bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations In Transcription Factor NF-E2 Confer Resistance To Interferon Treatment In MPN Patients
Authors: Jutzi Jonas S. , Gründer Albert , Kaiser Sandra , Nikoloski Gorica , Jansen Joop H. , Pahl Heike L.
Published: October 2019

Recently, we described insertion and deletion mutations in transcription factor NF-E2 in a subset of patients with Myeloproliferative Neoplasms (MPN), most frequently patients with primary or secondary myelofibrosis (PMF or sMF, respectively) as well as patients with polycythemia vera (PV). These mutations lead to the formation of truncated NF-E2 proteins that lack DNA binding activity. Nonetheless, mutant NF-E2 proteins augment wild-type NF-E2 activity both in vitro and in vivo in a murine bone marrow transplant model. Mice expressing mutant NF-E2 develop erythrocytosis and thrombocytosis.

Here, we demonstrate that, in contrast to mice expressing wild-type NF-E2, treatment of mice expressing mutant NF-E2 with interferon-alpha does not lower red blood cell numbers (RBC), white blood cell counts (WBC) or platelet numbers. While these parameters are significantly decreased by interferon-alpha (IFN) in mice expressing wild-type NF-E2, mice expressing mutant NF-E2 showed no response to interferon treatment in these counts over a four month period.

Two PV patients carrying truncating mutations in NF-E2 received IFN treatment. Blood samples were available both from before treatment as well as from 3 and 4 years after the beginning of therapy, respectively. Purified granulocytes from these samples were analyzed for mutant NF-E2 allele burden by GeneScan analysis. In both patients, the mutant NF-E2 allele burden rose during IFN treatment, increasing from 1% to 30% in one patient and from 24% to 100% in the second.

We have demonstrated that acquisition of an NF-E2 mutation confers a proliferative advantage on cells already carrying the JAK2V617F mutation. Using colony assays from primary MPN cells, we showed that cells carrying both mutations outcompete cells carrying only the JAK2V617F mutation in vivo. Cells expressing both mutant NF-E2 and JAK2V617F are statistically significantly more frequently in the S-phase of the cell cycle and express significantly higher levels of several proteins that promote G1/S transition, cyclin D3, CDK4 and CDK6. The data presented here suggest that IFN treatment is insufficient to counteract the proliferative drive conferred by mutant NF-E2.

Disclosures:

No relevant conflicts of interest to declare.
Identification Of FOXO3A As a Key Downstream Target For The Oncogenic JAK2V617F
Authors: Kobayashi Masaki , Araki Marito , Hironaka Yumi , Ohsaka Akimichi , Komatsu Norio
Published: October 2019

A gain-of-function mutant JAK2 has been demonstrated to play a causal role in myeloproliferative neoplasms (MPN) development. The mutant JAK2, typically a substitution of position 617 valine (V) to phenylalanine (F) (hereinafter called JAK2V617F), activates STAT, Akt, and ERK pathways, which subsequently promotes cell proliferation. Although these pathways are well documented, their downstream effectors in MPN are still poorly understood.

FOXO3A, a subfamily of Forkhead transcription factors, paradoxically regulates cell death and survival in a variety of cells. We and others have previously shown that an oncogene product Bcr-Abl tyrosine kinase suppresses FOXO3A activity by phosphorylation and subsequently represses cell death in chronic myelogenous leukemia (CML), proposing a potential therapeutic strategy for CML treatment. Thus, defining FOXO3A function and uncovering its regulation in MPN are important for the understanding of this disease and the development of a novel therapeutic strategy.

To demonstrate this, we employed a model cell line Ba/F3/EpoR that requires IL-3 for survival but becomes independent of the cytokine when JAK2V617F is expressed. When JAK2V617F-expressing Ba/F3/EpoR cells were treated with a JAK2 inhibitor, FOXO3A phosphorylation diminished in a dose-dependent manner. The trend of diminution paralleled to the reduction of phospho-STAT5, a major JAK2V617F substrate, suggesting that FOXO3A phosphorylation is regulated by JAK2V617F activity. Removal of JAK2 inhibitor from the media induced a massive FOXO3A phosphorylation within 20 minutes in JAK2V617F-expressing but not in the wild-type JAK2-expressing Ba/F3/EpoR cells. This observation indicates that at least in this model system, JAK2V617F positively modulates the phosphorylation of FOXO3A.

