMPHOMA

W. Ai¹ | K. Vu¹ | P. Porcu² | L. Pincus¹ |

M. Wieduwilt³ | L. Kaplan¹ | B. Andreadis¹ |

J. Brammer²

¹Department of Medicine, University of California, San Francisco, United States; ²Department of Medicine, Ohio State University, Columbus, United States; ³Department of Medicine, University of California, San Diego, United States

**Introduction:** Romidepsin and liposomal doxorubicin have single-agent activity in cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma (PTCL). In pre-clinical studies using lymphoma cell lines, we demonstrated that romidepsin had strong synergistic effects in combination with doxorubicin. The aim of this study is to determine the safety and feasibility of the combination of romidepsin and liposomal doxorubicin in patients with relapsed/refractory CTCL and PTCL.

**Patients and Methods:** This is a phase I study that employed a standard "3+3" dose-escalation design with a dose-expansion cohort to evaluate safety, maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and clinical activity of romidepsin in combination with liposomal doxorubicin. Patients with relapsed/refractory 1) CTCL (stage IB-IVB MF or SS) after ≥ 1 prior systemic therapy or ≥ 2 prior skin-directed therapies, or 2) PTCL of any stage or subtype after ≥ 1 prior systemic therapy, are eligible. The primary endpoint is to identify the MTD of romidepsin in combination with liposomal doxorubicin. The secondary endpoints included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Patients were treated with liposomal doxorubicin 20 mg/m² on day 1 and romidepsin at four dose levels (8, 10,

12 and 14 mg/m²) on days 1, 8 and 15, every 28 days, until 2 cycles beyond best response, 8 cycles, disease progression, or intolerability; whichever comes first. Response was determined by using the International Working Group and International Society for CTCL consensus criteria for PTCL and CTCL, respectively.

**Results:** Twenty-three patients (11 CTCL, 12 PTCL) were enrolled and received at least 1 dose of study drugs. Three DLTs occurred: grade 4 nonfebrile neutropenia lasting > 7 days in 1 PTCL patient, grade 3 thrombocytopenia lasting > 7 days in 2 PTCL patients. The MTD of romidepsin was determined to be 12 mg/m². There was a higher incidence of grade 3/4 treatment-related hematologic AEs in the PTCL vs. the CTCL cohort. The most frequent non-hematologic AEs were nausea (48%), fatigue (43%), vomiting (35%), and anorexia (30%).

Disease response was evaluable in 20 (10 CTCL, 10 PTCL) patients, with a ORR of 70% (1 CR, 6 PR, 2 SD, 1 PD) in the CTCL cohort and 20% (2 CR, 8 PD) in the PTCL cohort. The median reduction in mSWAT score was 81% in CTCL patients. After a median follow-up of 17.8 months, median PFS was 7.4 months for the CTCL cohort and 2 months for the PTCL cohort. Median OS was not reached for the CTCL cohort and 7.8 months for the PTCL cohort.

**Conclusions:** The combination of romidepsin and liposomal doxorubicin has an acceptable toxicity profile at an MTD of 12 mg/m² for romidepsin with mostly grade 1/2 fatigue and GI-related toxicities. The combination has promising activity in CTCL and merits further investigation.

**Keywords:** T-cell lymphoma (TCL).

## 284
### A PHASE 1B/2 STUDY OF ORAL NANATINOSTAT (N) AND VALGANCICLOVIR (VG) IN SUBJECTS WITH EPSTEIN-BARR VIRUS (EBV)-ASSOCIATED LYMPHOMAS

P. Porcu¹ | B. Haverkos² | E. Brem³ |

A. Vallurupalli⁴ | T. Feldman⁵ | O. Alpdogan¹ |

J. Brammer⁶ | L.J. Bryan⁷ | S. Barta⁸ |

A. Schriefer⁹ | S. Obrzut¹⁰ | H. Shen¹¹ |

R. Rochford¹² | R. Baiocchi⁶ | C. Casper¹³ |

J. Gutheil¹⁴ | T. Melink¹⁴ | C. Kearns¹⁴ |

D. Burner¹⁴ | R. McRae¹⁵ | P. Daniels¹⁵ |

M.S. Warren¹⁵ | J. Woody¹⁶ | I. Royston¹⁵ | D. Faller¹⁷

¹Medical Oncology and Hematopoietic Stem Cell Transplantation, Thomas Jefferson University, Philadelphia, United States; ²Hematology, University of Colorado School of Medicine, Aurora, United States;

³Hematology/Oncology, University of California Irvine, Orange, United States; ⁴Hematologic Malignancies and Cellular Therapeutics, University of Kansas Medical Center, Kansas City, United States; ⁵Hematology & Oncology, John Theurer Cancer Center, Hackensack, United States;

⁶Hematology, Ohio State University, Columbus, United States;

⁷Oncology, Augusta University, Augusta, United States; ⁸Hematology,
University of Pennsylvania, Philadelphia, United States; ⁹Oncologia, Clinica Cehon, Canela, Salvador, Brazil; ¹⁰Radiology, University of California San Diego, La Jolla, United States; ¹¹Microbiology, University of Pennsylvania, Philadelphia, United States; ¹²Immunology & Microbiology, University of Colorado, Aurora, United States; ¹³Research, Infectious Disease Research Institute, Seattle, United States; ¹⁴SciQuus Oncology Inc, La Jolla, United States; ¹⁵Viracta Therapeutics, Cardiff, United States; ¹⁶Latterell Venture Partners, San Francisco, United States; ¹⁷Hematology & Medical Oncology, Boston University School of Medicine, Boston, United States

**Introduction:** Nanatinostat (N; VRx-3996) is a Class I-selective oral hydroxamate histone deacetylase (HDAC) inhibitor active against HDAC1-3, but not HDAC6. N induces the expression of EBV thymidine kinase (BXLF1) and protein kinase (BGLF4) in EBV-positive lymphomas models. This study examines if N sensitizes tumors to antivirals (i.e., VG) and impacts EBV-induced T-cell exhaustion.

**Methods:** The study employs a 3+3 design with expansion in patients (pts) with relapsed/refractory EBV+ lymphomas. (determined by EBER-ISH). Objectives: Maximal Tolerated Dose (MTD), Recommended Phase 2 Dose (RP2D), and efficacy. Primary endpoint: safety. Secondary endpoints: PK, response rate. Exploratory endpoints: PBMC histone H3 acetylation (Ac), quantitative plasma EBV DNA (pEBVd), and immune biomarkers. Responses: investigator-assessed and centrally-reviewed by Lugano Classification.

**Results:** As of 07-Mar-2019, 18 pts (5F/13M, median age 60 yrs [19-79]) were enrolled with a median of 2.5 prior therapies and various lymphoma subtypes including anaplastic large cell (1), AITL (2), Gray Zone (1), DLBCL (6), Hodgkin's (2), lymphoplasmacytic (2), peripheral T-cell (4). Cohort 1 (C1: N 10 mg BID/VG 900 mg BID) exceeded MTD (DLTs: leukopenia, neutropenia and thrombocytopenia). PK demonstrated ~2-fold higher N exposure compared to a prior Phase I study. VG levels were consistent with published data.

Best Response by PET
(Normalized/corrected to liver SUV)

<figure><img src="image_174.png" /></figure>

Doses were reduced in Cohort 2 (C2: N 5 mg BID/VG 450 mg BID) with no DLTs observed to date and 2 active pts. Most non-hematologic treatment-related AEs (TRAEs) were G1-2. G3+ TRAEs were largely hematologic (neutropenia, leukopenia, thrombocytopenia). Increases in creatinine occurred early and responded to reductions in VG. 7 pts are evaluable for response (2 CR (DLBCL [in C1], AITL [in C1]), 2 PR (peripheral T-cell [C2] and DLBCL [C2]), 2 SD (Hodgkin's [C1], DLBCL [C1]), and 1 PD (Gray Zone [C1]) with all 7 patients showing reduction in PET avidity (range 9.7 - 100% reduction compared to liver update) as demonstrated in the figure. 8 pts had detectable baseline pEBVd with 7 demonstrating a reduction (median -54% [17 to -83%]). PBMC H3Ac levels, multi-cytokine expression panel and functional assessments in CD8 T cells are in progress.

**Conclusion:** N+VG appears well-tolerated at 5 mg and 450 mg BID, respectively with hematologic DLTs in the initial higher dose cohort. Responses are observed at all dose levels, in both B and T-cell lymphomas, with reductions in pEBVd levels and preliminary evidence of changes in T cell functionality. A RP2D will be defined prior to dose expansion. NCT03397706.

**Keywords:** epigenetics; Epstein-Barr virus (EBV); histone deacetylase inhibitors.

**Disclosures:** Porcu, P: Employment Leadership Position: Thomas Jefferson University; Consultant Advisory Role: Innate Pharma; Honoraria: Actelion, Celgene; Research Funding: Infinity Pharmaceuticals, Celgene, Millennium, Seattle Genetics, Oncomed. Haverkos, B: Consultant Advisory Role: Viracta Therapeutics. Brem, E: Consultant Advisory Role: Bayer, Pfizer, Celgene, Genentech, Janssen, Pharmacyclics. Feldman, T: Consultant Advisory Role: Seattle Genetics, Bayer, Bristol-Myers Squibb; Honoraria: Seattle Genetics, Pharmacyclics/Janssen, Abbvie, Bristol-Myers Squibb, Kite Pharma, Bayer, Takeda; Research Funding: Bristol-Myers Squibb, Seattle Genetics, Portola Pharmaceuticals, Eisai, Kyowa Hakko Kirin, Amgen, Viracta Therapeutics, Cell Medica, Roche, Trillium Therapeutics, Pfizer; Other Remuneration: Kite Pharma, Pharmacyclics, Abbvie, Janssen, Celgene, Takeda. Alpdogan, O: Consultant Advisory Role: Kyowa Hakko Kirin. Brammer, J: Honoraria: Seattle Genetics; Research Funding: Celgene. Barta, S: Consultant Advisory Role: Janssen Oncology; Research Funding: Seattle Genetics, Merck, Celgene, Takeda, Bayer. Schriefer, A: Employment Leadership Position: Cehon; Honoraria: Cehon. Obrzut, S: Other Remuneration: Viracta Therapeutics. Shen, H: Other Remuneration: Viracta Therapeutics. Rochford, R: Other Remuneration: Viracta Therapeutics. Baiocchi, R: Consultant Advisory Role: Viracta Therapeutics, Prelude Therapeutics; Research Funding: Prelude Therapeutics, Esanex. Casper, C: Consultant Advisory Role: Janssen, GlaxoSmithKline, Temptime, Viracta Therapeutics, Curevo Vaccines; Research Funding: Janssen. Gutheil, J: Employment Leadership Position: SciQuus Oncology; Consultant Advisory Role: Viracta Therapeutics; Stock Ownership: SciQuus Oncology. Melink, T: Employment Leadership Position: SciQuus Oncology; Stock Ownership: SciQuus Oncology. Kearns, C: Employment Leadership Position: SciQuus Oncology. Burner, D: Employment Leadership Position: SciQuus Oncology Inc. Warren, M: Employment Leadership Position: Viracta Therapeutics; Stock Ownership: Viracta Therapeutics. Woody,
J: Employment Leadership Position: *Latterell Venture Partners*; Consultant Advisory Role: *Viracta Therapeutics*; Stock Ownership: *Viracta Therapeutics*. Royston, I: Employment Leadership Position: *Viracta Therapeutics*; Stock Ownership: *Viracta Therapeutics, Biocept*. Faller, D: Employment Leadership Position: *Takeda, Array Biopharma, Phoenicia Biosciences*; Consultant Advisory Role: *Viracta Therapeutics, Briacell, Takeda*; Stock Ownership: *Array Biopharma, Phoenicia Biosciences, Viracta Therapeutics, Briacell, Takeda*.

285
IMPROVEMENT OF RITUXIMAB ADMINISTRATION BY PREMEDICATION WITH RUPATADINE, MONTELUKAST AND THEIR COMBINATION

R. Kotchetkov¹ | J. McLean² | L. Gerard¹ | D. Nay¹ | S. Hopkins³

¹Oncology, SMRCP, Barrie, Canada; ²Research, RVH Research Institute, Royal Victoria Regional Health Centre, Barrie, Canada; ³Provincial Drug Reimbursement Programs, Cancer Care Ontario, Toronto, Canada

**Background:** Rituximab is associated with frequent infusion reactions, in particular during the first administration. They carry significant burden to both patients and health care providers. Standard pre-medications (SP) do not prevent reactions sufficiently. Montelukast (M) and Rupatadine (R) are used for symptomatic treatment of chronic urticarial and allergic rhinitis. Their impact on Rituximab

infusion is unknown. We assessed whether addition of M, R and their combination (M+R) improves Rituximab delivery, infusion/rate and decreases rate, severity of reactions and cost of administration

**Methods:** Adult patients with lymphoproliferative disorders (LPD) treated at our cancer center between Jan 2018 to Jan 2019 with Rituximab-containing regiments were evaluated. Our study was limited to the initial Rituximab infusion. Patients received either SP with diphenhydramine/acetaminophen and additional M, R or M+R combination. Comparative analysis of infusion time/rate, severity of infusion reactions, number of rescue medications and cost of Rituximab infusions among groups was performed using one-way ANOVA with Tukey post-hoc or chi-square. The study was approved by our institutional REB

**Results:** Patients received either: 1) standard pre-medications (SP); 2) SP + Montelukast 10 mg, 12h and 30 min prior to infusion (M); 3 SP + Rupatadine (R) 10 mg; 12 h prior to infusion or 4) both (SP+M+R) given as the same schedule. Patient characteristics and therapy are shown in Table 1.

Compared to SP, the M, R and M+R groups had greater improvement in Rituximab delivery. Mean infusion time was 305 [range 235-441] min. in SP, 254 [105-390] in M, 265 [193-350] in R and 229 [196-342] in M+R groups, (p = 0.0001). Mean Rituximab delivery rate (mg/min) was 2.40 [1.78-3.23] (SP), 3.10 [2.12-6.67] (M), 2.85 [2.10-3.88] (R), and 3.12 [2.4-3.69] in M+R group. Infusion reactions occurred in 92% in SP vs. 38, 45, 31% in M, R and M+R groups (p = 0.0001). Median reaction grade was 2 in SP, 1 (M), 0 (R and M+R). Median number of rescue medications was 3 [0-10] in SP vs 0 [0-7]

<table><caption>TABLE 1 Patient characteristics and therapy received</caption><thead><tr><th></th><th>Standard Premedications</th><th>Montelukast</th><th>Rupatadine</th><th>Monteleukast + Rupatadine</th></tr></thead><tbody><tr><td>Number of patients</td><td>26</td><td>21</td><td>20</td><td>26</td></tr><tr><td>Age: median [min, max]</td><td>67 [45-87]</td><td>73 [35-91]</td><td>65 [51-88]</td><td>72 [50-86]</td></tr><tr><td>Male (%)</td><td>46</td><td>71</td><td>75</td><td>81</td></tr><tr><td>Hx of allergy (%)</td><td>39</td><td>33</td><td>50</td><td>31</td></tr><tr><td>LPD: pts (%)</td><td></td><td></td><td></td><td></td></tr><tr><td>Diffuse Large B-Cell Lymphoma</td><td>6 (23)</td><td>3 (14)</td><td>5 (20)</td><td>8 (31)</td></tr><tr><td>Follicular Lymphoma</td><td>9 (31)</td><td>2 (10)</td><td>5 (20)</td><td>5 (19)</td></tr><tr><td>Chronic Lymphocytic Leukemia</td><td>4 (16)</td><td>4 (19)</td><td>5 (20)</td><td>2 (8)</td></tr><tr><td>Other indolent Non-Hodgkin Lymphoma</td><td>7 (27)</td><td>12 (57)</td><td>5 (20)</td><td>11 (42)</td></tr><tr><td>Advance stage/bulky (%)</td><td>65</td><td>76</td><td>85</td><td>70</td></tr><tr><td>Tx naive</td><td>96</td><td>86</td><td>80</td><td>93</td></tr><tr><td>Chemotherapy regimen:</td><td></td><td></td><td></td><td></td></tr><tr><td>RCHOP</td><td>8 (31)</td><td>5 (24)</td><td>6 (30)</td><td>10 (39)</td></tr><tr><td>BR</td><td>15 (58)</td><td>9 (43)</td><td>6 (30)</td><td>10 (39)</td></tr><tr><td>FCR</td><td>2 (8)</td><td>2 (10)</td><td>5 (25)</td><td>2 (8)</td></tr><tr><td>Other</td><td>1 (4)</td><td>5 (24)</td><td>3 (15)</td><td>4 (14)</td></tr><tr><td>Rituximab dose (mg): Mean [min-max]</td><td>727 [600-1000]</td><td>750 [550-1000]</td><td>740 [550-950]</td><td>762 [550-1000]</td></tr></tbody></table>

Abbreviations: LPD, lymphoproliferative disorders; DLBCL, Diffuse Large B-Cell Lymphoma; FL, Follicular Lymphoma; CLL, Chronic Lymphocytic Leukemia; iNHL, indolent Non-Hodgkin Lymphoma; RCHOP, Rituximab/Cyclophosphamide/ Doxorubicin/Oncovin/Prednisone; BR, Bendamustine/Rituximab; FCR, Fludarabine/Cyclophosphamide/Rituximab.
in M, R and M+R groups. Cost of rescue medications (US$) was 41 [0-63] in SP group, 11 [0-50] (M), 17 [0-63] (R), 7 [0-58] (M+R) groups (p < 0.0001). Mean nursing cost (US$) per patient infusion was calculated as 269 [207-388] in SP vs 222 [92-343] (M), 233 [170-308] (R), 202 [174-301] (M+R) group.

**Summary/Conclusion:** Addition of Montelukast, Rupatadine, and particularly Montelukast+Rupatadine combination significantly shortened Rituximab infusion time, decreased the rate and severity of infusion reactions, and lowered total cost of Rituximab administration

**Keywords:** B-cell lymphoma; chemotherapy; rituximab.
PUBLICATION

BIOLOGY AND PATHOLOGY

286
MULTI-OMICS APPROACHES TO
UNDERSTAND GASTRIC MUCOSA-
ASSOCIATED LYMPHOID TISSUE (MALT)
LYMPHOMA

H. Song¹ | E. Kang² | H. Soh² | H. Chung² |

J. Chun² | S. Yoon² | U. Ijaz³ | Y. Koh²

¹Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea; ²Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea; ³School of Engineering, University of Glasgow, Glasgow, United Kingdom

Gastric Mucosa-Associated Lymphoid Tissue (MALT) lymphoma is one of non-Hodgkin lymphoma that occurs in digestive tracts. The main risk factor for gastric MALT lymphoma is *Helicobacter pylori* (Hp) infection. Pathogenesis of Hp encompasses four steps: 1) entrance into host and tolerance against acidic environment; 2) motility and chemotaxis; 3) interaction between adhesion-receptors and

colonization, and 4) release of toxins and damage to the host. Up until now, the pathogenic mechanism diverging from the bacterial infection to lymphomagenesis is largely unknown. We aim to characterize the carcinogenic mechanism of *Hp*-associated MALT lymphoma using multi-omics data analysis.

Gastric tissues of both tumor and adjacent normal were collected from gastric MALT lymphoma patients. Matched saliva samples from each donor were also collected. Whole exome sequencing (WES) data was generated using gastric lesion tissues and saliva samples whereas whole transcriptome sequencing (WTS) data was generated using gastric lesion and adjacent normal tissues. For WTS analysis (18 tumor and 5 normal samples), we aligned raw data, quantified the gene expression, and explored both significant genes and pathways using negative binomial GLM fitting and their function using GO ontologies. For WES analysis, we aligned raw data, performed variant calling, and detected a list of genes with somatic mutations (12 pairs).

Based on the comparison of expressed genes between tumor and nor-
mal samples from MALT lymphoma patients, biological pathways
associated with diverse immune responses were converged when fil-
tered using classical Fisher's test on the GO ontologies of markedly
regulated genes. The immune-associated pathways include
leukocyte/lymphocyte activation, immune response-regulating cell

<figure><img src="image_175.png" /><figcaption>Figure 1. Significant GO ontologies for enriched genes in rectangular boxes</figcaption></figure>
surface receptor signaling pathway, and regulation of immune system processes. In addition, both complement C3 and adenosine deaminase (ADA) genes were upregulated in tumor gastric tissues. *IFI30* gene encoded by Gamma-interferon-inducible lysosomal thiol reductase (GILT) whose function is closely associated with bacterial activities was also upregulated in the tumor tissue. WES data revealed that two recurrent missense mutations (K169R and N175S) of CDC27 were found in ~20% of MALT lymphoma patients.

Upregulated expression of complement C3 gene in tumor tissues suggests that the defense mechanisms of innate immunity have been activated due to the bacterial infection. Upregulated ADA agrees on the findings from previous studies showing higher ADA activities in *Hp*-infected patients. Upregulation of *IFI30* in tumor tissues supports evidence of enhanced bacterial hemolysis and bacterial replication/infection activities. On top of that, repeatedly observed mutations in a cell cycle-associated gene, CDC27, may have a role to play in cell growth inhibition.

