Abstract
177
LuLu-DOTA-NNV003, a radioimmunoconjugate targeting CD37, is developed as novel radioimmunotherapy (RIT) treatment for patients with B cell non-Hodgkin’s lymphoma (NHL). Since patients ...are at risk for developing hematological toxicities due to CD37 expression on normal B cells, we aimed to develop
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Zr-labeled NNV003 for positron emission tomography (PET) imaging as a surrogate tool to predict
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LuLu-DOTA-NNV003 RIT whole-body distribution and tumor uptake. NNV003 antibody was first radiolabeled with
89
Zr.
89
ZrZr-
N
-sucDf-NNV003 tumor uptake was evaluated by PET imaging of mice bearing human CD37-expressing REC1 B cell NHL or RAMOS Burkitt’s lymphoma xenograft tumors followed by ex vivo analysis. Finally, CD37-targeting of
89
ZrZr-
N
-sucDf-NNV003 and
177
LuLu-DOTA-NNV003 RIT were compared.
89
ZrZr-
N
-sucDf-NNV003 accumulated in REC1 tumors over time, which was not observed for non-specific,
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In-labeled IgG control molecule. In RAMOS tumor-bearing mice,
89
ZrZr-
N
-sucDf-NNV003 tumor uptake was higher than
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InIn-DTPA-IgG at all tested tracer protein doses (10 µg, 25 µg and 100 µg;
P
< 0.01), further confirming
89
ZrZr-
N
-sucDf-NNV003 tumor uptake is CD37-mediated.
89
ZrZr-
N
-sucDf-NNV003 and
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LuLu-DOTA-NNV003 RIT showed similar ex vivo biodistribution and tumor uptake in the RAMOS tumor model. In conclusion,
89
ZrZr-
N
-sucDf-NNV003 PET imaging can serve to accurately predict CD37-targeting of
177
LuLu-DOTA-NNV003. To enable clinical implementation, we established a good manufacturing practice (GMP)-compliant production process for
89
ZrZr-
N
-sucDf-NNV003.
The 2016 World Health Organization classification defines diffuse large B-cell lymphoma (DLBCL) subtypes based on Epstein-Barr virus (EBV) infection and oncogenic rearrangements of
as drivers of ...lymphomagenesis. A subset of DLBCL, however, is characterized by activating mutations in
We investigated whether
mutations could improve the classification and prognostication of DLBCL. In 250 primary DLBCL,
mutations were identified by allele-specific polymerase chain reaction or next-generation-sequencing,
rearrangements were analyzed by fluorescence
hybridization, and EBV was studied by EBV-encoded RNA
hybridization. Associations of molecular features with clinicopathologic characteristics, outcome, and prognosis according to the International Prognostic Index (IPI) were investigated.
and
mutations were identified in 29.6% and 12.3%,
, and
rearrangements in 10.6%, 13.6%, and 20.3%, and EBV in 11.7% of DLBCL, respectively. Prominent mutual exclusivity between EBV positivity, rearrangements, and
mutations established the value of molecular markers for the recognition of biologically distinct DLBCL subtypes.
-mutated DLBCL had a significantly inferior 5-year overall survival than wild-type
DLBCL (log-rank;
=0.019). DLBCL without any of the studied aberrations had superior overall survival compared to cases carrying ≥1 aberrancy (log-rank;
=0.010).
mutations retained their adverse prognostic impact upon adjustment for other genetic and clinical variables by multivariable analysis and improved the prognostic performance of the IPI. This study demonstrates the clinical utility of defining
-mutated DLBCL as a distinct molecular subtype with adverse prognosis. Our data call for sequence analysis of
in routine diagnostics of DLBCL to optimize classification and prognostication, and to guide the development of improved treatment strategies.
Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of ...PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL.
We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively.
MYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196.
The low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.