To further investigate the function of FOXO3A in JAK2V617F-expressing Ba/F3/EpoR cells, we knocked down FOXO3A expression by shRNA. When treated with a JAK2 inhibitor to induce apoptotic cell death, FOXO3A-knocked down cells were more resistant to the inhibitor than the control cells. This strongly suggests that when JAK2V617F activity is blocked and subsequently FOXO3A is dephosphorylated, FOXO3A induces apoptosis in JAK2V617F-expressing cells.

Finally, we elucidated that the inhibition of JAK2V617F activity results in a reduction of FOXO3A phosphorylation and the induction of apoptosis in CD34-positive cells obtained from MPN patients. Taken together, we propose a model that shows JAK2V617F induces FOXO3A phosphorylation and subsequently blocks FOXO3A activity to repress apoptosis in JAK2V617F positive MPN cells.

Disclosures:

Kobayashi: Sysmex Corporation: Employment. Komatsu:Sysmex Corporation: Research Funding.
Use Of 46/1 Haplotype Permits To Follow JAK2V617F Clonal Architecture In PV Patients: Clonal Evolution and Impact Of IFNα Treatment
Authors: Hasan Salma , Cassinat Bruno , Le Couédic Jean-Pierre , Favale Fabrizia , Monte-Mor Barbara , Lacout Catherine , Solary Eric , Villeval Jean-Luc , Casadevall Nicole , Kiladjian Jean-Jacques , Vainchenker William , Plo Isabelle
Published: October 2019

Somatic V617F oncogenic mutation of the protein kinase JAK2 is the most prevalent genetic abnormality in the three myeloproliferative neoplasms (MPNs), namely polycythemia vera (PV, 95%), essential thrombocythemia (ET, 55%) and myelofibrosis (MF, 50%). About 30% of PV patients are homozygous for this mutation due to mitotic homologous recombination (HR). JAK2 46/1 haplotype is strongly associated with the cis-aquisition of JAK2V617F mutation. Since, HR involves most of 46/1 haplotype, JAK2V617F and 46/1 tagging SNPs are also reduced to homozygosity. We hypothesized that 46/1 tagging SNPs, which are in complete linkage disequilibrium with JAK2, can serve as a measure of JAK2V617F homozygosity. 46/1 allele burden (X%) can be used to calculate the HR (HR%) that is a measure of JAK2V617F/V617F clones [(X%-50%)x2]. JAK2V617F/V617F frequency and JAK2V617F allele burden (Y%) can be then exploited to calculate the frequency of JAK2V617F/WT [(Y%-RH%)x2] and JAK2WT [100-(JAK2V617F/WT%+ JAK2V617F/V617F)] clones. The purpose of this study was to calculate the clonal frequency of WT, JAK2V617F/WT and JAK2V617F/V617F in progenitors compartments of PV patients.

Here, we have dissected the JAK2V617F clonal architecture in 9 PV patients heterozygous for the 46/1 haplotype. First, we measured the global JAK2V617F and 46/1 allele burden in CD34+ cells either by allele-specific PCR or by Ion Torrent sequencing in order to calculate the expected WT, JAK2V617F/WT and JAK2V617F/V617F clones. Next, we compared thees results with the experimental clonal frequency of WT, JAK2V617F/WT and JAK2V617F/V617F clones in individual colonies derived from the CD34+CD38+ compartment. This algorithm was validated in majority of patients. Moreover, we have exploited this formula to the terminally differentiated polynuclear neutrophils (PNN) and found that JAK2V617F/V617Fclones acquire strong amplification during differentiation.

Finally, we used this model to assess the therapeutic potential of IFNα in a cohort of 15 PV patients. IFNα exposure resulted in more impressive and rapid decrease of JAK2V617F allele burden of pure JAK2V617F homozygous patients than in pure JAK2V617F heterozygous patients. Calculations revealed this decrease in JAK2V617F is due to preferential targeting of JAK2V617F/V617Fclones in responding patients.