**Keywords:** Helicobacter pylori; immune system; Mucosa-Associated Lymphoid Tissue (MALT).

287

THE ROLE OF DUSP22 (DUAL SPECIFICITY PHOSPHATASE 22) GENE EXPRESSION IN THE PROGNOSIS OF LOW GRADE LYMPHOMAS

S. Paydas

E.K. Bagir

M. Ergin

G. Seydaoglu

A. Boz

Medical Oncology, Cukurova University, Adana, Turkey

**Introduction:** Dual specificity phosphatase 22 (DUSP22) is a novel phosphatase and has been demonstrated to be a cancer suppressor gene associated with various biological and pathological processes. Prognostic role of DUP22 expression in cases with ALK (-) anaplastic large cell lymphoma has been demonstrated but little is known about DUSP22 expression and its prognostic value in other lymphomas. Here clinical/prognostic importance of DUSP22 expression has been determined in cases with indolent lymphomas including follicular lymphomas, marginal zone lymphomas and chronic lymphocytic leukemia. The aim of this study to evaluate the prognostic importance of DUSP22 expression in low grade lymphomas according to clinical and demographic variables.

**Methods and patients:** Fluorescence in situ hybridization (FISH) was performed 3 micron sections of formalin-fixed, paraffin-embedded tissue. IRF4/DUSP22 (Cytotest, USA) break-apart FISH probe kit was used for rearrangement detection. Slides were analyzed using standard fluorescence microscopy techniques. 100 cell nuclei without overlapping were counted on each slide. The cutoff value for IRF4/DUSP22 was accepted %15. Eighty four cases with indolent lymphoma were evaluated for DUSP22 expression. The signal could not be detected in 8 cases and of the total 76 cases with indolent lymphoma were

<figure><img src="image_176.png" /></figure>

evaluated for DUSP22 expression. Female/male ratio was 31/45. Fifteen cases had stage I disease, 8 had stage II, 18 had stage III and 26 had stage IV disease. Thirty nine had follicular lymphoma (FL), 30 had marginal zone lymphoma (MZL) and 7 had chronic lymphocytic leukemia (CLL). Among FL, 5 had grade I, 10 cases had grade II, 24 had grade III disease: 13 had grade IIIA-11 had grade IIIB. Among MZL 4 had nodal and 26 had extranodal MZL. Complete response was achieved in 42 cases and partial response in 11 cases; 8 cases did not respond to treatment. During this analysis 43 cases were living without disease, 12 cases were living with disease and 21 cases died.

**Results:** DUSP22 expression was detected in 17 cases (22.3%). Expression was detected in 4 of 31 women and 13 of 45 men. DUSP22 expression was detected 10 of 39 cases with FL, 6 of 30 cases with MZL and 1 of 7 cases with CLL. Among grade I cases. The mean OS was found to be longer in cases without DUSP22 compared to cases with DUSP22 expression (126 vs 58 months p:0.036) (Figure 1). However no significant differences were found between the cases with and without DUSP22 expression according to mean of event-free survival (EFS) (58 vs 82 months p:0.717). The OS was found to be longer in DUSP22 (-) females than males (157 vs 104 months p: 0.018). In univariate analysis, OS was found to be longer in cases with early stage disease (p:0.0001) and in females (p:0.009). Sex (OR:3.5, %95CI:1.0-12.1, p:0.046), stage (OR:8.0, %95CI:1.736.7, p:0.008) and DUSP22 expression (OR:3.4, %95CI:1.2-9.3, p:0.018) were found to be independent prognostic factors according to Cox regression analysis.

**Conclusions:** Little is known about the functional roles of DUSP22 and the underlying mechanisms. The silencing of DUSP22 in peripheral T cell lymphomas especially in ALK (-) anaplastic large cell lymphoma suggest that this gene is a candidate tumor suppressor gene and its inactivation may contribute to the pathogenesis of peripheral T cell lymphoma subtypes. Some studies showed that of the 20-30% of ALK(-) anaplastic large cell lymphomas have chromosomal rearrangements of DUSP22. DUSP22 rearranged cases with ALK (-) subtype have favorable outcomes similar to ALK (+) anaplastic large cell lymphoma. There is no sufficient data about the clinical and/or prognostic significance of DUSP22 rearrangement in other
lymphomas. We found poor outcome in cases with DUSP22 expression. How can we define this controversy? It is known that DUSP22 regulates MAPK signal transduction but the effect of DUSP22 on MAPKs is controversial. Since there have been several conflicting reports regarding its substrate specificity. One report showed that DUSP22 dephosphorylates ERK2 in vitro, while other studies showed that DUSP22 enhances JNK activation but not p38 and ERK2. Therefore, further studies are required to clarify the physiological role of DUSP22. We found poor overall survival in cases with DUSP22 expression and we need further studies about the prognostic significance of DUSP22 expression in indolent lymphomas.

**Keywords:** non-Hodgkin lymphoma (NHL); prognostic indices.

## 288
GENE MUTATIONS AND SITES OF INVOLVEMENT IN DUODENAL-TYPE FOLLICULAR LYMPHOMA

K. Miyazaki¹ | T. Takeuchi¹ | M. Yamaguchi¹ |
N. Asano² | A. Sawaki¹ | H. Imai³ | R. Ono⁴ |
T. Nosaka⁴ | K. Kobayashi⁵ | K. Tanaka⁶ | N. Katayama¹

¹Department of Hematology and Oncology, Mie University Graduate School of Medicine, Tsu, Japan; ²Department of Molecular Diagnostics, Shinshu Medical Center, Suzaka, Japan; ³Pathology Division, Mie University Hospital, Tsu, Japan; ⁴Department of Microbiology and Molecular Genetics, Mie University Graduate School of Medicine, Tsu, Japan; ⁵Gastroenterology, Matsusaka Chuo General Hospital, Matsusaka, Japan; ⁶Department of Endoscopy, Mie University Hospital, Tsu, Japan

**Background:** Duodenal-type follicular lymphoma (DTFL) is a rare variant of FL characterized by duodenal involvement and favorable prognosis. A clinicogenetic risk model m7-FLIPI was originally proposed for the prognostication of nodal FL. There are few studies analyzing the m7-FLIPI score and gene mutations according to the sites of involvement in patients with DTFL.

**Methods:** This study included 11 patients with DTFL diagnosed according to the 2017 WHO classification between 2005 and 2016 at Mie University Hospital. Five patients with involvement of mesenteric lymph nodes were excluded in total 16 patients. Intestinal involvement was examined by double-balloon endoscopy and/or capsule endoscopes in addition to esophagogastroduodenoscopy and colonoscopy in all the patients. Genomic DNA was examined using the AmpliSeq Comprehensive Cancer Panel investigating the exonic regions of 409 genes and direct sequencing of MEF2 (exon 2 to 3).

**Results:** The median age was 59 years (42-84) and 6 patients were women. Sites of lymphomatous involvement were as follows: second part of the duodenum, 100% of the patients; third part of the duodenum, 82%; first part of the duodenum, 0%; jejunum, 72%; ileum, 36%; colon, 9%; stomach, 0%. Lymphoma cells by all the patients were positive for CD20, CD10, and BCL2 by immunohistochemistry. The t (14;18) translocation was confirmed in 10 of the cases by FISH. All

the patients were alive at follow up (median 5.7 years). The mutation frequencies of genes regarding m7-FLIPI were as follows: EP300, 36%; FOXO1, 0%; CREBBP, 64%; ARID1A, 45%; and EZH2, 36%. The CARD11 mutation was identified in 55% of the cases, whereas none of cases had the MEF2B mutation. One hundred percent of the patients were classified in the low risk group with the m7-FLIPI model. KMT2D was mutated in 45% of the cases. Of note, KMT2C mutation was detected in 64% of the cases. MYC, TP53 and MYD88 were not mutated in any of the cases. All 3 cases without the involvement of both ileum and jejunum had the NOTCH2 mutation.

**Conclusions:** Our results suggest that the m7-FLIPI model was valid with DHFL patients in this cohort. The incidence of KMT2C was higher compared with that of the other lymphoma subtypes (1-13%). KMT2C and NOTCH2 mutations may be associated with the diversity in the sites of intestinal involvement in DTFL.

**Keywords:** follicular lymphoma (FL).

**Disclosures:** Miyazaki, K: Honoraria: Chugai Pharma, Kyowa Hakko Kirin, Celgene; Research Funding: Teijin Pharma, Mochida Pharmaceutical Co. Ltd., Toyama Chemical Co, Ono Pharmaceutical, Takeda, Janssen, Astellas Pharma, Novartis, Shionogi Pharmaceutical, Novo Nordisk, Daiichi Sankyo, Eisai, Pfizer, Kyowa Hakko Kirin, Chugai Pharma, Nippon Shinyaku, Sumitomo Group. Takeuchi, T: Honoraria: Chugai Pharma; Research Funding: Teijin Pharma, Mochida Pharmaceutical Co. Ltd., Toyama Chemical Co, Ono Pharmaceutical, Takeda, Janssen, Astellas Pharma, Novartis, Shionogi Pharmaceutical, Novo Nordisk, Daiichi Sankyo, Eisai, Pfizer, Kyowa Hakko Kirin, Chugai Pharma, Nippon Shinyaku, Sumitomo Group. Yamaguchi, M: Honoraria: Chugai Pharma, Eisai, Kyowa Hakko Kirin, Janssen, Meiji Seika Kaisha, MSD, Teijin Pharma, Celgene, Takeda; Research Funding: Teijin Pharma, Mochida Pharmaceutical Co. Ltd., Toyama Chemical Co, Ono Pharmaceutical, Takeda, Janssen, Astellas Pharma, Novartis, Shionogi Pharmaceutical, Novo Nordisk, Daiichi Sankyo, Eisai, Pfizer, Kyowa Hakko Kirin, Chugai Pharma, Nippon Shinyaku, Sumitomo Group. Asano, N: Honoraria: Takeda, Chugai Pharma, Celgene. Sawaki, A: Honoraria: Chugai Pharma; Research Funding: Teijin Pharma, Mochida Pharmaceutical Co. Ltd., Toyama Chemical Co, Ono Pharmaceutical, Takeda, Janssen, Astellas Pharma, Novartis, Shionogi Pharmaceutical, Novo Nordisk, Daiichi Sankyo, Eisai, Pfizer, Kyowa Hakko Kirin, Chugai Pharma, Nippon Shinyaku, Sumitomo Group. Katayama, N: Honoraria: Bristol-Myers Squibb, Takeda, Astellas Pharma, Novartis, Shire, Shionogi Pharmaceutical, Novo Nordisk, Taisho Toyama Pharma, Sysmex, Celgene, Pfizer, Alexion Pharmaceuticals, Kyowa Hakko Kirin, Chugai Pharma, Nippon Shinyaku, Sumitomo Group; Research Funding: Teijin Pharma, Mochida Pharmaceutical Co. Ltd., Toyama Chemical Co, Ono Pharmaceutical, Takeda, Janssen, Astellas Pharma, Novartis, Shionogi Pharmaceutical, Novo Nordisk, Daiichi Sankyo, Eisai, Pfizer, Kyowa Hakko Kirin, Chugai Pharma, Nippon Shinyaku, Sumitomo Group.

## 289
PREVALENCE OF EPSTEIN BARR VIRUS IN FOLICULAR LYMPHOMA AND PROGNOSTIC VALUE OF FOXP1 AND P53 BIOMARKERS IN A MEXICAN POPULATION
<figure><img src="image_177.png" /></figure>

A.J. Salazar-Vargas<fnref n="1" /> | G. Soca-Chafre<fnref n="2" /> |
C. Lome-Maldonado<fnref n="1" /> | L. Moreno<fnref n="1" /> | D. Perez<fnref n="1" /> |
S. Rivas-Vera³ | A. Ramirez-Ibarguen³

¹Hemato-pathology, National Cancer Institute of Mexico, Mexico City, Mexico; ²Personalized Medicine Laboratory, National Cancer Institute of Mexico, Mexico City, Mexico; ³Hematology, National Cancer Institute of Mexico, Mexico City, Mexico

**Introduction:** Follicular lymphoma (FL) is the second most frequent neoplasm in B cells. Epstein Barr Virus (EBV) causes a latent infection in B-lymphocytes with transformation capacity; however in FL there is just one previous publication that documented 2.6% of prevalence and there are no studies in Latin America. Other factors related to worse overall survival (OS) are P53 mutations and the transcription factor FOXP1, who is down-regulated in presence of EZH2 mutations. In search of prognostic biomarkers for LF, and in the absence of molecular tests for TP53 and EZH2, we wanted to determine the prognostic value of FOXP1 and P53, as well as the association between EBV and LF in a Mexican population.

**Methods:** We conducted an observational, descriptive, analytical and retrospective study including cases with lymph node diagnosis of LF. We obtained clinical data from the electronic records and reviewed the slides selecting the representative area of the neoplasia for tissue microarrays (TMA, in situ hybridization (EBER-ISH) and immunohistochemical markers. The statistical analysis was performed using Kaplan-Meier curves, Log-rank test and Cox regression using IBM SPSS version 25.

**Results:** We analyzed 228 patients, with a median OS of 13.4 years; 16% of the cases were EBV+. We found association in EBV+ patients to low hemoglobin ≤12mg/L (p = 0.014) and albumin ≤4g / dL (p = 0.009) levels. EBV+ patients had shorter OS than the EBV- (7 vs. 13 years, p = 0.029). EBV+ combinations with Ki67 ≥ 40% and histological grade 3 were associated with OS of 3.6 and 3.2 years respectively (p = 0.052, p = 0.017). Concurrent expression of EBV+, FOXP1 ≥ 10% and Ki67 ≥ 40% reduces OS to 2.9 years (p = 0.004). P53

expression ≥ 1% was positive in 100% of EBV+ cases (p = 0.021) while p53 ≥ 20% was positive in 31% of them (p = 0.067). FOXP1 expression ≥ 85% was negatively associated with OS (p = 0.010) and relapse-free survival (RFS) (p = 0.051), as was p53 ≥ 10% (p = 0.035). FOXP1 ≥ 10% concurrent with EBV+ decreases OS to 6.7 years (p = 0.014). Multivariate analyses for OS confirmed FOXP1 ≥ 85% as independent prognostic factor.

**Conclusions:** The LF/EBV+ association has a high prevalence in Mexico with a significant prognostic value for OS. FOXP1 expression ≥ 85%, but not at 10% was an independent factor of poor prognosis. P53 had an impact on OS at a cut-off value of 10%, independently of EBV status. Those LF/EBV+ patients with concurrent Ki67 expression (≥ 40%) and histological grade 3 exhibited worse prognosis. We consider of particular importance to identify relevant prognostic factors in the Mexican population. These biomarkers should be both reproducible and accessible to our diagnostic capabilities allowing the development of better therapeutic strategies for high-risk EBV+ LF cases.

**Keywords:** Epstein-Barr virus (EBV); follicular lymphoma (FL).

290
INCREASED CCND1 FISH SIGNALS ARE
ASSOCIATED WITH WORSE PROGNOSIS IN
MANTLE CELL LYMPHOMA

Y. Le Bris<fnref n="1" /> | O. Theisen<fnref n="1" /> | C. Godon<fnref n="1" /> | M. Tiab<fnref n="2" /> |
A. Moreau³ | C. Bossard³ | F. Subiger⁴ |
D. Chiron⁵ | T. Gastinne⁶ | B. Tessoulin⁶ |
H. Maisonneuve<fnref n="2" /> | S. Le Gouill⁶ | M. Béné<fnref n="1" />

<footnote n="1">¹Hematology Biology, Nantes University Hospital, Nantes, France;</footnote>

<footnote n="2">²Hematology Clinic, Centre Hospitalier Départemental de Vendée, La Roche sur Yon, France; ³Pathology, Nantes University Hospital, Nantes, France; ⁴Biology, Centre Hospitalier Départemental de Vendée, La Roche sur Yon, France; ⁵INSERM_U1232, CRCINA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France; ⁶Hematology Clinic, Nantes University Hospital, Nantes, France</footnote>
**Background:** Mantle cell lymphoma (MCL) is a heterogeneous disease still lacking reliable prognostic factors. A recurrent feature of this disease is however the t(11;14) translocation, yielding an increased production of Cyclin D1 through activation by the IGH promoter. In a previous study based on a controlled randomized trial (LyMA study, #113303; presented at ASH 2017 and currently submitted) we observed that large gains on chromosome 11, identified by single nucleotide polymorphisms (SNP), in a region always including the CCND1 locus encoding for cyclin D1, were related to a worse prognosis. In this series of 94 lymph node biopsies, this anomaly was observed in 7 patients who displayed a significantly worse outcome (p=0.004).

**Aims:** Here we examined a real-life cohort of MCL patients who benefited from fluorescence in situ hybridization (FISH), to assess whether chromosome 11 gains indeed impacted outcome.

**Methods:** All patients intensively treated between 1995 and 2018 with an assessment of CCND1 gains by FISH analysis were included. Fifty-one patients were consolidated with an autograft similar to that applied in the LyMA trial. Patients with FISH results suggestive of tetraploidy were considered separately. Outcomes were established as the delay between the initiation of the first line treatment and first relapse, death or last news.

**Results:** A cohort of 65 MCL patients was included with a median age of 58 years old (range 34-74) and a M/F ratio of 2:1. Seventeen patients displayed a significant signal suggesting CCND1 or fusion gain. FISH signals were interpreted as normal wild type chromosome, fusion signals and extraneous (supernumerary) signals. When considering patients with extra CCND1 or fusion signals, compared with patients with an expected classical translocation signal, a significantly worse outcome was observed in terms of progression free survival (PFS) with respective medians of 30 vs 116 months (HR=2.4[1.0-5.7];p=0.05). Moreover, when removing patients likely to present tetraploidy (i.e. double translocation load associated with whole genome increase), the significance of these gains reached a significance of p=0.02 (25 vs 116 months; HR=3.2[1.2-8.7]).

**Conclusions:** Although the t(11;14) translocation is an undisputed hallmark of MCL diagnosis, in an integrated approach of morphological and immunophenotypic features, closer examination of FISH signals could provide an additional prognosis factor, as suggested by SNP analysis but not systematically requiring it.

**Keywords:** B-cell lymphoma; fluorescence in situ hybridization (FISH); mantle cell lymphoma (MCL).

Y. Wang¹ | S. Yu¹ | C. Chuang² | M. Yao¹ |

T. Huang¹ | J. Tang²

¹Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; ²Hematology, Tai Cheng Stem Cell Therapy Center, Taipei, Taiwan, Republic of China

**Introduction and aim of study:** Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphoma, and the mechanism of its development is still not fully elucidated. Most of mantle cell lymphoma did not undergo germinal center reaction such as somatic hypermutation. Interestingly, prior immunoglobulin sequencing has noted that there are several stereotypes of V(D)J recombination, hypothesizing that these stereotypes might result from unknown biased selection. This study took advantage of next generation sequencing (NGS) to characterize the detail of recombined VDJ sequences in mantle cell lymphoma in comparison of other lymphoma subtypes.

**Patient and method:** In this single-institute retrospective study, 112 patients (male: 90 vs. female: 22) were diagnosed as having MCL between 2006 and 2018. The median age at diagnosis was 62 years old. The Ann Arbor staging at diagnosis was mostly stage 4 (81.1 %), and the blastoid variant comprised of 13.4 %. Among them, 15 patients' lymphoma specimens were analyzed through LymphoTrack® Dx IGH/IGK assay coupled with Illumina MiSeq sequencer to characterize the post-recombine immunoglobulin sequences. As control, 53 patients with other lymphoma subtypes including diffuse large B cell lymphoma, follicular lymphoma, MALToma, Hodgkin lymphoma and other lymphoproliferative disorders were analyzed with the same platform.

**Results:** In line with the prior notion that mantle cell lymphoma developed independent of somatic hypermutation in the germinal center, the mutation rate of V gene in mantle cell lymphoma is much lower than other lymphoma subtypes (median: 1.32% vs. 7.27%, p<0.001, Figure 1). VH3-21 was the most prominent stereotype in mantle cell lymphoma (53.3%). Through conceptual translation for the recombined VDJ sequences in three reading frames, an 87-amino-acid peptide domain was obtained via alignment at the carboxyl terminal for comparison. Interestingly, a 10-amino-acid motif, which is hydroxyl and amine group-rich, differed strikingly between MCL and other lymphoma subtypes. Seven out of 10 amino acids in this motif were significantly enriched in mantle cell lymphoma (Figure 2).

**Conclusion:** VDJ recombination of immunoglobulin heavy chain in mantle cell lymphoma of this study underwent the lower somatic hypermutation rate compared with other lymphoma subtypes. The predominant stereotype involved VH3-21. A hydroxyl- and amine-rich motif was identified as mantle cell lymphoma-specific peptide. Implications of this motif in three dimension and its relationship with potential antigens warrant further investigation.