Antigen presentation by tumor cells in the context of Human Leukocyte Antigen (HLA) is generally considered to be a prerequisite for effective immune checkpoint inhibitor therapy. We evaluated cell ...surface HLA class I, HLA class II and cytoplasmic HLA-DM staining by immunohistochemistry (IHC) in 389 classical Hodgkin lymphomas (cHL), 22 nodular lymphocyte predominant Hodgkin lymphomas (NLPHL), 137 diffuse large B-cell lymphomas (DLBCL), 39 primary central nervous system lymphomas (PCNSL) and 19 testicular lymphomas. We describe a novel mechanism of immune escape in which loss of HLA-DM expression results in aberrant membranous invariant chain peptide (CLIP) expression in HLA class II cell surface positive lymphoma cells, preventing presentation of antigenic peptides. In HLA class II positive cases, HLA-DM expression was lost in 49% of cHL, 0% of NLPHL, 14% of DLBCL, 3% of PCNSL and 0% of testicular lymphomas. Considering HLA class I, HLA class II and HLA-DM together, 88% of cHL, 10% of NLPHL, 62% of DLBCL, 77% of PCNSL and 87% of testicular lymphoma cases had abnormal HLA expression patterns. In conclusion, an HLA expression pattern incompatible with normal antigen presentation is common in cHL, DLBCL, PCNSL and testicular lymphoma. Retention of CLIP in HLA class II caused by loss of HLA-DM is a novel immune escape mechanism, especially prevalent in cHL. Aberrant HLA expression should be taken into account when evaluating efficacy of checkpoint inhibitors in B-cell lymphomas.
Purpose Our aim was to capture the biology of classical Hodgkin lymphoma (cHL) at the time of relapse and discover novel and robust biomarkers that predict outcomes after autologous stem-cell ...transplantation (ASCT). Materials and Methods We performed digital gene expression profiling on a cohort of 245 formalin-fixed, paraffin-embedded tumor specimens from 174 patients with cHL, including 71 with biopsies taken at both primary diagnosis and relapse, to investigate temporal gene expression differences and associations with post-ASCT outcomes. Relapse biopsies from a training cohort of 65 patients were used to build a gene expression-based prognostic model of post-ASCT outcomes (RHL30), and two independent cohorts were used for validation. Results Gene expression profiling revealed that 24% of patients exhibited poorly correlated expression patterns between their biopsies taken at initial diagnosis and relapse, indicating biologic divergence. Comparative analysis of the prognostic power of gene expression measurements in primary versus relapse specimens demonstrated that the biology captured at the time of relapse contained superior properties for post-ASCT outcome prediction. We developed RHL30, using relapse specimens, which identified a subset of high-risk patients with inferior post-ASCT outcomes in two independent external validation cohorts. The prognostic power of RHL30 was independent of reported clinical prognostic markers (both at initial diagnosis and at relapse) and microenvironmental components as assessed by immunohistochemistry. Conclusion We have developed and validated a novel clinically applicable prognostic assay that at the time of first relapse identifies patients with unfavorable post-ASCT outcomes. Moving forward, it will be critical to evaluate the clinical use of RHL30 in the context of positron emission tomography-guided response assessment and the evolving cHL treatment landscape.
PARP inhibitors have been shown to increase the efficacy of radiotherapy in preclinical models. Radioimmunotherapy results in selective radiation cytotoxicity of targeted tumour cells. Here we ...investigate the combined effect of anti-CD37 β-emitting 177Lu-NNV003 radioimmunotherapy and the PARP inhibitor olaparib, and gene expression profiles in CD37 positive non-Hodgkin's lymphoma cell lines.
The combined effect of 177Lu-NNV003 and olaparib was studied in seven cell lines using a fixed-ratio ray design, and combination index was calculated for each combination concentration. mRNA was extracted before and after treatment with the drug combination. After RNA-sequencing, hierarchical clustering was performed on basal gene expression profiles and on differentially expressed genes after combination treatment from baseline. Functional gene annotation analysis of significant differentially expressed genes after combination treatment was performed to identify enriched biological processes.
The combination of olaparib and 177Lu-NNV003 was synergistic in four of seven cell lines, antagonistic in one and both synergistic and antagonistic (conditionally synergistic) in two, depending on the concentration ratio between olaparib and 177Lu-NNV003. Cells treated with the combination significantly overexpressed genes in the TP53 signalling pathway. However, cluster analysis did not identify gene clusters that correlate with the sensitivity of cells to single agent or combination treatment.
The cytotoxic effect of the combination of the PARP inhibitor olaparib and the β-emitting radioimmunoconjugate 177Lu-NNV003 was synergistic in the majority of tested lymphoma cell lines.
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