These results demonstrate that JAK2 46/1 haplotype can be used to estimate JAK2V617F clonal architecture in PV patients. This simple modeling can be useful to follow the efficacy and specificity of treatment on JAK2V617F clones in MPNs, without needing exploration at the unicellular level. In addition, it suggests that IFNα treatment more specifically targets the JAK2V617F/V617F clone in responding patients.

Disclosures:

No relevant conflicts of interest to declare.
CD8+ /ICOS+ /CXCR5+ Tumor Infiltrating T-CELLS (CD8fc) Sharing Functional Similarities With TFH CELLS ARE Present In Classical Hodgkin’S Lymphoma Tissues Displaying A Specific “MIXED NODULARITY” Pattern
Authors: Le Thi Suong , Broussais Florence , Bouabdallah Reda , gondois-Rey Françoise , Xerri Luc , Olive Daniel
Published: October 2019

We have previously reported that some classical Hodgkin’s Lymphoma (cHL) tissues display a gene signature evocative of a Th1 immune reaction. In order to better characterize this process, immune cell subsets were isolated from cHL tissue samples (n=21) using a powerful multicolor flow cytometry method, in parallel with cell sorting. Fresh tissue samples from follicular B cell lymphoma (FL, n=8), diffuse large cell B cell lymphoma (n=8) and reactive lymphadenitis (n= 5) were used as controls.

In 4 cLH cases, we observed a significant proportion of activated CD8+ T-cells expressing ICOS and CXCR5 at high levels. The presence of either CD8+/ICOS+/CXCR5- T cells or CD8+/ICOS +/ CXCR5+ T-cells was a specific feature of HL tissues since it was absent from B-cell lymphomas, T-cell lymphomas and reactive tissues. In contrast, CD8+/CXCR5+ T-cells were found not only in cHL, but also in most other samples analyzed.

Further phenotypic characterization showed that the CD8+/ICOS +/ CXCR5+ T cells expressed markers associated with CD4 TFH cells, like PD1, BTLA, bcl-6 and IL-21. Under stimulation, they expressed only low levels of IFNG, granzyme B and perforin, and thus do not fulfill the criteria of activated cytotoxic effectors. Co-culture experiments showed a dramatic enhancement of CD86 expression on stimulated B-cells in contact with CD8+/ICOS +/ CXCR5+ T cells. This effect was similarly observed after co-culture with CD4+TFH cells. The 4 cHL cases associated with CD8+/ICOS +/ CXCR5+ T-cells contained CD30+ CD15+ EBV+ Reed Sternberg cells (RSC). They were characterized a nodular non-sclerotic pattern reminiscent of the nodular lymphocyte-rich classical HL (NLRCHL) subtype, but also displayed a specific “mixed nodularity” feature. Various nodules were indeed observed, including reactive germinal centers (GC) partly colonized by RSC co-localizing with CD8+/ICOS+ T-cells, suggesting an early GC invasion triggering an intra-follicular CD8 T-cell reaction. Other nodules were composed of a high number of RS cells admixed with numerous CD8+/ICOS+ T-cells. This “mixed nodularity” pattern was absent in the other HL cases.

Altogether, our results point out a previously unrecognized intra- follicular CD8 T-cell subset sharing phenotypic and functional features with CD4 TFH, that we have thus considered as putative “follicular cytotoxic” CD8 T-cells (TFC). This cell subset appears to be specifically associated with EBV+ cHL tissues with unusual histo-phenotypic features, which may probably reflect a strong CD8 activation process.

Disclosures:

No relevant conflicts of interest to declare.
MCL-1 Critically Regulates Survival Of T-Lymphocyte Non-Hodgkin Lymphoma
Authors: Spinner Sabine , Gemma Kelly , Herold Marco , Pechloff Konstanze , Crispatzu Giuliano , Ruland Jürgen , Herling Marco , Peschel Christian , Strasser Andreas , Jost Philipp J.
Published: October 2019