**Keywords:** immunoglobulins (Ig); mantle cell lymphoma (MCL); molecular genetics.
<figure><img src="image_178.png" /><figcaption>Figure 2.</figcaption></figure>

<figure><img src="image_179.png" /></figure>

292
THE IMMUNE MICROENVIRONMENT AS A
PROGNOSTIC TOOL FOR MCL PATIENTS

J.M. Rodrigues<fnref n="1" /> | C. Freiburghaus<fnref n="1" /> | A. Carlsson<fnref n="2" /> |

S. Husby³ | C.W. Eskelund³ | L. Lokhande<fnref n="1" /> |

A. Kolstad⁴ | K. Grønbæk³ | M. Jerkeman⁵ | S. Ek<fnref n="1" />

**Introduction:** Breakthroughs in immunotherapy and targeted drugs have provided increased survival among patients with mantle cell lymphoma (MCL) but companion diagnostic tools remain to be developed.

Median overall survival (OS) was until recently 3-5 years but novel strategies such as the Nordic MCL2/MCL3 regiments combining high dose cytarabine with Rituximab and autologous stem cell transplantation (ASCT) have improved life-expectancy to more than 12 years for younger fit patients. Although promising, it is still not possible to stratify patients into different treatment protocols based on diagnostic markers. The molecular diversity has been explored but so far TP53 mutational status and Ki-67 expression are the only validated markers associated to poor treatment response. Based on the success of incorporating anti-CD20 antibodies and ASCT to MCL treatment, the role of an effective immune response has been pin-pointed. In this study, we aim to identify prognostic markers for patients treated within the Nordic MCL2/3 protocol with the long-term goal of developing validated predictive tools.

**Methods:** The data set combines clinicopathological information, TP53 mutational status and FFPE samples (total n=98;

<footnote n="1">¹Department of Immunotechnology, Lund University, Lund, Sweden;</footnote>

<footnote n="2">²Bionamic, Data Consulting, Lund, Sweden; ³Department of
Haematology, Rigshospitalet, Copenhagen, Denmark; ⁴Department of
Oncology, Division of Cancer Medicine, Oslo University Hospital, Oslo,
Norway; ⁵Department of Oncology, Lund University, Lund, Sweden</footnote>
Immunohistochemistry (IHC) n=52; Gene expression (GEX) n=72). The median follow-up time is 9 years. Tissue microarrays were stained with p53 and c-myc to distinguish classical MCL from more aggressive variants. Immune infiltration was accessed by staining with CD3, CD4, CD8, CD20, CD25, FoxP3 and PD1 antibodies. IHC results were digitally scored with HALO software (Indica Labs). Optimal cut-off points for immune cell numbers were selected using the maximally selected chi-square method (maxχ²). GEX was performed with Gene ST 1.0 arrays (Applied Biosystems) and analyzed with Qlucore Omics Explorer (Qlucore AB).

**Results:** Preliminary results showed that the percentage of CD4+ cells is negatively correlated with progression free survival (PFS) (p<0.05) in linear Cox model analysis. After dichotomization with maxχ² method, high infiltration of CD4, CD3 and CD8 cells was negatively correlated with PFS (p<0.05) and CD4, CD8 and FoxP3 high infiltration was associated to a worse OS. Efforts to expand the cohort by inclusion of material from additional MCL2/3 clinical sites are ongoing.

**Conclusions:** Immunotherapy is an effective therapy against many cancers and immune infiltration has been proved to be an important indicator of response to therapy. Efforts to validate these findings and develop companion diagnostic test are of major importance. Our findings show that MCL tumor immune-microenvironment, with particular emphasis on the CD4+ population, is prognostic for patients treated with combinatorial protocols, including anti-CD20 treatment and ASCT. Further studies to classify T cell subsets are warranted to further refine the understanding of the molecular events associated with response to therapy.

**Keywords:** immune system; immunochemotherapy; mantle cell lymphoma (MCL).

## 293
MUTATION OF PIM1 GENE IN PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA INHIBITS CELL DEATH THROUGH CHANGE IN SUBCELLULAR LOCALIZATION OF Pim-1 AND INCREASE OF BAD PHOSPHORYLATION

N. Sasaki<fnref n="1" /> | K. Ichimura² | M. Nagane³ | A. Tomiyama⁴

**Backgrounds:** In a study of Next Generation Sequencing in primary central nervous system lymphoma (PCNSL), we have previously reported several mutations of high frequency, in comparison with systemic diffuse large B cell lymphoma (DLBCL)s. Consequences of these specific mutations in PCNSL are unknown. In this study, we have analyzed the

functional consequence of mutations in the *PIM1* gene, observed in 100% of PCNSL patients, which encodes a serine/threonine kinase and is known to drive tumorigenesis in several malignancies.

**Methods:** Four most frequent mutations of *PIM1* in PCNSL, S77N, K115N, P216S, L275F, were chosen from our previous study, and each mutant was generated by site directed mutagenesis in *PIM1* cDNA cloned in an expression vector. Resulting vectors were transiently transfected into human cancer cell lines. Cell death of the cells expressing each mutant was evaluated by dye-exclusion method under treatment of chemotherapeutic agents. Alteration of molecular signaling was evaluated by immunoblotting.

**Results:** Among the four mutants, increased phosphorylation of BCL-2 associated death promoter (BAD) at Ser112, which is a phosphorylation target of Pim-1, was observed by expression of *K115N* mutant compared with wild type *PIM1* in Nagai and Hela cells expressing endogeneous BAD. Decreased cell death under camptothecin treatment was also observed in *K115N* mutant expressing Nagai cells compared with wild type *PIM1*-expressed cells. Moreover, we observed a significant shift in subcellular localization of Pim-1 carrying *K115N* mutant; from the nucleus, main sublocalization for wild type Pim-1, into the cytosol determined by immunocytochemistry and immunoblotting of nuclear and cytosolic fraction of the cells.

**Discussion:** It is suggested that *PIM1 K115N* mutant may drive chemoresistance through increased BAD phosphorylation that suppresses cell death compared with wild-type *PIM1* through modification of its subcellular localization.

**Keywords:** B-cell lymphoma; primary CNS lymphoma (PCNSL).

## 294
CLINICALLY INDOLENT PRIMARY HHV8-NEGATIVE EFFUSION-BASED LYMPHOMA IS CHARACTERIZED BY COMPLEX GENOMIC ALTERATIONS WITH AGGRESSIVE FEATURES

D. de Jong | M. Mendeville | M.G. Roemer | G.T. Los-de Vries | P. Stathi | N. Hijmering | B. Ylstra

Pathology, AmsterdamUMC/VUMC, Amsterdam, Netherlands

Human herpesvirus 8-negative effusion-based lymphoma (HHV8-negative EBL) is a distinct lymphoma entity related to fluid overload states caused by underlying medical conditions such as cardiac failure or renal insufficiency. Due to a phenotypical resemblance with secondary effusion of diffuse large B-cell lymphoma (DLBCL), HHV8-negative EBL is easily misdiagnosed. However, in contrast to DLBCL, HHV8-negative EBL follows a mild clinical course that is largely determined by comorbidity and high age at diagnosis, underpinning the importance to differentiate between the two entities. From the files of the Dept of Pathology, AmsterdamUMC/VUMC, we retrospectively identified 11 cases of HHV8-negative EBL. All patients presented with isolated pleural (n=10) or pericardial effusion (n=1).

<footnote n="1">¹Department of Neurosurgery, Kyorin University Graduate School of Medicine, Tokyo, Japan; ²Division of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan; ³Department of Neurosurgery, Kyorin University Faculty of Medicine, Tokyo, Japan; ⁴Department of Neurosurgery, National Defense Medical College, Saitama, Japan</footnote>
## HHV8-negative
Effusion Based Lymphoma

<figure><img src="image_180.png" /></figure>

<figure><img src="image_181.png" /></figure>

Age at presentation ranged from 60-92 years (median 85 years). All cases, except one, showed a mature B-cell immunophenotype (CD20, CD79a positive), one case showed plasmacytic differentiation (CD138 positive). All were EBER negative, 9/10 showed a non-GCB phenotype and 1/10 a GCB-phenotype (Hans/Tally alogorithms).

We performed extensive genomic profiling using a custom-made all-in-one assay (NimbleGen EZ SeqCap) to determine genome wide copy number alterations, mutations (369 genes) and translocations (12 target regions) on 8 HHV8-negative EBL cases. A mean number of 33.6 copy number gains and losses per case were found, with recurrent focal deletions of chromosome 3p14.2 and 6q21 in 6/8 cases, encompassing tumor suppressor genes *FHIT* and *PRDM1*, respectively. We observed a high rate of mutations across genes frequently involved in DLBCL, including patterns of somatic hypermutation (*PIM1*, *KLHL1*, *BCL2*). The most abundant hotspot mutation was found in *MYD88L265P* (3/8 cases). In addition, 5/8 cases had one or more translocations, involving *MYC* (n=2), *BCL2* (n=1), *BCL6* (n=2), *TP63* (n=1), *EXOC2* (n=1) and *KMT2D* (n=1), with Ig and non-Ig partners, of which one *MYC/BCL6* double hit context.

We conclude that HHV8-negative EBL is characterized by heterogeneous genomic features with frequent mutations, copy number aberrations and translocations, similar to aggressive B-cell lymphomas, but despite this follows an indolent clinical course.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); elderly; molecular genetics.

## 295
GENOTYPING PRIMARY MEDIASTINAL B-CELL LYMPHOMA (PMBCL) BY MEANS OF CIRCULATING TUMOR DNA ANALYSIS

A. Rivas-Delgado<fnref n="1" /> | F. Nadeu<fnref n="2" /> | A. Enjuanes<fnref n="2" /> |

L. Magnano³ | P. Mozas<fnref n="1" /> | M. Osuna³ | S. Martín³ |

T. Baumann<fnref n="1" /> | N. Castrejón de Anta⁴ | O. Balagué⁴ |

J. Delgado<fnref n="1" /> | N. Villamor³ | E. Campo⁴ | E. Giné<fnref n="1" /> |

A. López-Guillermo<fnref n="1" />

**Introduction:** Primary mediastinal B-cell lymphoma (PMBCL) is a rare subtype of non-Hodgkin lymphoma that predominantly occurs in adolescents and young adults. Biologically, PMBCL shares many similarities with classical Hodgkin lymphoma, including constitutive activation of the JAK-STAT and NF-kB pathways. High-throughput sequencing of circulating tumor DNA (ctDNA) in peripheral blood has emerged as a noninvasive approach in different types of neoplasms and it could be helpful in PMBCL when a biopsy specimen of the tumor is not available for molecular analysis. The aim of this study

<footnote n="1">¹Hematology Department, Hospital Clínic de Barcelona, Barcelona, Spain;</footnote>

<footnote n="2">²Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clínic, Barcelona, Spain; ³Hematopathology Unit, Department of Pathology, Hospital Clínic de Barcelona, Barcelona, Spain; ⁴Department of Pathology, Hospital Clínic de Barcelona, Barcelona, Spain</footnote>
<figure><img src="image_182.png" /></figure>

was to analyze the mutational profile at diagnosis and in the refractory/relapse settings in PMBCL patients using ctDNA and to compare, when available, with genotyping from tissue biopsy DNA.

**Methods:** We included 9 patients (M/F 4/5, median age 29 years) diagnosed with PMBCL in a single institution between 2015 and 2018 according to the WHO criteria. All patients were treated with chemoimmunotherapy. After initial treatment, 4 patients achieved complete remission (CR), 2 partial response (PR), 3 were refractory. Samples were obtained both at diagnosis and at progression. Tumor genomic DNA (gDNA) was isolated from formalin-fixed paraffin-embedded (FFPE) diagnostic tissue biopsies. A panel of 115 genes was amplified using a hybridization capture based protocol from 10-30 ng of ctDNA and 150 ng of gDNA (SureSelectXT-Agilent Technologies) and sequenced in a MiSeq instrument (Illumina).

**Results:** ctDNA was obtained from all patients at diagnosis and from 4/5 patients in PR or who were refractory after treatment. Median amount of ctDNA at diagnosis was 46 ng (range: 10-74 ng). In all the cases, at least one mutation could be detected, with a median of 10 mutations per sample (range 1-22 mutations). In 4 of the 5 cases with simultaneous tissue and peripheral blood samples, a similar number of mutations was detected with the same genes involved. The genes most frequently mutated at diagnosis were B2M, GNA13, SOCS1, STAT6, HIST1H1C, HIST1H1E,

MFHAS1, NFKBIE (figure). In 3 of the 4 cases with a ctDNA sample at diagnosis and at progression, changes in the allelic frequency of the mutations could be observed, whereas in the remaining case no mutation at progression could be detected. Of note, none of the 3 patients with NFKBIE mutation achieved a CR after first line treatment.

**Conclusions:** ctDNA could be used instead of tumor DNA to assess mutational landscape in patients with PMBCL.

**Keywords:** molecular genetics; primary mediastinal large B-cell lymphoma (PMLBCL).

296
ABNORMALITIES IN MICRORNA
EXPRESSION SIGNATURE CAN
CONTRIBUTE TO THE GENOME
INSTABILITY AND FAILURE OF DNA
REPAIR MECHANISMS IN CASE OF
DIFFUSE LARGE B-CELL LYMPHOMA

V. Halytskiy

Molecular Immunology Department, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kiev, Ukraine
**Introduction:** Genome instability and high mutation rate are typical features of diffuse large B-cell lymphoma (DLBCL), facilitating the rapid tumor evolution and acquisition of therapy resistance. This research aims to identify in what way the shifts in microRNA (miRNA) expression pattern can contribute to the DNA damage and genome instability.

**Methods:** MiRNA targets within gene transcripts were predicted in silico using the TargetScan software.

**Results:** MiRNAs miR-17, miR-18, miR-19, miR-20, miR-21, miR-29, miR-92, miR-106, miR-144, miR-146, miR-155, miR-221/222, miR-363, miR-500 and miR-574, hyperexpression of which is essential for DLBCL cells, can target transcripts of genes encoding many DNA repair enzymes as well as other DNA damage response proteins that are key elements of all DNA repair systems – base excision repair (UNG, SMUG1, MBD4, TDG, OGG1, NTHL1, NEIL1/2, APEX1, LIG3, APLF), direct reversal of damages (ALKBH3), repair of DNA-topoisomerase crosslinks (TDP1/2), mismatch excision repair (MSH2/3, MLH1/3), nucleotide excision repair (RAD23B, DDB1, RPA1/3, ERCC2, GTF2H1/2/3/5, CCNH, ERCC1/4/6/8, UVSSA), homologous recombination (RAD50/51/52, RAD51B/D, XRCC2, RAD54B, MRE11A, NBS1, RBBP8, EME1, GEN1) and non-homologous end-joining (XRCC6, PRKDC, LIG4, DCLRE1C, NHEJ1). Targets of the up-regulated miRNAs are also revealed in transcripts of genes encoding ATM kinase and Fanconi anemia proteins (FANCA/C/D2/E/F/G/I/M, BRCA2, BRIP1, RAD51C, BTBD12, FAAP20).

In addition, down-regulation of miRNA miR-150 can contribute to the aberrant reactivation and hyperexpression of AICDA gene, encoding cytidine deaminase AID, because its transcript carries targets of this miRNA. Also, down-regulation of anti-onco-miRNAs (e.g. miR-15/16, miR-34, miR-145 and miR-150) allows overexpression of genes encoding the factors NF-kB, SP1, HoxC4, TCF3, Stat6, Id2/3, which are AICDA transcription activators. On the contrary, tumors have indolent course if AID cannot be reactivated. For instance, MALToma cells hyperexpress miRNA miR-150, high conservative binding sites of which are revealed in transcript of AICDA gene. It can be assumed, that AID can cause even the target damage (endogenous gene knock-out) of tumor suppressor genes (incl. genes of anti-onco-miRNAs) in result of mutations in single-stranded R-loop that arises from DNA duplex during the transcription.

**Conclusions:** MiRNAs, hyperexpression of which is essential for abnormal proliferation and surviving of DLBCL cells, silence also genes encoding DNA repair enzymes as well as other key elements of the DNA damage response network. Therefore, shifts in miRNA signature can contribute to increase of genomic instability and mutation rate, leading on the whole to oncogene abnormalities and damage of tumor suppressor genes. This process underlies the tumor evolution as well as drug resistance acquisition.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); microRNA.

297
COMPARISON OF OUTCOMES BETWEEN
PATIENTS WITH MYC REARRANGED DLBCL

AND DOUBLE/TRIPLE HIT HIGH-GRADE B
CELL LYMPHOMA: A PAN-LONDON
RETROSPECTIVE REVIEW

D. El-Sharkawi<fnref n="1" /> | S. Sharma<fnref n="1" /> | L. Cook<fnref n="2" /> |
B. Hanley<fnref n="2" /> | R. Johnston<fnref n="3" /> | A. Arasaretnam<fnref n="3" /> |
I. Lazana<fnref n="4" /> | P. Greaves<fnref n="5" /> | A. Parkinson<fnref n="5" /> | Y. Peng<fnref n="6" /> |
S. Kassam<fnref n="4" /> | V. Peacock<fnref n="4" /> | R. Kaczmarski⁷ |
M. Bower⁸ | B. Cheung⁹ | C. De Lord¹⁰ |
M. Cross<fnref n="1" /> | K. Vroobel¹¹ | A. Wotherspoon¹¹ |
F. Aldridge¹² | J. Khwaja¹³ | B. Sharma¹⁴ |
K. Cwynarski¹³ | R. Pettengell<fnref n="6" /> | I. Chau¹⁵ |
D. Cunningham¹⁵ | K. Naresh¹⁶ | S. Iyengar<fnref n="1" />

**Introduction:** Diffuse large B cell lymphoma (DLBCL) is a clinically heterogeneous disease. Increasing emphasis is being placed on the prognostic impact of tumour biology to identify groups at highest risk of treatment failure. Chromosomal rearrangements enhancing the activity of the MYC proto-oncogene (MYCr) have been linked to adverse outcomes although some studies suggest that cases harbouring additional translocations affecting BCL2, BCL6 or both, have the worst outcomes. There is currently no consensus on the management of MYCr, 'double-hit' (DH) and 'triple-hit' (TH) cases. Retrospective data suggests that whilst treatment intensification is associated with an improved PFS, it does not translate to an improved overall survival (OS). Moreover recent evidence suggests that the MYC translocation partner may be important in prognostication. We aimed to capture the trends in current management of patients with these high risk alterations in a pan-London retrospective study.

**Methods:** DLBCL patients with MYCr, DH or TH were identified at 9 London centres. Demographics, cytogenetics, treatment and outcomes were obtained from patient records and anonymised data was submitted for analysis. OS was defined as survival from date of diagnosis until death, censored at last follow-up. Median OS and follow-up time were calculated using the Kaplan Meier (KM) and reverse KM

<footnote n="1">¹Haematology, Royal Marsden Hospital, Sutton, United Kingdom;</footnote>

<footnote n="2">²Haematology, Hammersmith Hospital, London, United Kingdom;</footnote>

<footnote n="3">³Haematology, Royal Sussex County Hospital, Brighton, United Kingdom;</footnote>

<footnote n="4">⁴Haematology, King's College Hospital, London, United Kingdom;</footnote>

<footnote n="5">⁵Haematology, Queen's Hospital, Romford, United Kingdom;</footnote>

<footnote n="6">⁶Haematology, St George's University Hospitals NHS Foundation Trust, London, United Kingdom; ⁷Haematology, Hillingdon Hospital, Uxbridge, London, United Kingdom; ⁸Haematology, Chelsea and Westminster Hospital, London, United Kingdom; ⁹Haematology, Croydon University Hospital, Croydon, United Kingdom; ¹⁰Haematology, St Helier Hospital, Carshalton, United Kingdom; ¹¹Histopathology, Royal Marsden Hospital, Sutton, United Kingdom; ¹²Clinical Cytogenetics, Royal Marsden Hospital, Sutton, United Kingdom; ¹³Haematology, University College Hospital, London, United Kingdom; ¹⁴Radiology, Royal Marsden Hospital, Sutton, United Kingdom; ¹⁵Department of Medicine, Royal Marsden Hospital, Sutton, United Kingdom; ¹⁶Histopathology, Hammersmith Hospital, Hammersmith, United Kingdom</footnote>
<figure><img src="image_183.png" /><figcaption>Figure</figcaption></figure>

method respectively. Log-rank test was used to assess differences in
median OS.

**Results:** 101 patients (pts) have been evaluated to date. MYC rearrangement was the sole abnormality in 51 cases. 34 had DH (22 with BCL2 and 12 BCL6 rearrangement) and 16 TH. Median age was 65 yrs (Range 18-93). IPI was evaluable in 95 cases (0-2 in 28 and 3-5 in 67 pts). There was no significant difference in median age or IPI comparing sole MYCr cases to DH/ TH cases. Of the 101 pts, 27 (17 MYCr, 10 DH/TH) received either R-CODOX M/ R-IVAC or DA R-EPOCH. A further 53 pts received R-CHOP or similar regimens with an equal split between MYCr and DH/TH cases. The remaining had alternative treatments and a minority received only palliation.
With a median follow-up of 25 months, the median OS for pts with MYCr was not reached (NR) versus 15 months for pts with DH/ TH, p= 0.2 (Figure 1a). In pts under 70 years, median OS for pts with DH/ TH (n=36) was 13 months versus NR in the MYCr pts (n=33), p=0.02 (Figure 1b). In these younger pts with DH/ TH OS was 13 months in pts who received R-CHOP like chemotherapy (n= 23) and 15 months in those who had R-CODOX M/ R-IVAC or DA R-EPOCH (n=10).

**Conclusions:** Patients with DH/TH lymphomas in our cohort had a dismal outcome irrespective of age and IPI emphasizing the need for newer treatment strategies. The data shows a trend towards better outcome in patients with sole MYCr compared to those with DH/TH. This difference is striking in patients under 70, with a significantly better outcome in favour of those with sole MYCr. Data collection is ongoing to investigate the outcomes according to MYC translocation partner.

**Keywords:** "double-hit" lymphomas; MYC.

D. Sermer¹ | S. Bobillo¹ | V. Seshan² | A. Dogan³ |

A. Younes¹

¹Medicine, Memorial Sloan-Kettering Cancer Center, New York, United States; ²Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, United States; ³Pathology, Memorial Sloan-Kettering Cancer Center, New York, United States

**Introduction:** Patients with DLBCL and "double hit" cytogenetics (MYC plus BCL2 and/or BCL6 translocations) have been shown to have inferior outcomes and appear to do better with first-line intensive chemotherapy regimens such as R-EPOCH compared to R-CHOP. In contrast, the clinical significance of EC of these genes is unknown. The current retrospective study investigates clinical characteristics and outcomes of DLBCL patients with 5 different cytogenetic profiles.

**Methods:** The study population consisted of adult patients diagnosed with DLBCL between 2001-2015 and available FISH reports, who were then categorized into the following groups: 1) MYC EC+ BCL2/6 EC (DEC), 2) MYC EC +BCL2/6 TL (EC), 3) MYC TL+BCL2/6 TL (DHL), 4) MYC TL+BCL2/6 EC (TL) and 5) Normal FISH (NL). To be eligible for inclusion, all were treated with rituximab and anthracycline-based chemotherapy and had at least 12 months of follow-up. Patients with a history of prior indolent lymphomas were excluded. EC was defined by ≥10% of nuclei in the FISH report. The primary objective was 2-year progression-free survival (PFS) and secondary objectives were complete remission rate (CRR), event-free survival (EFS) and overall survival (OS). In addition, outcomes based on type of induction (R-CHOP-based regimens vs intensive therapy) and number of ECs (1-2 versus ≥3) were analyzed (excluding NL).

**Results:** Out of 267 patients with available FISH reports, 98 were categorized into the 5 designated groups with 5-35 patients in each. Patients with DHL were most likely to have high IPI score, GCB-subtype, and be treated with intensive therapy. Relevant clinical outcomes are presented in Table 1. Across all groups, the CRR ranged from 74% (DHL) to 100% (EC, TL). Only DHL was found to be significantly associated with worse
<figure><img src="image_184.png" /><figcaption>PFS curves with X-axis representing time from treatment initiation (months). Fig 1A (Top): PFS of 5 cytogenetic groups of interest. Black line represents remaining 169 uncategorized patients as reference. Fig 1B (Bottom): PFS of 4 groups categorized by type of induction therapy and by DHL vs non-DHL status.</figcaption></figure>

CRR (p=0.031). 2-yr PFS ranged from 69% (DEC) to 100% (TL). There was no significant difference in PFS (p=1.0), OS (p=0.5), or EFS (p=0.9) between groups by log-rank analysis (Fig 1A). Use of intensive vs R-CHOP-based therapies resulted in 2-yr PFS of 77% vs 70% in the DHL group and 70% vs 81% in a pooled non-DHL group. However, differences in survival curves were not significantly significant in either DHL (PFS, p=0.9; OS, p=0.7; EFS, p=0.7) or non-DHL groups (PFS, p=0.2; OS,

p=0.09; EFS, p=0.3). (Fig 1B). Lastly, there was no association between number of ECs (1-2 versus ≥3) and outcomes.

**Conclusions:** A retrospective analysis of 5 different cytogenetic profiles of uncertain clinical significance in DLBCL only revealed an association between DHL and inferior CRR rate.

**Keywords:** "double-hit" lymphomas; cytogenetics; diffuse large B-cell lymphoma (DLBCL).

<table><caption>TABLE 1</caption><thead><tr><th></th><th>DEC</th><th>EC</th><th>DHL</th><th>TL</th><th>NL</th><th>p</th></tr></thead><tbody><tr><td>Total Number</td><td>13 (4.9%)</td><td>18 (6.7%)</td><td>27 (10%)</td><td>5 (1.8%)</td><td>35 (13%)</td><td></td></tr><tr><td>CR</td><td>11 (85%)</td><td>18 (100%)</td><td>20 (74%)</td><td>5 (100%)</td><td>29 (83%)</td><td>0.031</td></tr><tr><td>SD or PD</td><td>1 (7.7%)</td><td>0 (0%)</td><td>6 (22%)</td><td>0 (0%)</td><td>5 (14%)</td><td>0.007</td></tr><tr><td>Relapse</td><td>2 (15%)</td><td>3 (17%)</td><td>1 (3.7%)</td><td>1 (20%)</td><td>3 (8.6%)</td><td>NS</td></tr><tr><td>2-yr EFS</td><td>69%</td><td>82%</td><td>77%</td><td>100%</td><td>74%</td><td>NS</td></tr><tr><td>2-yr PFS</td><td>69%</td><td>78%</td><td>74%</td><td>100%</td><td>74%</td><td>NS</td></tr><tr><td>2-yr OS</td><td>77%</td><td>89%</td><td>78%</td><td>100%</td><td>79%</td><td>NS</td></tr></tbody></table>
299
CLINICAL, IMMUNOPHENOTYPIC AND
GENETIC CHARACTERISTICS OF
AGGRESSIVE (NON-BURKITT) B-CELL
LYMPHOMA IN A REAL LIFE COHORT

L. Roulin¹ | J. Jais² | E. Poullot³ | C. Robe³ |
E. Scherman⁴ | F. Lemonnier¹ | F. Le Bras¹ |
A. Maarek¹ | K. Belhadj¹ | J. Dupuis¹ |
M. Hammoud¹ | T. El Gnaoui¹ | S. Mule⁵ | E. Itti⁶ |
M. Delfau-Larue⁷ | P. Gaulard³ | C. Haioun¹ |
C. Copie-Bergman³

¹Lymphoid malignancies Unit, Henri Mondor Hospital, APHP, Créteil,
France; ²Biostatistics, Necker Hospital, APHP, Paris, France; ³Pathology
Department, Henri Mondor Hospital, APHP, Créteil, France;

⁴Haematology, Nimes Hospital, Nimes, France; ⁵Radiology Department,
Henri Mondor Hospital, APHP, Créteil, France; ⁶Nuclear Medicine
Department, Henri Mondor Hospital, APHP, Créteil, France;

⁷Immunobiology and Haematology Department, Henri Mondor Hospital,
APHP, Créteil, France

**Introduction:** Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease characterized by recurrent genetic alterations. Several adverse prognostic factors are well known as International Prognostic Index (IPI) parameters or MYCand BCL2 and/or BCL6 gene rearrangements (High-Grade B-cell Lymphoma double/triple hit, HGBL-DH/TH, according to the 2016 WHO classification). Other factors are controversial as previous history of indolent lymphoma, cell of origin (COO), co-expression of MYC and BCL2 proteins (double expressor (DE) status), MYC partner gene if rearranged. The aim of this study is to describe a « real life » cohort of patients treated in our institution for aggressive (non-Burkitt) B-cell lymphoma and to evaluate the prognostic impact of phenotypic and genetic alterations.

**Methods:** We collected clinical and histological characteristics of patients uniformly treated between January 2009 and June 2017 with rituximab R-CHOP/CHOP-like chemotherapy. Treatment was reinforced with high dose methotrexate in case of central nervous system (CNS) localization or HGBL subtype. All tumor samples were analysed for MYC and BCL2 protein expression by immunohistochemistry. BCL2, BCL6 and MYC break were analysed by Interphase Fluorescence In Situ Hybridization (FISH) with breakapart probe. When MYC was rearranged MYC partner gene was analysed with double fusion probes (MYC/IgH, MYC/IgL, MYC/IgK).

**Results:** 242 patients were studied. Baseline characteristics were: median age: 61 y (18-88), IPI 3-5: 49.6%, CNS localization: 6.6%. With a median follow-up of 4.4 years, 5y-OS and 5y-PFS were 72% (66.0-78.5) and 65% (58.5-71.3), respectively. Histological diagnoses were DLBCL-NOS: 57%, transformed DLBCL: 26%, HGBL: 7.9%, PMBL: 4.1% and others: 5%. Using Hans algorithm, 48.8% were classified as germinal center (GC), 47.9% as non-GC, 3.3% could not be

evaluated and 38% were DE. MYC rearrangement was detected in 36 cases (14.9%) with an immunoglobulin partner gene (IgH, IgL or IgK) in 24 (77.4%) of 31 evaluable cases. BCL2 and BCL6 rearrangement were observed in 45 cases (18.6%) and 62 (25.6%) cases respectively. Fifteen cases were HGBL DH/TH including 7 DH MYC/BCL2, 5 DH MYC/BCL6 and 3 TH MYC/BCL2/BCL6. Four cases were HGBL-NOS. IPI score 3-5 (p<0.0001) and HGBL (p=0.03) were significantly associated with inferior OS. IPI score 3-5 (p<0.0001), DE status (p=0.03), MYC-R (p=0.05) and BCL2-R (p=0.0014) were significantly associated with inferior PFS. Previous indolent lymphoma, COO and MYC partner gene had no impact on OS or PFS.

**Conclusions:** This monocentric study in a large cohort of aggressive B-cell lymphoma patients treated in real life conditions confirms previous reports on the negative prognostic impact of DE status, MYC and BCL2 translocations and HGBL subtype. These genetic and immunohistological characteristics should have implications in the design and interpretation of future clinical trials.

**Keywords:** "double-hit" lymphomas; diffuse large B-cell lymphoma (DLBCL); fluorescence in situ hybridization (FISH).

**Disclosures:** Belhadj, K: Honoraria: Amgen, Celgene, Takeda, Janssen China. Dupuis, J: Other Remuneration: Celgene (travel Grant). Gaulard, P: Research Funding: Takeda. Haioun, C: Consultant Advisory Role: Roche, Celgene, Janssen-Cilag, Gilead Sciences, Takeda; Honoraria: Novartis, Amgen, Servier-Pfizer, Gilead Sciences; Other Remuneration: Roche, Celgene, Amgen (Travel, Accommodations, Expenses).

300
REAL-WORLD PROGNOSTIC IMPACT OF
BCL2 AND MYC EXPRESSION AND
TRANSLOCATION AMONG DIFFUSE LARGE
B-CELL LYMPHOMA PATIENTS TREATED
WITH FIRST-LINE R-CHOP

M.K. Downer¹ | N. Chaudhary¹ | E. Szafer-Glusman² |
M. Breuleux³ | E.D. Hsi⁴ | J.M. Biondo⁵ |
A. Bazeos⁶ | Y. Jiang²

¹Personalized Healthcare Data Science, Genentech Inc., South San Francisco, United States; ²Oncology Biomarker Development, Genentech Inc., South San Francisco, United States; ³Global Product Development/Medical Affairs Oncology, F. Hoffmann-La Roche Ltd, Basel, Switzerland; ⁴Department of Laboratory Medicine, Cleveland Clinic, Cleveland, United States; ⁵US Medical Affairs, Genentech Inc., South San Francisco, United States; ⁶PD Clinical Science, Oncology, Roche Products Ltd, Welwyn Garden City, United Kingdom

**Introduction:** Patients with diffuse large B-cell lymphoma (DLBCL) and overexpression of both BCL2 and MYC proteins (double-expressor; DE) or translocations of both MYC and BCL2 genes (double-hit; DH) have inferior outcomes. Given heterogeneity of testing across laboratories, we investigated outcomes of DE and DH patients
<table><caption>**TABLE 1** Univariate Cox proportional-hazards model analyses comparing OS and TTNT among DLBCL patients treated with 1L R-CHOP according to BCL2 and MYC status</caption><thead><tr><th rowspan="2"></th><th rowspan="2">N</th><th colspan="3">OS</th><th colspan="3">TTNT</th></tr><tr><th>Deaths</th><th>HR (95% CI)</th><th>p</th><th>Events</th><th>HR (95% CI)</th><th>p</th></tr></thead><tbody><tr><td colspan="8"><strong>Assessed by FISH</strong></td></tr><tr><td colspan="8"><strong>DH Status</strong></td></tr><tr><td>Non-DH*</td><td>644</td><td>126</td><td>1.00 (ref)</td><td></td><td>186</td><td>1.00 (ref)</td><td></td></tr><tr><td>DH</td><td>44</td><td>18</td><td>3.22 (1.96, 5.29)</td><td>&lt;0.0001</td><td>23</td><td>2.75 (1.78, 4.24)</td><td>&lt;0.0001</td></tr><tr><td colspan="8"><strong>BCL2 Translocation</strong></td></tr><tr><td>Negative</td><td>570</td><td>114</td><td>1.00 (ref)</td><td></td><td>163</td><td>1.00 (ref)</td><td></td></tr><tr><td>Positive</td><td>198</td><td>42</td><td>1.13 (0.79, 1.61)</td><td>0.50</td><td>70</td><td>1.39 (1.05, 1.84)</td><td>0.02</td></tr><tr><td colspan="8"><strong>MYC Translocation</strong></td></tr><tr><td>Negative</td><td>761</td><td>136</td><td>1.00 (ref)</td><td></td><td>206</td><td>1.00 (ref)</td><td></td></tr><tr><td>Positive</td><td>101</td><td>41</td><td>3.19 (2.24, 4.55)</td><td>&lt;0.0001</td><td>52</td><td>2.74 (2.02, 3.72)</td><td>&lt;0.0001</td></tr><tr><td colspan="8"><strong>Assessed by IHC</strong></td></tr><tr><td colspan="8"><strong>DE Status</strong></td></tr><tr><td>Non-DE†</td><td>251</td><td>33</td><td>1.00 (ref)</td><td></td><td>50</td><td>1.00 (ref)</td><td></td></tr><tr><td>DE</td><td>232</td><td>46</td><td>1.51 (0.96, 2.36)</td><td>0.07</td><td>79</td><td>1.87 (1.31, 2.66)</td><td>0.001</td></tr><tr><td colspan="8"><strong>BCL2 Expression</strong></td></tr><tr><td>Negative</td><td>396</td><td>70</td><td>1.00 (ref)</td><td></td><td>83</td><td>1.00 (ref)</td><td></td></tr><tr><td>Positive</td><td>1429</td><td>317</td><td>1.33 (1.03, 1.72)</td><td>0.03</td><td>466</td><td>1.71 (2.16, 1.36)</td><td>&lt;0.0001</td></tr><tr><td colspan="8"><strong>MYC Expression</strong></td></tr><tr><td>Negative</td><td>203</td><td>28</td><td>1.00 (ref)</td><td></td><td>43</td><td>1.00 (ref)</td><td></td></tr><tr><td>Positive</td><td>317</td><td>55</td><td>1.23 (0.78, 1.94)</td><td>0.38</td><td>95</td><td>1.48 (1.03, 2.12)</td><td>0.03</td></tr></tbody></table>

tested in the real world and treated with first-line (1L) standard-of-care immuno-chemotherapy: rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). If outcomes reflect those in large multi-center trials by central testing, local laboratories may provide reliable and rapid diagnosis, and allow faster trial enrollment and treatment.

**Methods:** We identified DLBCL patients treated with 1L R-CHOP in the Flatiron Health electronic health record (EHR)-derived database with BCL2/MYC expression by immunohistochemistry (IHC) and cytogenetic data by fluorescence in situ hybridization (FISH), based on values extracted by Flatiron Health (2011–18). Due to nonstandard reporting, no standard cutoff was used to assign IHC positivity. Univariate Cox proportional-hazards models estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS) and time to next treatment (TTNT).

**Results:** Of 483 patients with both BCL2 and MYC expression available, 232 (48%) were DE. Of 688 patients with both BCL2 and MYC translocation available, 44 (6%) were DH, similar to prior findings. Inferior OS and TTNT were observed among DH and DE patients, and those with high BCL2 expression and MYC translocation. Inferior TTNT was observed among patients with high MYC expression and BCL2 translocation. Multivariate analyses will be presented.

**Conclusions:** BCL2 expression and MYC translocation had strong prognostic ability in this population. Despite missing data inherent to EHRs, and varied testing methods used in practice, DE and DH patients had worse prognosis than non-DE and non-DH patients, comparable with results from single-center trials. This demonstrates a robustness of BCL2 and MYC biomarkers to identify high-risk 1L DLBCL patients. Improving technical, interpretive, and reporting accuracy would further strengthen confidence in these markers.

**Acknowledgments:** This study was funded by F. Hoffmann-La Roche–Genentech. Third party editorial assistance was provided, funded by F. Hoffmann-La Roche.

This abstract has been previously submitted to EHA 2019.

**Keywords:** BCL2; diffuse large B-cell lymphoma (DLBCL); MYC.

**Disclosures:** Downer, M: Employment Leadership Position: Genentech; Stock Ownership: Genentech; Other Remuneration: Travel, Accommodations, expenses: Genentech. Chaudhary, N: Employment Leadership Position: Genentech; Stock Ownership: Genentech. Szafer-Glusman, E: Employment Leadership Position: Genentech; Stock Ownership: Roche. Breuleux, M: Employment Leadership Position: Roche; Stock Ownership: Roche, Gilead. Hsi, E: Employment Leadership Position: Cleveland Clinic; Consultant Advisory Role: Celgene, Jazz, Seattle Genetics; Research Funding: Abbvie, Eli Lilly. Biondo, J: Employment

<footnote>*Non-DH includes those who were either BCL2+/MYC-, BCL2-/MYC+, or BCL2-/MYC-, tested via FISH. Those who were unknown for either BCL2 or MYC translocation were not included in the DH and non-DH classification.</footnote>

<footnote>†Non-DE includes those who were either BCL2+/MYC-, BCL2-/MYC+, or BCL2-/MYC-, tested via IHC. Those who were unknown for either BCL2 or MYC expression were not included in the DE and non-DE classification.</footnote>
**Leadership Position:** Genentech; Stock Ownership: Genentech. Bazeos, A: Employment Leadership Position: Roche; Stock Ownership: Roche. Jiang, Y: Employment Leadership Position: Genentech; Stock Ownership: Genentech.

## 301 DIFFUSE LARGE B-CELL LYMPHOMA SURVIVAL PROGNOSTICATION, A COMPARATIVE ANALYSIS OF CELL OF ORIGIN VS. MYC/BCL2 EXPRESSION

M. Rodriguez¹ | I. Fernandez-Miranda² | R. Mondejar³ |
J. Capote⁴ | S. Rodriguez-Pinilla¹ | L. Cereceda¹ |
R. Alonso¹ | R. Cordoba⁵ | M. Provencio² |
P. Martin-Acosta⁶ | A. Sanchez² | L. Pedrosa² |
S. Gómez² | M. Piris-Villaespesa⁷ | M. Garcia-Cosio⁷ |
C. Quero⁸ | M. Llanos⁹ | C. Barcena¹⁰ |
M. Fraga¹¹ | F. Camacho¹² | Y. Castro¹³ |
J. Garcia¹⁴ | M. Mollejo¹⁵ | F. Climent¹⁶ |
E. Mayordomo¹⁷ | E. Bacalari¹⁸ | G. Olmedilla¹⁸ |
M. Sánchez-Beato² | M. Piris¹

¹Pathology, Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Investigación Fundación Jiménez Díaz, Madrid, Spain; ²Medical Oncology, Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana, Madrid, Spain; ³Haematology, Hospital Universitario Virgen del Rocío, Sevilla, Spain; ⁴Medical Oncology, Instituto de Investigación Fundación Jiménez Díaz, Madrid, Spain; ⁵Haematology, Instituto de Investigación Fundación Jiménez Díaz, Madrid, Spain; ⁶Pathology, Instituto Investigación Sanitaria Puerta de Hierro-Segovia de Arana, Madrid, Spain; ⁷Haematology, Instituto de Investigación Hospital Ramón y Cajal, Madrid, Spain; ⁸Medical Oncology, Hospital Universitario Virgen de la Victoria, Málaga, Spain; ⁹Medical Oncology, Hospital Universitario de Canarias, Santa Cruz de Tenerife, Spain; ¹⁰Pathology, Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain; ¹¹Pathology, Hospital Clínico Universitario de Santiago-CHUS, Santiago de Compostela, A Coruña, Spain; ¹²Pathology, Hospital Universitario de Getafe, Madrid, Spain; ¹³Pathology, Hospital Universitario Príncipe de Asturias, Madrid, Spain; ¹⁴Pathology, Hospital MD Anderson Cancer Center, Madrid, Spain; ¹⁵Pathology, Hospital Virgen de la Salud, Toledo, Spain; ¹⁶Pathology, Hospital Universitario de Bellvitge, Barcelona, Spain; ¹⁷Pathology, Hospital Universitario y Politécnico de La Fe, Valencia, Spain; ¹⁸Pathology, Hospital Universitario La Paz, Madrid, Spain

**Introduction:** Multiple conflicting prognostic markers have been proposed for R-CHOP treated diffuse large B-cell lymphoma (DLBCL) cases. Aggressive behaviour has been shown to be associated with ABC-like phenotype, Double Hit (DH) or Double Expression (DE) phenotype, CD5 expression and other molecular or immunohistochemical markers.