The heterogeneous subgroup of T-lymphocyte non-Hodgkin lymphoma (T-NHL) exhibits an aggressive growth pattern and is relatively insensitive to chemotherapy. The molecular mechanisms of survival and resistance to therapy remain largely undefined. We hypothesized that BCL-2 proteins with anti-apoptotic properties protect T-NHL cells from cell death, cause resistance to therapy and therefore represent an attractive target for therapy. MCL-1 potently protects physiologically healthy T-lymphocytes from cell death, but its function in transformed T-cells is incompletely understood. We therefore dissected the role of MCL-1 using in silico meta-analyses on available gene expression data on human primary T-NHL samples. We identified MCL-1 as the primary BCL-2 family protein to be highly expressed across most major T-NHL subsets. Expression of MCL-1 was restricted to its anti-apoptotic full-length splice variant as opposed to its pro-apoptotic short isoform. To functionally characterize the requirement for MCL-1, we utilized a T-NHL mouse model, which is based on four consecutive low-dose whole-body γ-irradiations in 4 week-old mice resulting in the development of T-NHL mimicking human peripheral T-cell lymphoma. We utilized mice harbouring loxP-flanked Mcl-1 or loxP-flanked Bcl-x(L) in combination with inducible Cre recombinase to conditionally delete the gene of interest after lymphoma induction. Interestingly, conditional deletion of only one allele of Mcl-1 in fully established primary T-NHL cells ex vivo led to a significant and specific loss of viability, whereas survival remained unaffected by full deletion of Bcl-x(L). Reduced MCL-1 levels resulted in substantially elevated sensitivity to standard chemotherapeutics such as anthracyclins, cyclophosphamide and etoposide. In addition, mono-allelic deletion of Mcl-1 in vivo prolonged survival of lymphoma-bearing mice. Together, these data argue that anti-apoptotic MCL-1 is the single most important BCL-2 family member involved in the sustained survival of T-NHL.

Disclosures:

Strasser: Genentech Inc: Consultancy.
A New Prognostic Index In Diffuse Large B-Cell Lymphoma Using Serum Albumin: A Pilot Study Evaluating The Albumin Adjusted-International Prognostic Index (A-IPI)
Authors: Dalia Samir , Chavez Julio C , Bello Celeste M. , Chervenick Paul A. , Little Bryan J , Al Ali Najla H , Sokol Lubomir , Sotomayor Eduardo M. , Lee Ji-Hyun , Fisher Kate , Thompson Zachary , Choi Bong-jin , Shah Bijal D.
Published: October 2019

Introduction

Low serum albumin (SA) has been identified as a prognostic marker in multiple hematological malignancies including diffuse large B-cell lymphoma (DLBCL). Low SA may be an indicator of worsening disease biology, increased inflammation, or increased co-morbidities. Changing demographics and improved care of the elderly may abrogate some of the risk attributable to age. SA, however, may not be influenced by such changes. Currently the Revised International Prognostic Index (R-IPI), the currently best accepted model for prognosis in DLBCL, does not account for albumin. We therefore explored the use of albumin as a prognostic variable in conjunction with the covariates included in the R-IPI.

Methods

Patients with DLBCL treated between 2007-2010 with RCHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) were retrospectively identified using the Moffitt Total Cancer Care platform. Age, ECOG performance status, LDH, extrandoal sites of disease, stage, and albumin were collected and analyzed using Random Forest Modeling. Both age and extranodal involvement failed to retain statistical significance and were dropped from the model, leading to the generation of a new algorithm, the Albumin Adjusted IPI (A-IPI). This score assigned a point for SA<3.7g/dL, LDH >the upper limit of normal, ECOG performance status >=2, and Ann Arbor stage III-IV. Progression free and overall survival was compared by the risk groups using Kaplan-Meier curves along with the log rank test. Statistical analysis was done using R statistical software.

Results

A total of 124 patients were identified. Median age was 58 years with 62% male. 46% were over the age of 60 at diagnosis and 63% were Ann Arbor stage 3 or 4. The A-IPI score identified three groups of patients (Very good: A-IPI=0, Good: A-IPI=1-2, Poor: A-IPI=3-4), similar to the R-IPI. PFS and OS at 4 years were compared using the A-IPI and R-IPI (Table 1).

Conclusions

In comparison to the R-IPI, the A-IPI appears to better define poor risk patients while retaining the ability to discriminate well between low (“Very Good”) and intermediate (“Good”) risk groups. Confirmation in a larger and prospective cohort is indicated, however, these preliminary data suggest SA may be a better surrogate for co-morbid status, pro-inflammatory states, and worse disease biology than age in patients with DLBCL.