**Methods:** Here we have analyzed the survival probability in a series of chemoimmunotherapy-treated 100 DLBCL cases comparing COO

vs. MYC/BCL2 gene expression vs. the expression of other prognostic markers such as CD30, PDL1, CD5, p53 and Ki67, using a NanoString custom assay that includes COO and other 7 genes. Data have been also correlated with immunohistochemical and other cytogenetic studies. Univariable and multivariable Cox proportional hazard regression models were used to evaluate the proposed prognostic factors. For Kaplan Meier analysis the continuous series were divided taken into account the median value and quartiles of the series.

**Results:** Double Expression (DE) (MYC/BCL2) was found in 26 cases and related with a shorter time to progression (TTP) (HR: 1.95, P<0.05). COO analysis identified 53 cases as GC-type; 27 as ABC-type and 19 unclassified. ABC-phenotype was associated with shorter overall survival (OS) (HR:2.5; p<0.05).

Association between ABC and DE identified 11 cases with double expression and ABC-phenotype, with a HR: 2.82, n<0.05.

Clinical variables integrated into the IPI were significantly associated with both TTP and OS.

All the three parameters were basically independent prognostic markers. When associated, multivariate analysis allowed to integrate IPI, DE and COO into a single model, identifying an integrated risk score for each sample that stratifies the series into four groups from risk quartiles (0.50, 0.74 and 1.56) with very strong differences in TTP and DSS. The relapse probabilities at 36 months for the risk groups created were 0.90, 0.67, 0.43 and 0.35 from down risk to high risk, while the OS probabilities at 36 months were 0.96, 0.71, 0.64 and 0.52.

**Conclusions:** DE (MYC/BCL2), COO and IPI are independent prognostic markers in CHOP-R treated DLBCL cases. An integrated risk score identified quartiles with significant differences in OS, TTP and DSS, and could eventually be used for selecting risk-stratified therapeutic strategies.

**Keywords:** "double-hit" lymphomas; activated B-cell-like (ABC); diffuse large B-cell lymphoma (DLBCL).

## 302 A NEW PROPOSAL FOR IDENTIFICATION OF DOUBLE-HIT DIFFUSE LARGE B-CELL LYMPHOMA BASED ON THE PREDOMINANCE OF DOUBLE HITS INVOLVING BCL6 REARRANGEMENT IN TAIWAN

Y. Su¹ | C. Lee² | S. Hsieh³ | M. Liu³ |
C. Fang⁴ | C. Chen⁵ | W. Lee³ | Y. Yen⁶ |
J. Whang-peng⁷ | T. Chao¹

¹Hematology/oncology, Shuang-Ho Hospital, New Taipei, Taiwan (Republic of China); ²Anatomic Pathology, Taipei Institute of Pathology, Taipei, Taiwan (Republic of China); ³Pathology, Shuang-Ho Hospital, New Taipei, Taiwan (Republic of China); ⁴Pathology, Wan-Fang Hospital, Taipei, Taiwan (Republic of China); ⁵Pathology, Taipei Medical University Hospital, Taipei, Taiwan (Republic of China); ⁶The Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan (Republic of China)
<table><caption>Table 1</caption><thead><tr><th rowspan="2">No.</th><th colspan="3">FISH</th><th colspan="4">IHC</th><th rowspan="2">Phenotype</th></tr><tr><th>MYC</th><th>BCL2</th><th>BCL6</th><th>MYC</th><th>BCL2</th><th>BCL4</th><th>CD10</th></tr></thead><tbody><tr><td>1.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>2.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>3.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>non-GCB</td></tr><tr><td>4.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>5.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>6.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>7.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>8.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>non-GCB</td></tr><tr><td>9.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>non-GCB</td></tr><tr><td>10.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>GCB</td></tr><tr><td>11.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>non-GCB</td></tr><tr><td>12.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>non-GCB</td></tr></tbody></table>

<figure><img src="image_185.png" /><figcaption>Figure 1</figcaption></figure>

<figure><img src="image_186.png" /><figcaption>Figure 2</figcaption></figure>

China); ⁷Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan (Republic of China)

**Background:** Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of the non-Hodgkin's lymphoma all over the world and it is extremely heterogeneous both clinically and pathologically. Concurrent MYC and BCL2/BCL6 translocation, the so-called double hits (DH) has been identified as an important reverse prognostic factor in DLBCL. For BCL2-rearranged DH DLBCL, it has been believed that double expression of Myc and BCL2 detected by the immunohistochemical(IHC) stains with a GCB phenotype is the most important surrogate to screen DH. Chang and his colleagues have published a manuscript to describe the immunophenotypic and genetic characteristics of DLBCL in Taiwan in 2016. In their study, BCL6, rather than BCL2, rearrangement seemed predominant in DH DLBCL although only 3 patients were identified. BCL6-rearranged DH

DLBCL has been less discussed because of its minority in the Western countries. The frequency and predominant rearrangement type of DH DLBCL in Taiwan should be explored.

**Aims:** We aim to establish the predominance of BCL6 rearrangement in DH DLBCL and figure out the phenotypes and the status of Myc, BCL2 and BCL6 expression in DH DLBCL in Taiwan. Finally, we correlate DH with their IHC features to propose a flow chart to screen DH DLBCL.

**Results:** Among the 154 patients, 25 of them had MYC translocation (16.2%). Of the 25 MYC-rearranged patients, concurrent BCL2 or BCL6 translocation were detected in 12 patients. Two-thirds of them (8 patients) had simultaneous MYC and BCL6 translocation while the other 3 were MYC- and BCL2-rearranged (Figure 1). We had one patient with concurrent MYC, BCL2 and BCL6 translocation, the so-called triple hits (TH). Taken together, 75% of DH (including TH) DLBCL patients had BCL6 rearrangement and therefore we confirmed the predominance of BCL6 rearrangement in Taiwan. Among
DH (including TH) DLBCL patients, one did not have enough residual tissue to confirm his phenotype and consequently, only 11 can be further analyzed. Only 6 out of 11 (54.5%) has Myc overexpression in the IHC stains. Among BCL6-rearranged DH (including TH) DLBCL patients, only 3 out of 9 (33.3%) has concurrent over-expression of Myc and BCL6 in the IHC stains (Table 1) and consequently, simultaneous overexpression of Myc and BCL6 proteins was not a good surrogate to screen BCL6-rearranged DH DLBCL. Similarly, only one-third has concurrent overexpression of Myc and BCL2 proteins in the BCL2-rearranged counterpart. Five patients (55.6%) were non-germinal center B-cell (non-GCB) phenotype in 9 BCL6-rearranged DH(including TH) patients, indicating the screening of DH with BCL6 rearrangement should include the non-GCB phenotype of DLBCL.

As a result, we propose an IHC flow chart to screen DH for GCB and non-GCB DLBCL, respectively. For GCB DLBCL, we would like to test MUM1 first. If it is positive, we will do MYC FISH directly. If not, further BCL6 IHC stain is mandatory. For patients with positive BCL6, MYC FISH will be done to screen DH (Figure 2). Among non-GCB DLBCL, MUM1 test is still our first step. For MUM1-positive non-GCB DLBCL, Myc IHC stain is the next gate keeper. If Myc IHC is positive, we will do MYCFISH directly. If not, FISH will only be done in patients with positive either BCL2 or BCL6 protein (Figure 3). According to the flow charts, we can find 10 patients with DH DLBCL and the detection rate is 90.9%. Furthermore, we can detect DH DLCBL with either BCL2 or BCL6 rearrangement presented with either GCB or non-GCB phenotype.

**Conclusions:** 1. The majority (75%) of DH (including TH) DLBCL in Taiwan had BCL6 rearrangement and it was quite different from data of the Western countries.

2. More than half (55.6%) of BCL6-rearranged DH (including TH) DLBCL were non-GCB phenotype, indicating the screening of DH with BCL6 rearrangement should include the non-GCB phenotype of DLBCL in Taiwan.

3. The screening of BCL6-rearranged DH DLBCL was mandatory in Taiwan.

4. According to our flow charts, we can detect DH DLCBL with either BCL2 or BCL6 rearrangement, presented with either GCB or non-GCB phenotype with a detection rate of 90.9%.

**Keywords:** BCL2; BCL6; MYC.

303
OUTCOME OF PATIENTS WITH C-MYC REARRANGED DIFFUSE LARGE B CELL LYMPHOMA ASSOCIATED OR NOT WITH BCL2 AND/OR BCL6 REARRANGEMENT: A MULTICENTRIC AND RETROSPECTIVE STUDY

S. Favre¹ | C. Botella-Garcia¹ | F. Bijou² |

B. Deau-Fisher³ | A. Banos⁴ | P. Rispal⁵ |

A. Saint-Lézer⁶ | O. Fitoussi⁷ | F. Liffermann⁸ |

G. Labouré⁹ | C. Dagada¹⁰ | N. Milpied¹ |

K. Bouabdallah¹

¹Hematology, University Hospital of Bordeaux, Pessac, France;

²Hematology, Bergonié Institute, Bordeaux, France; ³Hematology, Hôpital Cochin, APHP, Paris, France; ⁴Hematology, Centre Hospitalier de la Côte Basque, Bayonne, France; ⁵Medecine, Centre Hospitalier Agen-Nérac, Agen, France; ⁶Medecine, Centre Hospitalier de Mont-de-Marsan, Mont-de-Marsan, France; ⁷Hematology, Polyclinique Bordeaux Nord Aquitaine, Bordeaux, France; ⁸Medecine, Centre Hospitalier de Dax, Dax, France;

⁹Hematology, Centre Hospitalier de Libourne, Libourne, France;

¹⁰Medecine, Centre hospitalier de Pau, Pau, France

**Introduction:** Myc rearrangement plays an important role in diffuse large B cell lymphoma (DLBCL). It is often associated with Bcl2 and/or Bcl6 rearrangements, known as double or triple hit lymphoma (DH/TH) and seems to have a bad outcome after standard immunochemotherapy treatments. Some more intensive regimens have been associated with better outcomes, but to date, there is no consensus for a standard of care. We evaluated the impact of first-line treatment on the outcome of newly diagnosed DLBCL patients with C-myc alone or in combination with Bcl2 and/or Bcl6 rearrangements.

**Methods:** It is a retrospective and multicenter study of C-myc rearranged DLBCL patients who were treated between 2012 and 2017 in 3 European centers. Patients received different induction regimens with or without front-line transplant (auto or allo SCT). Clinical and biological characteristics of the patients were analyzed, as well as the type of chemotherapy, the response rate and the survival. All the patients had a PET-CT at baseline and for response evaluation.

**Results:** Sixty-three patients were included. Median age was 60 (26,8 - 86) with 71% of male. All patients had a DLBCL with C-myc rearrangement by FISH. C-myc was associated with Bcl2 (23 patients (38%)), Bcl6 (13 pts (21%) or both (6 pts (10%). AA stage was ≥ 3 in 56 patients and 46/58 patients had an IPI ≥ 2. Chemotherapy regimens consisted in R-CHOP (47 pts), R-Da-EPOCH or Burkitt-like regimens (16 pts). 8 pts received an auto-SCT and 2 pts an alloSCT . The overall response rate (complete and partial response) after front line therapy was 60,6%, (CR=52,4%). Twenty-four pts had a refractory disease. After a median follow-up of 26.3 months (0.3-68.5) the progression free survival and overall survival were 48% and 63% respectively. PFS and OS were 32% / 53%, 59% / 70%, and 60% / 60% for patients with isolated C-myc, DH and TH respectively. After R-CHOP, PFS and OS were 46% and 61,9% respectively. After intensive chemotherapy and SCT, PFS and OS were 60% and 66,7% respectively.

**Conclusion:** In this retrospective but multicenter study, we confirm that DLBCL pts with C-myc alone or associated with Bcl2 and/or Bcl6 rearrangement have a poor outcome after R-CHOP. In the absence of a randomized trial with a large number of patients, it is difficult to conclude on the best chemotherapy regimen.
**Keywords:** "double-hit" lymphomas; diffuse large B-cell lymphoma (DLBCL); MYC.

304
DIGITAL SPATIAL PROFILING OF IMMUNE MARKERS IN R-CHOP TREATED DIFFUSE LARGE B-CELL LYMPHOMA REVEALS A DOMINANT PROGNOSTIC SIGNIFICANCE OF M2 MACROPHAGE INFILTRATION

A.D. Jeyasekharan<fnref n="1" />

M.M. Hoppe<fnref n="1" />

S. Fan²

A. VanSchoiack³

L. Xin⁴

S. De Mel⁴

M. Poon⁴

E. Chan⁴

J. Lee⁴

Y.L. Chee⁴

W.J. Chng⁴

S.B. Ng²

**Introduction:** Understanding the relative prognostic significance of distinct immune infiltrates in the micro-environment of Diffuse Large B-Cell Lymphoma (DLBCL) is essential to appropriately develop

immunotherapy for front line treatment. Traditional immunohisto-
chemical approaches are limited by the number of markers that can
be simultaneously assessed within formalin-fixed paraffin embedded
(FFPE) samples of DLBCL. In this study, we have used a novel
technology- Digital Spatial Profiling (DSP) by Nanostring, to study the
immune contexture of DLBCL samples through a customized panel of
36 antibodies to markers of immunological significance covering sub-
populations of lymphocytes, macrophages and key immune
checkpoints.

**Methods:** 81 cases of DLBCL, treated with R-CHOP chemo-immunotherapy and with at least 3 months of follow up were included in this study. The samples were arrayed on a Tissue Microarray (TMA). The antibodies in the panel were conjugated to proprietary oligonucleotide sequences (barcodes), and simultaneously applied to a single TMA slide. Regions of interest (ROI) were selected using a PAX5/CD20 fluorescent counterstain. Barcoded antibodies were digitally quantified, and normalized for area of the ROI. The prognostic significance of each immunological marker for Progression Free Survival (PFS) and Overall Survival (OS) was then evaluated using both univariate and multivariate models. The TMA was also evaluated for PDL1 expression using quantitative immunofluorescence (qIF) (OPAL-Vectra), which was compared to PDL1 estimation in the Nanostring immune panel.

<figure><img src="image_187.png" /></figure>

<figure><img src="image_188.png" /></figure>

<figure><img src="image_189.png" /><figcaption>Figure 1. A, Kaplan-Meier plots for PFS stratified according to median PDL1 DSP score. B, OS analysis according to CD163 DSP score. C, OS analysis according to CD68 DSP score. Log-rank test.</figcaption></figure>

<footnote n="1">¹Cancer Science Institute, National University of Singapore, Singapore, Singapore; ²Pathology, National University of Singapore, Singapore, Singapore; ³Digital Pathology, Nanostring Technologies Inc, Seattle, WA, United States; ⁴Haematology-Oncology, National University Hospital Singapore, Singapore, Singapore</footnote>
**Results:** PDL1 estimation by the qIF assay and the DSP assay showed concordance (r=0.50, p< 0.001) and PDL1 DSP score was a statistically significant predictive marker for PFS in multivariate analysis (HR 2.6, 95% CI 1.1-6.5, p=0.035)- Figure 1A. Among the markers in the Nanostring immune panel, tumour infiltration by M2 macrophages (CD163 and CD68) showed the highest negative prognostic value for OS and was independently statistically significant on multivariate analysis that included IPI score and cell-of-origin data (CD163 HR 3.5, 95% CI 1.1-10.8, p=0.029; CD68 HR 4.4, 95% CI 1.4-13.6, p=0.012)- Figure 1B/C. Other markers including those of T-cell infiltration and non-PDL1 immune checkpoints on tumour cells did not approach statistical significance.

**Conclusion:** Highly multiplexed immune profiling demonstrates the relative significance of PDL1 expression and M2 macrophage infiltration towards survival of R-CHOP treated DLBCL. These findings suggest possible value in strategies incorporating PDL1 inhibition with

therapeutics targeting tumour promoting macrophages, to enhance first line responses to R-CHOP in DLBCL. The Nanostring DSP assay is an FFPE-compatible method for immune profiling in DLBCL, of potential applicability to the analysis of clinical trial samples.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); immunophenotype; macrophages.

**Disclosures:** Jeyasekharan, A: Honoraria: AstraZeneca, MSD; Research Funding: Janssen; Other Remuneration: Travel funding- Perkin Elmer.

305
A2aR AS ONE OF NOVEL IMMUNE
CHECKPOINTS, AND TUMOR-
INFILTRATING LYMPHOCYTES IN DIFFUSE
LARGE B-CELL LYMPHOMA

<figure><img src="image_190.png" /><figcaption>**Figure 1.** Localization of CD73 on tumor cells, A2aR+ TILs and CD8+ TILs, and their associations with survival in DLBCL.</figcaption></figure>
X. Wang¹ | T. Zhang¹ | B. Meng² | Q. Zhai² |

X. Ren³ | L. Li¹ | P. Wang⁴ | H. Zhang¹

¹Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; ²Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; ³Department of Immunology/Biotherapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; ⁴Department of Radiation Oncology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China

**Introduction:** CD73/A2a adenosine receptor (A2aR) adenosine signaling has been identified as one of the most attractive immunosuppressive pathways, as it dampens T cell-mediated immune responses and promotes tumor immune escape. Thus, A2aR is a novel immune checkpoint. Several anti-CD73 or anti-A2aR antibodies are being evaluated in different types of malignancies in clinical trials. However, the expression characteristics and clinical significance of the CD73/A2aR adenosine immunosuppressive axis, as well as tumor-infiltrating lymphocytes (TILs) in DLBCL remain unclear.

**Methods:** Multiplexed immunofluorescence staining, and a professionally and automatically assessed computer-assisted platform were applied to localize and quantify the different markers from the DLBCL tumor and microenvironment cells. Gene expression status was also analyzed according to the microarray data. The associations among marker expression patterns or their correlations with clinicopathological characteristics were estimated with the χ² test and two-tailed Spearman analyses.

**Results:** CD73 expression on DLBCL tumor cells, rather than the total protein and gene levels of CD73, was associated with survival of DLBCL patients. Patients with CD73⁺/Pax-5⁺ (median survival, 57.8 months; 95% CI, 46.4-69.3) experienced significantly poorer outcomes than those with CD73⁻/Pax-5⁺ (median survival, 73.5 months; 95% CI, 65.9-81.2). Additionally, A2aR expression on both total TILs and CD8⁺ TILs was correlated with survival of DLBCL patients. Patients with A2aR⁺ TILs (median survival, 53.3 months; 95% CI, 40.6-66.0) had a significantly shorter survival time than patients with A2aR⁻ TILs (median survival, 74.5 months; 95% CI, 67.5-81.5). Spearman's rank test showed that CD73 expression on DLBCL tumor cells was positively correlated with A2aR expression on TILs (R=0.395, p = 0.001). We further found that DLBCL patients could be more precisely stratified through the combination of CD73 tumor cell expression and A2aR TILs expression, and patients with CD73⁺/Pax-5⁺ and A2aR⁺ TILs experienced the worst outcome. We also revealed that DLBCL patients with CD73⁺/Pax-5⁺ and low CD8⁺ TILs or low absolute lymphocyte counts had unfavorable outcomes.

**Conclusions:** Our findings uncovered that DLBCL patients with CD73⁺ on tumor cells as well as A2aR⁺ on TILs or low CD8⁺ TILs exhibited inferior survival, supporting potential combination strategies using CD73/A2aR immunosuppressive blockades as treatment options for DLBCL patients.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); immunosuppression.

306
MORPHOLOGICAL EVALUATION OF THE
IMMUNE PROFILE IN A SERIES OF DIFFUSE
LARGE B CELLS LYMPHOMA IN TISSUE
MICROARRAYS: PRELIMINARY RESULTS

P. Cervera | M. Dupeux | D. Tran | A. Scriva |

S. Gimenez de Mestral | B. Fabiani | P. Coppo

75, APHP, Sorbonne University, Paris, France

**Introduction:** Immunotherapy with checkpoint inhibitors is emerging as a promising new option of antitumor treatment in solid tumor but also in lymphomas. The tumoral microenvironment is regarded as playing a prognostic role and may be correlated to the good therapeutic response to checkpoint inhibitors in Hodgkin lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. 60% of DLBCL patients are cured using standard chemotherapy (R-CHOP). However, 30-40% of DLBCL patients will develop relapse or have refractory disease that cannot be cured with the standard R-CHOP therapy or will suffer of adverse and toxic chemotherapy effects, indicating the need for more effective therapies for patient subsets. The establishment of predictors of treatment response and the understanding of the immunomodulation have become a priority. Therefore, the purpose of this study is to assess the morphological immune profile by density of known immune key players, taking into account the immune checkpoints.

**Methods:** A series of 189 consecutive DLBCL cases (68 female age from 20 to 94 years old with a median of 76 years old and 121 male, age from 20 to 92 years old, with a median of 66 years old, with biopsies carried out between 2010 and 2016 is investigated. Tissue microarray (TMA) samples have been collected in parallel embedded tumor tissue. They were immunostained with cytotoxic and immune modulators antibodies, first of all with CD8 and PDL1, antibodies. A software dedicated to TMA morphometric analysis with virtual slides ("ExploraNova"; La Rochelle, France) already developed as been used, allowing to study the density of the immunostained cells.

**Results:** this study evidence an inverse correlation between the density of CD8 immunostained cells and the age, (Pearson: r= -0,23; p: 0,01). There was a non significant tendance of correlation between the age, and the density of PDL1 heavily immunostained lymphocytes cells (Pearson, r= -0,16, p: 0,053). But no significant correlation was found between age and the density or relative area of faint PDL1 immunostained non lymphocytes mononuclear cells (Pearson, r= 0,12, p: 0,147 and r= -0,05, p: 0,536, respectively).

**Conclusions:** In accordance to the progressive alterations to the immune system associated with increased mortality in the very elderly, we demonstrated the loss of cytotoxic response in the
<figure><img src="image_191.png" /></figure>

microenvironment of DLBCL, related to the age. The blockade of PDL1 immunecheckpoint seems less clearly correlated to age, according to the cells on which it is expressed.

These preliminary results evidenced the variable spectrum of the immune response in DLBCL and the need to investigate thoroughly the different parameters of the microenvironnement according to clinical, morphological and biological parameters, with the advent of new therapeutic paradigms.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); immune system; immunohistochemistry (IHC).

## 307
EPSTEIN-BARR VIRUS-POSITIVE DIFFUSE LARGE B-CELL LYMPHOMA FEATURES DISRUPTED ANTIGEN CAPTURE/PRESENTATION AND HIJACKED T-CELL SUPPRESSION

X. Li<fnref n="1" /> | X. Jiang<fnref n="1" /> | W. Wang<fnref n="1" /> | J. Lee² | X. Zhou<fnref n="1" />

**Background:** B-cells can function as antigen presenting cells by presenting antigens captured by the B-cell receptor (BCR) on Class II Major Histocompatibility Complex (MHCII) to T-cells. In addition, B-cells can also maintain immune homeostasis by expressing PD-L1 and suppress T-cell activity. Epstein-Barr virus (EBV) infection can disrupt B-cell function and lead to B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Here we show that EBV-positive DLBCL (EBV+ DLBCL) has decreased BCR and MHCII expression, but over-expressed PD-L1, which may lead to immune evasion.

**Design:** An EBV+ DLBCL cohort (n=30) and an EBV-negative DLBCL control cohort (n=83) were established. Immunostaining of PD-L1,

MHCII, MHCII Transactivator (CIITA) and pBTK were performed on automated stainer. H-score was used to denote the results of staining of PD-L1 and pBTK. Break apart and deletion of CIITA locus was studied by fluorescent *in situ* hybridization. Surface immunoglobulin mean fluorescent insensitivity (MFI) was detected by flow cytometry to demonstrate the level of BCR.

**Results:** EBV+ DLBCL showed significantly lower expression of CIITA and MHCII compared to EBV-negative DLBCL. Genetic aberrations involving CIITA were also more common in EBV+ DLBCL, with 23% break apart events and 6% deletion events, compared to 2% break apart and 0% deletion in EBV-negative DLBCL. In addition to the loss of antigen presenting molecules, the antigen capturing receptor, BCR, was also down-regulated in EBV+ DLBCL. Accordingly, BCR signaling was also significantly decreased in EBV+ DLBCL as denoted by the respective pBTK levels. Finally, EBV+ DLBCL showed over expression of the T-cell inhibitory ligand, PD-L1.

**Conclusions:** Antigen capture and presentation system were disrupted, and T-cell inhibitory molecule was hijacked in EBV+ DLBCL, which may contribute to immune escape in this high risk disease. Therapies targeting these aberrations may improve the outcome of patients with EBV+ DLBCL.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); Epstein-Barr virus (EBV).

## 308
LATENT MEMBRANE PROTEIN 2A (LMP2A) MIMICS B-CELL RECEPTOR SIGNALING AND PROMOTES IMMUNE ESCAPE IN EPSTEIN-BARR VIRUS (EBV)-POSITIVE DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL)

X. Li | X. Jiang | W. Wang | D. Sheng | W. Yan

Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China

**Background:** EBV+ DLBCL is an aggressive malignancy that is largely resistant to current therapeutic regimens. LMP2A is expressed during different latency stages of EBV-infected B cells in which it triggers activation of cytoplasmic protein tyrosine kinases. Early studies have revealed that an immunoreceptor tyrosine-based activation motif in the cytoplasmic N-terminus of LMP2A can trigger a transient increase of the cytosolic $Ca^{2+}$ concentration similar to that observed in activated B cells. Meanwhile, tumor cells often express PD-L1 in EBV+ DLBCL, providing a possible mechanism for immune escape. We thus explored the correlations between LMP2A expression, B-cell receptor (BCR) signaling, and PD-L1 expression in EBV+ DLBCL.

**Design:** Two cohorts of DLBCL cases, respectively EBV-positive (n=28) and EBV-negative (n=32), were selected. LMP2A, PD-L1 and

<footnote n="1">¹Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China; ²Department of Pathology, University of Chicago, Chicago, United States</footnote>
<figure><img src="image_192.png" /></figure>

<figure><img src="image_193.png" /></figure>

the BCR signaling-related molecule, phosphorylated form of SYK (pSYK), were immunohistochemically evaluated on formalin-fixed, paraffin-embedded tumor tissues. The expression status of LMP2A, pSYK and BCR were further validated by a flow cytometry analysis using fresh tissues from 3 EBV-positive and 5 EBV-negative DLBCL patients.

**Results:** The EBV-positive cases, with a median age of 61yrs, were more frequently associated with a high-risk IPI score and a non-GCB phenotype (P=0.029). Compared with EBV-negative ones, patients with EBV-positive tumors showed a worse response to the RCHOP therapy and a shorter median survival (P = 0.041). Twenty-one EBV-positive cases (75 %) expressed LMP2A, whereas none of the EBV-negative cohort expressed this protein. The expression level of pSYK was significantly lower (P = 0.0313) in EBV+ DLBCL cases than those EBV-negative ones, and the pSYK level correlated negatively with LMP2A level (P = 0.119). The expression level of PD-L1 was significantly higher (P = 0.042) in EBV+ DLBCL, which correlated positively with the LMP2A level (P=0.184). Flow cytometry confirmed the LMP2A expression in EBV+ DLBCL, which correlated with a down-regulated BCR and pSYK expression.

**Conclusions:** EBV+ DLBCL seems to feature an inactive BCR signaling, which may be related to the expression of LMP2A.

Besides, LMP2A may function and promote the PD-L1 expression. These findings indicate that targeting immune checkpoints including PD-1/PD-L1 instead of BCR signaling molecules seems to be more reasonable and promising for the treatment of EBV+ DLBCL.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); Epstein-Barr virus (EBV).

309
PROGNOSTIC IMPACT OF TUMOR-ASSOCIATED MACROPHAGES, LYMPHOCYTE-TO-MONOCYTE AND NEUTROPHIL-TO-LYMPHOCYTE RATIO IN NEWLY DIAGNOSED DIFFUSE LARGE B-CELL LYMPHOMA PATIENTS

E. Cencini<fnref n="1" /> | A. Fabbri<fnref n="1" /> | L. Schiattone<fnref n="1" /> |

V. Mancini² | S. Lazzi² | L. Leoncini² | M. Bocchia<fnref n="1" />

<footnote n="1">¹Hematology, Azienda Ospedaliera Universitaria Senese & University of Siena, Siena, Italy; ²Human Pathology and Oncology, University of Siena, Siena, Italy</footnote>
**Introduction:** diffuse large B-cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphomas (NHLs). Standard chemoimmunotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) is able to cure a significant proportion of patients; however, 30% of total cases experience relapse or have refractory disease. The international prognostic index (IPI) represents the most useful prognostic score, but it is exclusively based on clinical parameters. Previous studies showed the microenvironment has a prognostic influence in DLBCL cases; among its cellular components, tumor-associated macrophages (TAM) surely play a leading role. TAM can be classified into 2 distinct types, M1 (anti-tumor activity) and M2 (pro-tumor activity). Another prognostic factor could be represented by lymphopenia, measured as lymphocyte-to-monocyte and neutrophil-to-lymphocyte ratio (LMR and NLR), that reflects a reduced host systemic immunity.

**Methods:** in this study we would like to evaluate the prognostic impact of total TAM, M1 and M2 subtypes, LMR and NLR in newly diagnosed DLBCL patients. The primary objective is represented by progression-free survival (PFS). Secondary objectives are complete remission (CR) rate and overall survival (OS). We have retrospectively analyzed a cohort of 37 consecutive newly diagnosed DLBCL patients, treated between 2009 and 2013. Out of 37 patients, 28/37 (75.6%) received R-CHOP or CHOP-like regimens, 9/37 (24.4%) were treated with less intensive therapies. Treatment response was assessed according to 2007 revised criteria. Immunohistochemistry stainings were performed on paraffin-embedded sections with antibodies against CD68 (PG-M1 clone, 1:50, Dako) and CD163 (1:200, Novocastra-Leica). We have evaluated total TAM (CD68+ and CD163+) and different subtypes (CD68+, CD163+, CD68+/CD163+). We have divided our cohort in 2 categories as previously published by Steidl and colleagues; score 1-2 represented the group with low expression, while score 3 represented the group with high expression. TAM with the expression of both CD68 and CD 163 were considered as M2. For LMR and NLR we used previously published cut-off of 2.71 and 2.81, respectively.

**Results:** CR rate was 70.3% (26/37 cases), 11 cases did not achieve an adequate response (5 PR, 4 SD, 2 PD). We did not report a significant correlation between total TAM, CD68+ TAM, CD163+ TAM, CD68+/CD163+ TAM, LMR, NLR and CR. We observed an association between lower expression of M2 TAM, higher expression of CD68+ TAM and improved PFS and OS, even if it did not reach a statistical significance.

**Conclusion:** our study suggests TAM CD68+ and especially M2 TAM could have a prognostic role for DLBCL cases receiving R-CHOP; the lack of statistical significance could be due to the small sample size. Multicenter prospective studies are needed to compare and standardize all the different available scoring methods, with the aim to perform in the future TAM evaluation at diagnosis in daily clinical practice.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); macrophages; prognostic indices.

310
THE SIGNIFICANCE OF PD-L1 AND PD1 RECEPTORS EXPRESSION IN PATIENTS WITH DIFFUSE B-CELL LARGE-CELL LYMPHOMA

S. Samarina¹ | A. Luchinin¹ | N. Minaeva¹ |

N. Semenova² | S. Gritsaev²

¹Diagnostics department, Federal State Budget Institution of Science "Kirov Scientific Research Institute of Hematology and Blood Transfusion of the Federal Medical-Biological Agency" (KSRIHandBT FMBA Russia), Kirov, Russian Federation; ²Morphology, Federal State Budget Institution Russian Research Institution of Hematology and Transfusiology of Federal Medical Biological Agency, St. Peterburg, Russian Federation

**Introduction:** The treatment response of patients with diffuse B-cell large-cell lymphoma (DLBCL) depend on clinical and immunohistochemical parameters as during of diagnostics as a therapy. Such as the promising prognostic markers may be the expression of PD-L1 and PD1 receptors on tumor cells.

**Material and methods:** 35 patients in age from 42 to 83 years (the median was 62 years) with DLBCL have been included in analysis. All patients received 6 cycles of induction R-CHOP therapy. The period of observation was from 1 to 110 months (the median - 20 months). For PD-L1 and PD1, staining in more than 5% of tumor cells was estimated as positive expression.

**Results:** The positive expression of PD-L1 and PD1 was detected in 7 (20.0%) and 17 patients (51.0%) accordingly. There were no significantly age differences between groups of patients with positive and negative expression of PD-L1 (Wilcoxon rank test, p=0.2) and PD-1 (Wilcoxon rank test, p=0.29). In the group with positive PD-L1 expression all of patients (100%) had a non-GCB subtype according to the Hans algorithm. Among patients with negative PD-L1 expression the GCB subtype was detected in 8 (33%), and the non-GCB subtype in 16 (67%). So there were no statistically significant differences (Fisher test, p = 0.146). In the group with positive PD-1 expression 4 (29%) patients had a GCB subtype and 10 (71%) - a non-GCB subtype of DLBCL. In negative PD-1 expression group the GCB subtype was detected in 4 (27%) and non-GCB - in 11 (63%) patients (Fisher test, p=0.146). Also we did not find statistical significantly associations between PD-L1 or PD1 positive expression and risk-groups by International Prognostic Index (Fisher test, p=0.94 [PD-L1 - IPI], p=0.54 [PD-1 - IPI]). The median of progressive-free survival (PFS) in patients with positive PD1 expression was 13 months and was not achieved in negative expression group (Hazard Ratio=1.4, 95% CI 0.4-4.84, p=0.6). In patients with
positive PD-L1 expression PFS was 100%, but in group with negative PD-L1 expression the median of PFS was 13 months (Log-rank test, p=0.6)

**Conclusion:** We did not find any significantly associations between PD-L1 and PD1 receptors expression on tumor cells and other known clinical and immunohistochemical parameters in patients with DLBCL as also as PFS. Maybe it is result of small sample of patients included in analysis. Further research work can show final conclusions.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); PD-1; PD-1L.

## 311
CLINICAL PRESENTATION AND
MOLECULAR CHARACTERISTICS OF
CUTANEOUS DLBCL

J. Bloehdorn<fnref n="1" /> | S.E. Weissinger² | A. Sindrilaru<fnref n="3" /> |

T. Peters<fnref n="3" /> | P. Möller² | R. Marienfeld² | A. Viardot<fnref n="1" />

**Introduction:** DLBCL with cutaneous manifestation shows a highly heterogeneous clinical course. We aimed to characterize molecular and clinical differences helping to assess the patient's individual risk and potential treatment options.

**Methods:** We identified 24 patients with cutaneous DLBCL treated at our center, 15 patients with leg-type DLBCL (LT-DLBCL) and 9 patients with cutaneous DLBCL at other anatomic sites (DLBCL-OS). Targeted sequencing was conducted for CD79B,MYD88, CARD11 and BTK. Besides routine markers, we further analyzed CD30, PD-1/ PD-L1 expression and aberrations of 9p24.1.

**Results:** Age at diagnosis was 48 years in DLBCL-OS vs. 81 years in LT-DLBCL (p=0.001) (range 22-94 years). 92% of cases from both entities were classified as non-GCB based on the Hans Classifier. DLBCL-OS showed manifestations on the head and back, but no other region. LT-DLBCL had a higher median ki67-index with 82% vs. 67% (p=0.007). Further, 66.6% of LT-DLBCL patients had MYD88 mutations, compared to 25% of DLBCL-OS patients. 75% of CD79B mutations were observed in LT-DLBCL along with the only BTK mutation. No CARD11 mutations were observed in the whole cohort. Patients received a median of 2 (0-7) treatment regimen showing a highly heterogeneous response to last therapy. Patients with DLBCL-OS showed a complete remission (CR) in 89% of cases, with only 11% being refractory. LT-DLBCL showed a CR in 45%, partial remission in 9%, stable disease in 9% and refractory disease in 37%. Median overall survival (OS) was significantly different for both subtypes with LT-DLBCL patients showing a much shorter OS compared to patients

with DLBCL-OS (21 months vs. not reached, p=0.01). 77% of DLBCL-OS patients were alive at last observation.

We further evaluated markers pointing to alternative treatment options. CD20 was expressed in all cases. While PD-L1 was negative in 89% of cases and no amplification of 9p24.1 was detected, 44% of cases showed positive staining for PD1 (5-90%). CD30 expression is reported in approximately 15-30% of DLBCL, with predominance in the non-GCB subtype, but was not observed in analyzed cases of our cohort. Distinct mutational profiles in DLBCL were reported to indicate BCR pathway addiction and potential sensitivity towards ibrutinib. 43% of cases with MYD88 mutation showed an accompanying CD79B mutation and in one case a BTK mutation. 27% exhibited a CD79B wild type together with a MYD88 mutation, previously associated with BCR independence. Patients exposed to ibrutinib (n=3) showed variable responses including 2 CR, with one lasting till date.

**Conclusion:** LT-DLBCL presents at older age and shows a poor clinical course, in contrast to DLBCL-OS. MYD88 mutations are frequent in LT-DLBCL. Mutational profiles and ibrutinib response were in line with previous models for sensitivity to BCR inhibition. CD30 negativity does not advocate the use of brentuximab vedotin as salvage treatment option in this setting.

**Keywords:** B-cell receptor (BCR); diffuse large B-cell lymphoma (DLBCL); molecular genetics.

## 312
EVALUATION OF BONE MARROW
INFILTRATION BY MULTIDIMENSIONAL
FLOW CYTOMETRY IN PATIENTS WITH
DIFFUSE LARGE B-CELL LYMPHOMA: SUB-
STUDY OF A PHASE 2 GELTAMO CLINICAL
TRIAL

M. Baile<fnref n="1" /> | S. Barrena² | J. Sancho<fnref n="3" /> | C. Grande<fnref n="4" /> |

R. Fernández<fnref n="5" /> | A. Batlle⁶ | M. Peñarrubia<fnref n="7" /> |

F. Peñalver<fnref n="8" /> | J. Hernández-Rivas⁹ | M. Guinea¹⁰ |

J. Pérez<fnref n="1" /> | M. García-Álvarez<fnref n="1" /> | M. Alcoceba<fnref n="1" /> |

B. Vidriales<fnref n="1" /> | A. Orfao² | A. Martín<fnref n="1" />

<footnote n="1">¹Department of Internal Medicine III, University Hospital Ulm, Ulm, Germany; ²Institute of Pathology, University Hospital Ulm, Ulm, Germany; ³Department of Dermatology, University Hospital Ulm, Ulm, Germany</footnote>

<footnote n="1">¹Hematology Department, Hospital Universitario de Salamanca / IBSAL, Salamanca, Spain; ²Cytometry (NUCLEUS), Cancer Research Center and University of Salamanca (IBMCC-USAL-CISC), Salamanca, Spain;</footnote>

<footnote n="3">³Hematology, Hospital Germans Trias i Pujol / ICO-IJC, Barcelona, Spain;</footnote>

<footnote n="4">⁴Hematology, Hospital Universitario 12 de Octubre, Madrid, Spain;</footnote>

<footnote n="5">⁵Hematology, Hospital de Cabueñas, Guijón, Spain; ⁶Hematology, Hospital Universitario Marqués de Valdecilla, Santander, Spain;</footnote>

<footnote n="7">⁷Hematology, Hospital Clínico de Valladolid, Valladolid, Spain;</footnote>

<footnote n="8">⁸Hematology, Hospital Universitario Fundación de Alcorcón, Madrid, Spain; ⁹Hematology, Hospital Universitario Infanta Leonor, Madrid, Spain; ¹⁰Hematology, Hospital Universitario de Araba, Vitoria-Gasteiz, Spain</footnote>
<table><caption>**TABLE 1** Prognostic impact of bone marrow infiltration by multidimensional flow cytometry</caption><thead><tr><th></th><th>n</th><th>CR (%)</th><th>p</th><th>ORR (%)</th><th>p</th><th>3-y PFS (%)</th><th>p</th><th>3-y OS (%)</th><th>p</th></tr></thead><tbody><tr><td>Concordant</td><td>6</td><td>50</td><td></td><td>83</td><td></td><td>17</td><td></td><td>33</td><td></td></tr><tr><td>Discordant</td><td>14</td><td>57</td><td></td><td>93</td><td></td><td>37</td><td></td><td>57</td><td></td></tr><tr><td>Not infiltrated</td><td>34</td><td>71</td><td>0,29</td><td>83</td><td>0,39</td><td>51</td><td>0,28</td><td>74</td><td>0,16</td></tr></tbody></table>

Abbreviations: CR, complete remission rate; MFC, multidimensional flow cytometry; OS, overall survival; PFS, progression-free survival.

**Introduction:** Multidimensional flow cytometry (MFC) has not yet shown a high clinical value in patients with diffuse large B-cell lymphoma (DLBCL), probably due to the phenotypic heterogeneity of these lymphomas. In the present study, we have evaluated bone marrow (BM) infiltration in patients with DLBCL using MFC with the following objectives: 1) evaluating its prognostic impact compared to other techniques such as histology or PET; 2) determine the specific phenotypic pattern of the tumor cell of each patient for subsequent follow-up of minimal residual disease (MRD) after treatment.

**Methods:** We centrally evaluated BM samples from patients included in the multicenter randomized phase 2 clinical trial GEL-R-COMP-2013 (EudraCT: 2013-001065-17), that compares R-CHOP versus R-COMP in patients with newly diagnosed DLBCL or follicular lymphoma (FL) grade 3b. High resolution (8 colors) and high sensitivity (more than 1 million cells analyzed) direct immunofluorescence techniques were used, following the protocols defined by EuroFlow. An

automatic analysis was carried out in addition to the manual analysis, using the Compass III tool, available in the Infinicyt analysis software (Cytognos, S.L.).

**Results:** From a total of 91 patients included in the clinical trial, 54 participated in the present study (48 DLBCL and 6 FL grade 3b). The median age was 75 years (61-86). BM infiltration was detected by MFC in 20 patients (37%) at diagnosis and 0 out of 13 at post-treatment evaluation. Regarding the phenotypic characterization, concordant BM infiltration by DLBCL was detected in only 6 cases. In contrast, discordant infiltration was detected in 14 (70%) patients, with a very heterogeneous phenotype: 3 low grade FL, 2 chronic lymphocytic leukemia, 5 marginal zone lymphoma and 4 small cell lymphoma with non-specific phenotype. Molecular studies showed monoclonal rearrangements in 14 out of 17 (82.3%) samples tested. Preliminary analysis of the prognostic impact of BM infiltration by MFC is shown in Table 1. The presence of concordant BM infiltration

<figure><img src="image_194.png" /><figcaption>**Image 1: Prognostic impact of bone marrow infiltration by MFC and R-IPI**</figcaption></figure>
was associated with worse survival, although the differences were not statistically significant, possibly due to the low number of cases. On the other hand, discordant BM infiltration had no significant prognostic influence.

**Conclusions:** High resolution MFC allowed the detection of small clonal populations in BM in a very high proportion of patients with newly diagnosed DLBCL. In most cases, infiltration was discordant with tumor histology and, apparently, had no prognostic relevance. MRD was not detected in any of the cases. We plan to correlate MFC findings with those of histology and PET, also centrally reviewed, as well as to perform molecular studies on lymph node and BM samples to assess their clonal relationship.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); flow cytometry.

**Disclosures:** Sancho, J: Honoraria: Roche, Janssen, Gilead, Khen Fharma, Celgene, Sanofy, Servier, Mundipharma; Research Funding: Gil-ead. Martín, A: Consultant Advisory Role: Celgene, Roche.; Honoraria: Celgene, Roche, Servier, Janssen.

## 313
ACTIVATION OF THE AhR/ARNT PATHWAY IS ASSOCIATED WITH OUTCOME OF DLBCL PATIENTS AFTER TREATED WITH RITUXIMAB

F. Jin<fnref n="1" /> | X. Yu<fnref n="1" /> | X. Liu<fnref n="1" /> | Y. Liu<fnref n="1" /> | H. Ma<fnref n="2" /> |

L. Qu<fnref n="2" /> | Y. Dai³

**Introduction:** As DLBCL is genetically and clinically heterogeneous, there is an increasing interest in identifying patients who gain the maximum benefit from rituximab. However, a few molecular markers have been characterized so far. Here, we sought to identify potential biomarkers that might predict outcome after rituximab.

**Methods:** Using the R2 Platform, a data-mining analysis was performed to sort out the genes correlated with CR to rituximab. An IHC analysis was then carried out to examine expression of these genes in biopsies of DLBCL patients who had received frontline rituximab-based regimens, as well as its relationship with PFS and OS.

**Results:** The data-mining analysis revealed two genes (AhR, SPARCL1) that were the most significantly expressed in DLBCL patients who achieved CR after rituximab, compared to those who had PD. While SPARCL1 is a secreted soluble protein related to DLBCL microenvironment, we thus focused on AhR, a member of the PAS superfamily of transcription factors involved in sensing environmental signals such as hypoxia (HIF-1α, HIF-2α, HIF-3α), and its partner ARNT encoding HIF-1β (the regulatory subunit of the HIF complexes). In a cohort of 93 DLBCL patients (extranodal excluded), IHC showed high nuclear

expression (>60%) of AhR and ARNT in 68% and 81% cases, respectively. With cut-off of 80% (for both genes), ARNT expression was significantly related to male, increased LDH level, and high IPI score (>2), but AhR was not. Interestingly, both of them were positively associated with expression of BCL-6 and Ki67, and ARNT also with MUM-1, but not BCL-2, CD10, and MYC. AhR expression was markedly associated with shorter PFS (HR=0.458 [0.169-0.920], P=0.034), while a similar trend was observed for ARNT although not significantly (HR=0.650 [0.314-1.359], P=0.260). Of note, the high level of AhR, rather than ARNT, was able to override the favorable effect of CR on PFS (P=0.652 vs P=0.002), as well as the adverse influence of high IPI score although to a lesser extent (P=0.075 vs P=0.027), but likely not on OS. In the subgroups, the unfavorable role of AhR was observed exclusively in GCB (P=0.044), rather than non-GCB (P=0.139). BCL-2, a marker for poor prognosis in DLBCL, no more predicted worse PFS in patients with high AhR (P=0.650 vs P=0.036), arguing that AhR expression might impair the value of BCL-2 for risk stratification. But this phenomenon was not observed in the case of ARNT. However, both PFS (78.6%) and OS (64.3%) of patients with high BCL-2 but low ARNT were obviously longer than the median PFS and OS of those with both high Bcl-2 and ARNT, suggesting a BCL-2-independent prognostic role of ARNT. Median OS for virtually all (sub)groups was not reached yet.

**Conclusions:** Together, these findings argue that the AhR/ARNT pathway is constitutively activated in DLBCL, suggesting a novel signal that might drive DLBCL. However, AhR, other than ARNT, might play important role in outcome prediction after rituximab, likely by interacting with other prognostic factors.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); prognostic indices; rituximab.

## 314
GENETIC PREDISPOSITION OF FAMILIAL HODGKIN LYMPHOMA

E. Khoury<fnref n="2" /> | A. Mendola<fnref n="2" /> | S. Choquet³ |

J. Landman-Parker⁴ | M. Veiga da Cunha<fnref n="2" /> |

C. Besson<fnref n="5" /> | N. Limaye<fnref n="2" /> | H.A. Poirel<fnref n="1" />

**Introduction:** Hodgkin lymphoma (HL) is a B-cell lymphoproliferative neoplasms that affects 2.7 individuals per 100,000/yr. Risk factors include EBV infection, especially in the context of acquired or innate immunodeficiency. Despite evidence that genetic predisposition plays

<footnote n="1">¹Department of Hematology, The First Hospital of Jilin University, Changchun, China; ²Department of Pathology, The First Hospital of Jilin University, Changchun, China; ³Laboratory of Cancer Precision Medicine, The First Hospital of Jilin University, Changchun, China</footnote>

<footnote n="1">¹Hematology and Genetics, Belgian Cancer Registry, Brussels, Belgium;</footnote>

<footnote n="2">²De Duve Institute, UCLouvain, Brussels, Belgium; ³Service</footnote>

<footnote>d'Hématologie, CHU La Pitié Salpétrière, Paris, France; ⁴Service</footnote>

<footnote>d'Hématologie et d'Oncologie Pédiatrique, Hôpital Armand Trousseau, Paris, France;</footnote>

<footnote n="5">⁵Unité d'Hémato-Oncologie - INSERM Unit 1018, Centre Hospitalier de Versailles - Université Versailles Saint Quentin en Yvelines, Versailles, France</footnote>
an important role in a subset of HL, there is a dearth of functionally validated disease-causative genes.

**Methods:** We aim to identify germline genetic "drivers" of HL by applying Whole Exome Sequencing (WES) to rare, familial HL samples, followed by functional validation of prioritized candidate genes with rare, potentially pathogenic variants that co-segregate with disease within families.

**Results:** A pilot study on 20 affected individuals from 10 families identified between 24 and 96 such altered genes per family, 23 of which were identified in more than one. Intriguingly, they include several proteins that regulate mitosis and mitotic checkpoints, suggesting this may be an important pathway in disease. Variants in the top two of these candidate genes (i) one involved in the first step of glycolysis and (ii) the other a cell-cycle regulated E3-ubiquitin ligase- were in vitro tested for their effects on protein function and showed a loss of function. We are further expanding the WES on an additional series of 18 families to confirm the recurrence of altered genes in familial HL and to look for somatic-second hit locally eliminating the wild-type allele, according to the classical paradigm for tumor-suppressor gene.

**Conclusions:** Rather than limiting ourselves to identifying a set of genes that cause disease in a few families each, we propose to use pathway-analysis to identify shared molecular disease mechanisms.

(*Nisha Limaye and Hélène A. Poirel: Equal contribution)

**Keywords:** Hodgkin lymphoma (HL); molecular genetics.

315

'NOT SO CLASSICAL': A STUDY OF THE
IMMUNOPROFILE IN CLASSICAL HODGKIN
LYMPHOMA, AN EXPERIENCE FROM A
REFERRAL CENTER

M. Das | R. Duggal | A. Dewan | S. Sharma |

D. Jain | A. Narrendran | S. Prasad | S. Sethi |

L. Kini | S. Mohanty

Department of Pathology, Core Diagnostics, Gurugram, India

**Introduction:** According to WHO classification, Classical Hodgkin Lymphoma (CHL) expresses CD15 and CD30 antigens in majority of cases, and CD20 in a small subset of cases. Expression of CD20 may affect the patients' prognosis, relapse, and refractoriness to the therapy. We come across a subset of cases of CHL in our lymphoma consultation cases that exhibit a lack of the 'positive' diagnostic marker CD15 and express CD20 in the Hodgkin and Reed-Sternberg (HRS) cells. This observation has important prognostic implication, based on the western studies from the more developed world, and furthermore prompted us for a systematic evaluation of the immunoprofile of South-East Asian CHL patients and to compare those with the Western data.

**Methods:** In this retrospective analysis, 111 cases of CHL diagnosed over a period of six months were studied. Demographics, clinical details, histopathological (including unusual morphologic patterns),

and immunohistochemical (IHC) parameters were recorded. The diagnoses were made according to the 2016 WHO classification of lymphoid neoplasms. The cases were further subtyped. The IHC panel included CD45, CD15, CD30, CD3, CD20, PAX5, ALK1, and EBV (LMP-1) in all cases and MUM1, CD21, EMA, PD1, OCT2, BOB.1, and BCL6, in a subset of cases, wherever necessary. A case was designated CD20+, if >10% of HRS cells were strongly positive.

**Results:** The cases of CHL fit into 'textbook phenotype' (WHO 2016 update) of CD15+, CD30+, CD20- in 54% cases. HRS cells were positive for CD30 in all, CD15 in 56%, PAX5 in 96%, CD20 in 18% cases and EBV (LMP1) in 58.3% cases. In majority of instances, morphology and an initial panel of CD3, CD20, CD15, CD30, CD45, and PAX5 were sufficient for diagnosis. When IHC pattern discordance was noted or mimics of CHL were in the differential diagnostic consideration, additional markers supported the diagnosis of CHL.

**Conclusions:**

1. Our study results broaden the understanding of the demographic, histopathologic, and IHC characteristics of CHL cases.

2. The second peak of bimodal distribution occurs two decades earlier as compared to those of the western population in majority cases in our cohort.

3. 56% of our cases expressed CD15 that is lower than the reported positivity rates (75% to 85%).

4. Significant CD20 positivity in HRS cells as seen in our cohort can create diagnostic dilemma. However, CD45 negativity in the presence of weak to barely perceptible PAX5 and moderate to strong expression for EBV aid in arriving at a diagnosis of CHL.

5. Future directions: The management of CHL is essentially based on radiotherapy and chemotherapy, due to lack of information about the clinical outcomes with anti-CD20 antibody therapy or combined drug therapy using a classic regimen. The prognostic impact of lack of CD15 and positive CD20 expression in the cases of CHL requires further research and clinical follow-up studies in a larger cohort of patient to assess the role of CD20 in the pathophysiology and progression of CHL.

**Keywords:** CD20; classical Hodgkin lymphoma (cHL); immunohistochemistry (IHC).

316

PRE-T CELL RECEPTOR SIGNALING DRIVES
LEUKEMOGENESIS AND IS A
THERAPEUTIC TARGET IN T CELL ACUTE
LYMPHOBLASTIC LEUKEMIA

F. Kuhnert

Oncology, Regeneron Pharmaceuticals, Tarrytown, United States

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid neoplasm that results from the malignant transformation of T-cell progenitors. The biology of T-ALL is strongly influenced by its developmental origins. For example, mutations in *NOTCH1*, a critical regulator
of thymocyte development, are found in approximately 60% of patient tumors. Less is known, however, about the extent to which other determinants of thymocyte development participate in the leukemogenesis of T-ALL. Using a thymus transplantation-based, spontaneous mouse model of T-ALL, we found that multiple β-selection checkpoint factors were upregulated in leukemic T cells, including Ptcrα, a subunit of the pre-T cell receptor (pre-TCR) complex. Genetic ablation of Ptcrα in the mouse model dramatically reduced the occurrence of T-ALL. In human T-ALL cell lines, CRISPR/Cas9 knockout of PTCRA reduced in vitro proliferative capacity and the ability to form tumors in vivo. Analysis of clinical T-ALL datasets and patient samples demonstrated that PTCRA is highly and specifically expressed in leukemic T cells but not in normal, mature T cells, suggesting an appropriate therapeutic window for targeted therapy in T-ALL. Cumulatively, our findings highlight an important role for pre-TCR signaling in driving and sustaining T-ALL and support the further evaluation of PTCRA-directed therapies in T-ALL.

**Keywords:** Notch pathway; T-cells.

**Disclosures:** Kuhnert, F: Employment Leadership Position: Regeneron Pharmaceuticals Inc.

317

# THE EVALUATION OF THE CLINICOPATHOLOGIC CHARACTERISTICS OF T-CELL NON-HODGKIN'S LYMPHOMA: THE SINGLE CENTER EXPERIENCE

N. Sayınalp | E. Aladag | M. Okay | R. Ciftciler |

S. Aksu | H. Demiroglu | Y. Buyukasik | H. Goker |

I.C. Haznedaroglu | O.I. Ozcebe

Hematology, Hacettepe University, Ankara, Turkey

**Aim:** T-cell lymphoma is a subgroup of non-Hodgkin's lymphoma with different morphological, immunological and clinical features that constitute approximately 10-15% of all lymphomas. In this study, we aimed to evaluate the clinical stages, histopathological diagnosis, treatment and response of T cell lymphoma patients in Hacettepe University Department of Hematology.

**Method:** 62 T cell lymphoma patients who followed between 2002 and 2018 in Hacettepe University Faculty of Medicine Department of Hematology were included in the study. Patients were evaluated retrospectively with imaging studies and pathological reports and classified according to the National Comprehensive Cancer Network (NCCN Guidelines Version 4.18) classification. Overall survival was analysed by evaluating the treatments which the patients were taken and response of the treatment.

**Results:** The number of patients diagnosed with T-cell lymphoma (n = 62) were 8% of all lymphoma cases followed in our department at this period. Of the 62 patients enrolled in the study, 8% were stage I, 17% were stage II, 29% were stage III and 46% were stage IV according to Ann Arbor staging. 9 patients with anaplastic type,

4 angioimmunoblastic type, 3 extranodal NK / T cell lymphoma nasal type, 4 enteropathy related T cell lymphoma, 2 follicular type, 6 hepatosplenic type, 10 T cell large granular lymphocytic leukemia (T-LGL) and 22 unclassifiable peripheral T cell lymphoma. Sixty patients (96.7%) were treated. 52 patients out of T-LGL, 18 (34.6%) received CHOEP chemotherapy as the firstline therapy and 27 (51.9%) received CHOP chemotherapy however, hematopoietic stem cell transplantation was performed in 18 of 52 patients (34.6%). Complete remission was achieved in 63.2% of the patients who received CHOP chemotherapy and this rate was 72% in CHOEP chemotherapy (p: 0.23). The median survival of patients who underwent hematopoietic stem cell transplantation (98.5 months) was higher than the patients who did not (71.8 months) but the difference was not statistically significant (p = 0.160). Median overall survival was 126.7 months [1 - 168.5 months], 5-year survival was 64.3% and 3-year survival was 74% in the whole study group. The group having the lowest overall survival was hepatosplenic T-cell lymphoma whose survival was 18.9 [1 - 89.3] months. It was significantly lower than the other groups (p = 0.020). In cox regression analysis, non-response to initial therapy was an independent prognostic factor for overall survival (OR = 11.6, p = 0.001).

**Conclusion:** T-cell lymphoma is a type of non-hodgkin lymphomas, consisting of several subgroups with different pathologies and different clinical features. Pathology and clinical difference inevitably affect treatment and prognosis. Each case of T cell lymphoma should be approached by considering these differences.

**Keywords:** R-CHEOP; R-CHOP; T-cell lymphoma (TCL).

318

# POTENTIAL METABOLITES WITH DIAGNOSTIC VALUE IN PLASMA FOR ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA BY LC-MS BASED UNTARGETED METABONOMICS STUDY

H. Yang¹ | T. Lei¹ | C. Li¹ | H. Yu¹ | Z. Chen²

¹Chemotherapy Center, Zhejiang Province Cancer Hospital, Hangzhou, China; ²Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Hangzhou, China

**Introduction:** Angioimmunoblastic T-cell lymphoma (AITL) is an uncommon subtype of mature peripheral T-cell lymphoma (PTCL). And its characteristics is not fully understood. Because it is a rare form of lymphoma, there is difficulty in diagnosis based on current traditional method. Metabolic reprogramming is a major cancer hallmark, which provides cancer cells with vital energy and metabolites. Mass spectrometry-based metabolomics has emerged as an informative technique for profiling metabolic features associated with cancers. This study aims to discover metabolites with potential diagnostic value in plasma of AITL patient.
**Methods:** Plasma was collected from 27 patients newly diagnosed with AITL in Zhejiang Cancer Hospital and 30 age- and gender-matched healthy controls. An aliquot of 50 µL plasma was added with 150 µL of chilled acetonitrile, vortexed and centrifuged. The supernatant was dried, reconstituted for metabolomics. Mass spectrometry analysis was performed on a UPLC coupled with a Q Exactive Orbitrap mass spectrometer. "XCMS" and "MetaX" were used to analyze LC-MS data. Significant features were selected as candidate for identification by matching their MS and MS/MS data with online databases HMDB and METLIN.

**Results:** Eighteen altered metabolites (choline, betaine, L-leucine, creatine, 2-butyl-1H-benzimidazole, hypoxanthine, methionine sulfoximine, L-carnitine, 2-hydroxycinnamic acid, L-tyrosine, D-tryptophan, indoleacrylic acid, lysoPC(P-18:0), 5-aminopentanoic acid, uric acid, sucrose, L-lactic) were identified (VIP value>1, p<0.05) in AITL patient plasma. The altered metabolites contributed to different

metabolic pathways including glycine, serine and threonine metabolism (P=0.003), glycerophospholipid metabolism (P=0.024), Phenylalanine metabolism (P=.030).

**Conclusion:** This study revealed metabolic reprogram and potential diagnostic metabolites in plasma for AITL, and gave more metabolic information for carcinogenesis of AITL.

**Keywords:** angioimmunoblastic T-cell lymphoma (AITL).

319
PROGNOSTIC SIGNIFICANCE OF PD-L1
AND PHOSPHORYLATED ERK EXPRESSION
IN T-CELL LYMPHOMA: PD-1/PD-L1 AXIS
ACTIVATES INTRACELLULAR ERK
SIGNALING IN TUMOR CELLS

<figure><img src="image_195.png" /><figcaption>**Fig.1.** Expression of PD-L1, p-ERK, overall survival, and PD-1/PD-L1 binding directly activates the intracellular ERK1/2 oncogene signaling in T-cell lymphoma.</figcaption></figure>
X. Wang¹ | L. Liu¹ | B. Meng² | S. Zhou¹ |

Z. Qian¹ | L. Qiu¹ | L. Li¹ | X. Liu¹ | K. Fu³ |

H. Zhang¹

¹Department of Lymphoma, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; ²Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; ³Department of Pathology, University of Nebraska Medical Center, NE, United States

**Introduction:** Programmed death ligand-1 (PD-L1) engages with PD-1, which leads to an exhaustion of antitumor T-cell responses. PD-L1 is a type I transmembrane protein with 290 amino acids, including immunoglobulin V-like and C-like domain. Structural analysis also shows that there is a protein kinase c (PKC) on PD-L1 cytoplasmic domain, suggesting that it may be involved in the activation of intra-cellular signal transduction. The purpose of this study was to investigate the prognostic significance of PD-L1 and phosphorylated ERK (p-ERK), and their interactions in T-cell lymphoma (TCL).

**Methods:** Multiplexed immunofluorescence staining was performed to visualize the expression of PD-L1 and p-ERK using the Opal immunostaining. Flow cytometry was used to quantify the membrane PD-L1 (mPD-L1) expression on TCL cell lines. Total PD-L1 expression in TCL cell lines, and the level of ERK and p-ERK were measured using western blotting. The associations among PD-L1, p-ERK expression and clinicopathological characteristics were estimated with two-tailed Spearman analyses. Kaplan-Meier analyses were used to compare survival curves.

**Results:** We found that the expression rate of PD-L1 and p-ERK were 52% and 28% in TCL cases, respectively. Spearman test showed that PD-L1 expression was correlated with stage (R=0.217, P=0.037), and p-ERK expression was correlated with stage (R=0.283, P=0.009), IPI score (R=0.321, P=0.004) and subtype (R=0.211, P=0.041). Meanwhile, PD-L1 expression was positively correlated with p-ERK expression (R=0.200, P=0.049). Survival analysis showed that TCL patients with PD-L1 and p-ERK positivity had significantly poorer outcome than those with their negativity (P=0.008 and P<0.001), respectively, and those patients with co-expression of PD-L1 and p-ERK had the worst OS (P<0.001). In three TCL cell lines with PD-L1 expression, we further found that the expression of p-ERK was upregulated after stimulation of PD-1, suggesting that ERK signaling was activated.

<table><caption>**TABLE 1** Prognostic factors affecting the DSS of ATLL patients</caption><thead><tr><th rowspan="2">Characteristics</th><th colspan="2">Univariate analysis</th><th colspan="2">Multivariate analysis</th></tr><tr><th>HR (95% CI)</th><th>P</th><th>HR (95% CI)</th><th>P</th></tr></thead><tbody><tr><td>miPD-L1<sup>+</sup></td><td>0.4209 (0.2165-0.8310)</td><td>0.0134*</td><td>0.2541 (0.1077-0.5766)</td><td>0.0011*</td></tr><tr><td>miCD134L<sup>+</sup></td><td>0.4090 (0.1525-0.9238)</td><td>0.0303*</td><td>0.2815 (0.0782-0.8333)</td><td>0.0207*</td></tr><tr><td>nGalectin-9<sup>+</sup></td><td>3.2327 (1.1847-7.5349)</td><td>0.0244*</td><td>3.7671 (1.2946-9.6958)</td><td>0.0172*</td></tr><tr><td>Age (&gt;70)</td><td>3.0763 (1.5750-6.1601)</td><td>0.0001*</td><td>4.5924 (2.1352-10.3178)</td><td>&lt;0.0001*</td></tr><tr><td>JCOG-PI (high)</td><td>1.8401 (0.9038-3.6389)</td><td>0.0911</td><td>1.4582 (0.6681-3.1094)</td><td>0.3366</td></tr><tr><td>Ann Arbor Stage (III or IV)</td><td>1.0509 (0.4967-2.6114)</td><td>0.9058</td><td>0.9710 (0.3711-3.0010)</td><td>0.9557</td></tr><tr><td>cleaved LDH</td><td>1.7837 (0.7943-4.7616)</td><td>0.1703</td><td>1.7862 (0.7113-5.2146)</td><td>0.2265</td></tr></tbody></table>

**Conclusions:** Our findings demonstrated that PD-1/PD-L1 axis activated intracellular ERK signaling in tumor cells, and PD-L1, p-ERK or their combination were potential biomarkers to predict prognosis for TCL patients.

**Keywords:** ERK; PD-1; T-cell lymphoma (TCL).

320
COMPREHENSIVE
IMMUNOHISTOCHEMICAL ANALYSIS OF
IMMUNE CHECKPOINT MOLECULES IN
ADULT T-CELL LEUKEMIA/LYMPHOMA

M. Takeuchi | K. Yamada | M. Seto | K. Ohshima |

H. Miyoshi

Department of Pathology, Kurume University, Kurume, Japan

**Introduction:** Adult T-cell leukemia/lymphoma (ATLL) is an aggressive hematopoietic malignancy with poor prognosis. As immune checkpoint inhibitors have improved the clinical outcome of some refractory tumors, understanding their role in ATLL is of interest. We previously reported that programmed cell death ligand 1 (PD-L1) expression on neoplastic and/or microenvironmental stromal cells could predict prognosis of ATLL. However, other immune checkpoint molecules have not been well studied in ATLL. We performed comprehensive immunohistochemical analysis of immune checkpoint molecules in ATLL and examined the clinical outcome.

**Methods:** Formalin-fixed, paraffin-embedded biopsy samples from 71 ATLL patients were reviewed. Immunohistochemistry was performed on a DAKO autostainer with the following antibodies: PD-L1, CD134, CD134L, CD137, CD137L, Galectin-9, Tim-3, CTLA-4, LAG-3, CD80, CD86, PD-L2, GITR, GITRL, and PD-1. Neoplastic expression of immune checkpoints was considered positive at ≥30% positive neoplastic cells, and microenvironmental expression was defined as positive in patients with ≥10 positive stromal cells per high power field.

**Results:** Immune checkpoint molecules were variably expressed on ATLL neoplastic and/or stromal cells. Neoplastic PD-1, CD134, galectin-9, GITR, and PD-L1 expression was nearly mutually exclusive, suggesting that immune checkpoint pathways differ in patients.
Patients with microenvironmental PD-L1 (miPD-L1+) or CD134L (miCD134L+) expression showed significantly better disease-specific survival (DSS) than that in miPD-L1- or miCD134L- groups (P = 0.00807 and 0.0407, respectively). Conversely, patients with neoplastic galectin-9 expression (nGalectn-9+) had significantly inferior DSS than nGalectin-9- patients (P = 0.0072). Univariate and multivariate analyses with clinical prognostic factors identified miPD-L1, miCD134, and nGalectin-9 expression and age (>70 y) as significant prognostic factors.

**Conclusions:** This is the first comprehensive analysis of ATLL immune checkpoint molecules. Our results may provide information on new therapeutic strategies in ATLL.

**Keywords:** human T-lymphotropic virus (HTLV); immunohistochemistry (IHC); PD-1L.

## 321
SAFETY AND EFFICACY OF ULTRASOUND-GUIDED COARSE NEEDLE BIOPSY IN THE DIAGNOSIS OF SUSPECTED LYMPHOMA

J. Xu¹ | J. Chen² | J. Zhang³ | B. Chen¹ |
J. Wang¹ | X. Xu¹

¹Clinical Hematology Department and Bone Marrow Transplantation, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, Nanjing, China; ²Pathology, Nanjing Drum Tower Hospital, Nanjing, China; ³Ultrasonic, Nanjing Drum Tower Hospital, Nanjing, China

**Background:** Accurate lymph node staging and sampling through biopsy is critical for diagnosis and accurate staging of lymphoma patients. The current study aims to investigate the safety and value of standardized a minimally-invasive Ultrasound-guided Core Needle Biopsy (UGCNB) in patients with suspicion of lymphoma.

**Methods:** UGCSB was performed in 367 patients suspected of lymphoma from January 2017 to July 2018. Biopsies were performed using 16-G or 18-G needle collecting At least 2 and up to four 4 tissue samples were collected. All needle adequate biopsies (at least two connected tissue longer than 0.5 cm) were formalin-fixed, paraffin-embedded, and hematoxylin-eosin (HE) stained according to regular protocol.

**Results:** Out of 367 patients underwent UGCNB, there were 58 patients (16%) diagnosed with lymphoma, 107 patients (29%) diagnosed with metastatic cancer, 99 cases (27%) with benign tumour, and 103 cases (28%) with unclear diagnosis. Out of the 58 patients diagnosed with lymphoma, there were 23 cases of Diffuse large B-cell lymphoma (DLBCL), NOS, 9 cases of (Follicular lymphoma, 7 cases of Classic Hodgkin lymphoma, 6 cases of small lymphocytic lymphoma, 3 cases of Mantle cell lymphoma, 2 cases of Angioimmunoblastic T-cell lymphoma, and 2 cases of Peripheral T-cell lymphoma, NOS. There were also 1 case each of Anaplastic large cell lymphoma, Plasma cell myeloma, Plasmablastic lymphoma Nodal marginal zone

lymphoma, and T-lymphoblastic leukaemia/lymphoma. Up until February 28, 2019 no patients of 99 patients diagnosed as benign tumour have been diagnosed with lymphoma.

**Conclusion:** The current study demonstrates that standardized UGCNB is a safe and effective method of diagnosing lymphoma. Our protocol help optimizes the sampling procedure and preserves the minimally-invasive nature of the protocol. Further studies using UGCNB in combination with molecular diagnostic tools such as immunohistochemistry and/or in-situ hybridization could provide even more diagnostic information.

**Keywords:** fine-needle aspirate (FNA); non-Hodgkin lymphoma (NHL).

## 322
IMAGE-GUIDED CORE BIOPSY IS SAFE AND EFFECTIVE FOR THE DIAGNOSIS OF LYMPHOMA

A. Gonzalez-Rodriguez | I. Amigo Fernandez |
J. Zanabili | P. Palomo | A. Gonzalez-Huerta |
S. Gonzalez-Muñiz | C. Sordo Bahamonde |
S. Lorenzo Herrero | S. Gonzalez Rodriguez |
S. Fernandez Garcia

Hematology, Hospital Universitario Central de Asturias, Oviedo, Spain

**Introduction:** Excisional biopsy (EB) from nodal or extranodal sites is the standard technique to reach an accurate histological classification and to establish the definitive diagnosis of lymphomas. Advances in knowledge in flow cytometry, genetics and molecular analyses, image-guided core biopsy (IGCB) could be an alternative to EB in most cases.

**Methods:** In this study, we compare the safety and efficacy for the diagnosis of lymphoma between image-guided core biopsy (IGCB) and

<table><caption>TABLE 1</caption><thead><tr><th></th><th>Image-guided core biopsy (IGCB)</th><th>Excisional biopsy</th><th>P</th></tr></thead><tbody><tr><td>Age (years, median)</td><td>66 (19-93)</td><td>58 (10-87)</td><td>0.026</td></tr><tr><td>Tree or more comorbidities</td><td>72 (80.9%)</td><td>30 (60%)</td><td>0.007</td></tr><tr><td>Interval between suspicion and biopsy (days, median)</td><td>9 (0-119)</td><td>23 (5-109)</td><td>&lt;0.0001</td></tr><tr><td>Time from diagnosis to treatment (median)</td><td>14 (0-364)</td><td>27 (4-436)</td><td>0.029</td></tr><tr><td>Complications (n° of patients)</td><td>Haemorrhage (1)</td><td>Severe pain that needed morphine treatment (1) Delayed healing process (1)</td><td></td></tr></tbody></table>
excisional biopsy in 117 patients who have suspicious radiological findings of lymphoma and 32 hematology patients with a possible recurrence.

149 consecutive lymphoma patients diagnosed between January 2016 and December 2017 were analysed in this study. IGCB was carried out in 89 patients. Among them, ultrasonic-guided IGCB was performed in 45 patients and CT-guided IGCB in 44. EB was performed in 50 patients. The efficacy and safety of both techniques were analysed by standard statistical methods.

**Results:** Nodal localization was mediastinal or abdominal (non-palpable) in 47 IGCB-diagnosed patients (52.8%), but only in 7 EB-diagnosed patients (14%). IGCB-analysed patients were older and had more comorbidities. Compared with EB, the diagnosis was faster and the treatment was initiated sooner in IGCB-analysed patients (table 1). Sample enough to be sent to pathology exam was obtained by IGCB in 89 patients (100%), to flow cytometry in 86 (96.6%), and to genetic analysis in 84 (94.4%). There was a complete concordance (100%) among the results obtained by flow cytometry, genetic and molecular analyses in those patients diagnosed by excisional biopsy, but only in 81 IGCB-diagnosed patients (91%). In 4 of those non concordant cases, the definitive diagnosis was further established by EB. The rate of complications was low with both techniques and no statistical differences were observed.

**Conclusions:** IGCB is an effective and safe alternative to EB for lymphoma diagnosis. Compared with EB, IGCB may be even less risky for older patients that have associated comorbidities. Another advantage of image is that non palpable lymph nodes can be visualized and biopsied. IGCB is also faster, since avoids the waiting list for surgery, and allows starting the treatment sooner. Inconclusive diagnoses using IGCG were infrequent. In those ambiguous cases, EB may be used to definitely diagnose the patients.

**Keywords:** flow cytometry.

## 323
### VALIDATION OF REAL-TIME MULTIPLEX LIGATION PROBE AMPLIFICATION (RT-MLPA) TO ANALYZE LYMPHOMA TRANSCRIPTOME FROM FORMALIN-FIXED PARAFFIN EMBEDDED SAMPLES

Y. Le Bris<fnref n="1" /> | D. Chiron<fnref n="2" /> | A. Moreau³ |

C. Bossard³ | A. Papin<fnref n="2" /> | C. Bellanger<fnref n="2" /> |

L. Bouard⁴ | A. Menard<fnref n="1" /> | S. Le Gouill⁴ |

C. Pellat-Deceunynck<fnref n="2" /> | M. Bene<fnref n="1" />

**Introduction:** Real-time multiplex ligation probe amplification (RT-MLPA) is a new technique allowing for the concomitant amplification

of several RNA from various tissues. It uses gene-specific probes targeting regions supposed to be close together on RNA molecules such as fusion transcripts but also normally coded exons. In the latter case, it can provide a precise idea of the landscape of gene expression in a given condition. Recent data have suggested that it may also be applied to poor-quality RNA extracted from formalin fixed paraffin embedded biopsies. This could allow to investigate for new markers in lymphomas.

**Methods:** With the aim of applying this methodology to lymph nodes from patients with mantle cell lymphoma (MCL), we first tested its feasibility on 7 MCL cell lines. The latter were obtained from the American Tissue Cell Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), graciously provided by Pr Chen Kiang or derived locally from patients. A panel of oligonucleotides was designed targeting RNA from CCND1, SOX11, Ki-67 and genes of the TP53 pathway. According to the principle of RT-MLPA, these oligonucleotides, tagged with size markers, were mixed with extracted RNA previously retrotranscribed in cDNA. After a time allowing for potential hybridization, ligase was added to link adjacent probes. Denaturation then removed the initial cDNA and ligated probes were amplified by PCR. The amplicons were then analyzed by capillary electrophoresis (3500XL). Hybridized probes yielded a size-specific signal and the height of each peak was proportional to the amount of RNA initially present in the preparation. In parallel, 6 of the cell lines were tested in gene expression profiling (GEP) (n=6) or sequenced by RNA-Seq (Illumina, n=3). Finally, RT-MLPA was also applied to 16 MCL FFPE lymph node samples and compared to 17 from diffuse large B-cell lymphomas (DLBCL).

**Results:** An expected high signal for CCND1, and significant levels of Ki67, were obtained in all cell lines while 6 had variable levels of SOX11. All results satisfactorily compared to RNA Seq and GEP data of the same cell lines. Exploration of the TP53 pathway disclosed a highly heterogeneous pattern among cell lines. Signals were interpretable in all RNA samples from FFPE lymph nodes but one, disclosing also between-patient heterogeneity. However, of interest, significantly different patterns were observed between MCL and DLBCL, with an overexpression of the TP53 pathway in the latter.

**Conclusion:** This work confirms the feasibility of the RT-MLPA technique to properly detect and semi-quantify MCL-related transcripts. The method was validated on representative MCL cell-lines. Its application to FFPE samples was demonstrated to be also feasible. This paves the way to further investigations of the transcriptome of lymphomas.

**Keywords:** diffuse large B-cell lymphoma (DLBCL); expression arrays; mantle cell lymphoma (MCL).

## 324
### CeVi: A UNIQUE CRYOPRESERVED HUMAN VIABLE CELL COLLECTION FROM LYMPHOMA PATIENTS, A CALYM INITIATIVE TO ACCELERATE INNOVATION AND ITS TRANSFER TO LYMPHOMA FIELD

<footnote n="1">¹Hematology Biology, Nantes University Hospital, Nantes, France;</footnote>

<footnote n="2">²INSERM_U1232, CRCINA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France; ³Nantes University Hospital, Pathology, Nantes, France; ⁴Nantes University Hospital, Hematology Clinic, Nantes, France</footnote>
<table><caption>TABLE 1</caption><caption>CeVi_Collection samples a March 2019</caption><thead><tr><th rowspan="2">Subtypes</th><th rowspan="2"></th><th colspan="3">Origin</th></tr><tr><th>Cases</th><th>%</th><th>Vials</th></tr></thead><tbody><tr><td rowspan="5">B-NHL</td><td>Follicular Lymphoma</td><td>309</td><td>19.6</td><td>1143</td></tr><tr><td>Diffuse Large B-cell Lymphoma</td><td>203</td><td>12.9</td><td>690</td></tr><tr><td>Marginal Zone Lymphoma</td><td>197</td><td>12.5</td><td>835</td></tr><tr><td>Mantle Cell Lymphoma</td><td>131</td><td>8.3</td><td>518</td></tr><tr><td>Other B NHLs*</td><td>234</td><td>14.9</td><td>1044</td></tr><tr><td>T-NHL</td><td></td><td>85</td><td>5.4</td><td>275</td></tr><tr><td>Hodgkin Lymphoma</td><td></td><td>160</td><td>10.2</td><td>586</td></tr><tr><td>Others**</td><td></td><td>14</td><td>0.9</td><td>78</td></tr><tr><td>Non Tumoral</td><td></td><td>240</td><td>15.3</td><td>1076</td></tr></tbody><tfoot><tr><td>TOTAL</td><td></td><td>1573</td><td>100</td><td>6245</td></tr></tfoot></table>

* e.g. Waldenström, lymphocytic lymphoma, ...

** e.g. Castelman, post-transplant lymphoma diseases

NHL: Non Hodgkin lymphoma

CeVi collection comprises a median 10M viable cells/vial (SD = 4.2), with a median 4 vials/sample. All samples are clinically and biologically annotated. Dry pellets and plasma are available for 70% and 12% cases, respectively. 180 lymph nodes are paired with PBMC +/- BM.

E. Mollaret<fnref n="1" /> | E. Gomez<fnref n="1" /> | B. Nadel<fnref n="1" /> | L. Naiglin<fnref n="1" /> | M. Anquetin² | C. Barau² | L. Baseggio³ | C. Bret⁴ | G. Brisou⁵ | P. Brousset⁶ | V. Camara-Clayette⁷ | G. Cartron⁸ | K. Da Cunha⁹ | P. Dartigues¹⁰ | J. Dupuis¹¹ | B. Ghaleh² | P. Gravelle⁶ | R. Houot¹² | O. Karmous-Gadacha⁴ | C. Laurent⁶ | J. Moreaux⁴ | C. Pangault¹³ | V. Ribrag⁷ | G. Salles¹⁴ | V. Szablewski¹⁵ | V. Thouault¹⁶ | G. Uze¹⁷ | V. Verge¹⁸ | L. Ysebaert¹⁹ | P. Gaulard²⁰ | T. Fest¹³

Rennes - Pontchaillou, Rennes, France; ¹⁷UMR 5235, CNRS / University Montpellier, Montpellier, France; ¹⁸Département de Biologie et Pathologie Médicales - Laboratoire d'hématologie, Gustave Roussy, Villejuif, France; ¹⁹Hématologie, IUC-Toulouse Oncopole, Toulouse, France; ²⁰Department of Pathology, AP-HP, Créteil, France

**Introduction:** Lymphomas are among the most heterogeneous diseases. Over 80 subtypes have been defined on the basis of clinical, morphological, phenotypical, genetic and molecular criteria. Today, the fine functional characterization of tumors within their microenvironment appears highly recommended, but remains poorly addressed due to the lack of comprehensive viable cell collections. Here, the CALYM Carnot Institute* reports the generation of a unique and comprehensive collection of viable cells from over 1000 clinically annotated patient samples.

*LYSA, LYSARC & 13 top French academic labs www.calym.org

## Materiel and Methods

* CeVi collection is built around 6 CeVi platforms supported by local Biological Resource Centers under CALYM's umbrella

* CeVi collects lymph nodes (LN), peripheral blood (PBMC), bone marrow (BM) and other lymphoma proliferation sites (e.g. spleen)

* Both, sample processing and storage are standardized & certified NF S96-900 (French QA policy), CeVi management is certified ISO 9001

* All viable cell samples are annotated, after anonymization, with clinical and biological data (authorized by French data protection authority, CNIL)

* Informed consent is available and validated by ad hoc ethical committee

* Sample requests are evaluated by a steering committee and granted based on scientific interest and sample subtype rarity.

<footnote n="1">¹R&D, Carnot CALYM Institute, Pierre-Bénite, France; ²Plateforme de Ressources Biologiques, APHP - Hôpitaux Universitaires Henri Mondor, Créteil, France; ³Laboratoire d'Hématologie, Groupement Hospitalier Sud/Hospices Civils de Lyon, Pierre-Bénite, France; ⁴Hématologie Biologique, CHU Montpellier, Montpellier, France; ⁵Hématologie Clinique, Groupement Hospitalier Sud/ Hospices Civils de Lyon, Pierre-Bénite, France; ⁶Pathology and Cytology Department, UMR1037 Centre de Recherche en Cancerologie de Toulouse, CHU Toulouse, IUCT Oncopole / Toulouse III Paul Sabatier University / Inserm, Toulouse, France; ⁷Laboratoire RT Hématologie (AMMICa INSERM US23/CNRS UMS3655), Gustave Roussy, Villejuif, France; ⁸Département d'Hématologie Clinique, CHU Montpellier, Montpellier, France; ⁹CRB Sud, Groupement Hospitalier Sud/ Hospices Civils de Lyon, Pierre-Bénite, France; ¹⁰Département de Biologie et Pathologie Médicales - Service de pathologie Morphologique, Gustave Roussy, Villejuif, France; ¹¹Unité Hémopathies Lymphoïdes, APHP - Hôpitaux Universitaires Henri Mondor, Créteil, France; ¹²Hématologie, CHU Rennes - Pontchaillou, RENNES, France; ¹³UMR-S 1236 / Laboratoire Hématologie, Université de Rennes I / CHU Rennes, Rennes, France; ¹⁴Hématologie Clinique, Hospices Civils de Lyon / Université Claude Bernard, Pierre Bénite, France; ¹⁵Pathologie et Oncobiologie, CHU Montpellier, Montpellier, France; ¹⁶Laboratoire d'hématologie, CHU</footnote>