Disclosures:

No relevant conflicts of interest to declare.
A “Universal” Lectin-Mediated Interaction Drives B-Cell Receptor Signaling In Primary Follicular Lymphoma Cells
Authors: Packham Graham , Krysov Serge , Coelho Vania , Johnson Peter , Stevenson Freda K.
Published: October 2019

B-cell receptor (BCR) signaling has been identified as a critical driver of B-cell malignancies and as a target for therapeutic attack. Clinical responses to novel inhibitors of BCR-associated kinases have been relatively modest in follicular lymphoma (FL) and a more detailed knowledge of BCR function in these cells is required. Surface Ig (sIg) is unusual in FL since variable regions contain N-linked glycosylation sites which are introduced by somatic mutation. These are rarely found in normal B cells, indicating strong positive selective pressure in malignant cells. Remarkably, added sugars terminate with high mannose suggesting a novel function for FL BCRs in binding to, and perhaps receiving stimulation via, microenvironmental mannose-binding lectins. In previous studies we demonstrated that candidate mannose-binding lectins, including DC-SIGN, promote intracellular calcium mobilization in primary FL cells, but not normal B cells. In this work, we characterized in more detail the response of FL cells to DC-SIGN and its inhibition by BCR-targeted kinase inhibitors.

Initial studies using immunoblotting demonstrated that, like anti-Ig antibodies, DC-SIGN caused increased phosphorylation of the downstream kinases AKT and ERK in primary FL samples. DC-SIGN treatment also resulted in increased expression of the MYC oncoprotein in a subset of samples. In contrast to FL samples, DC-SIGN did not increase AKT or ERK phosphorylation in normal B cells although anti-IgM induced strong responses in these cells. Overall, responses to DC-SIGN were similar in IgM+ and IgG+ FL samples. Flow cytometry demonstrated that DC-SIGN also increased phosphorylation of proximal signaling kinases (SYK and BTK), as well as phosphorylation of PLCγ2 in FL samples, and that DC-SIGN-induced signaling occurred within the CD19+BCL2+ malignant cells. Flow cytometry also revealed intraclonal variation in responses to DC-SIGN and, like responses to anti-Ig, DC-SIGN responses were strongest in a sub-population of malignant cells with high CD20 expression. Finally, we demonstrated that tamatinib, the active form of the SYK inhibitor pro-drug fostamatinib, significantly inhibited phosphorylation of ERK and PLCγ2 induced by either anti-Ig or DC-SIGN.

Overall our results are consistent with the hypothesis that N-linked glycosylation sites, introduced into BCRs by somatic mutation, are selected for in FL since they confer signaling responsiveness following binding of environmental lectins. Like canonical antigen signaling, lectin-mediated signaling via the BCR appears to be susceptible to therapeutic blockade using kinase inhibitors. However, further analysis of this novel lectin-mediated pathway may reveal novel targets for optimal therapeutic attack.

Disclosures:

No relevant conflicts of interest to declare.
Lack Of Clinical Benefit For Routine Surveillance Imaging For Diffuse Large B-Cell Lymphoma In First Complete Remission
Authors: Vose Julie M , Pingali Ravi , Wagner-Johnson Nina , Loberiza Fausto R. , Fenske Timothy S. , Jewell Sarah , Bast Martin , Bartlett Nancy L. , Armitage James O.
Published: October 2019

Background

The use of routine surveillance imaging (RSI) for patients in first complete remission (CR1) following front-line rituximab (R) based anthracycline therapy remains controversial. We compared patients with diffuse large B-cell lymphoma (DLBCL) who received an R-CHOP or a similar regimen, obtained a CR and then were followed by either RSI or clinical surveillance (CS) in which scans were only performed for signs or symptoms.

Methods

Patients from three tertiary care center from 2001-2011, who achieved a CR1 with frontline R-CHOP or similar therapy for DLBCL, and had a minimum follow up of 1 year were analyzed. Patients with HIV-related lymphoma, transformed lymphoma, and post-transplant lymphoproliferative disorders were excluded. Patients with composite lymphoma were included only if the DLBCL component was >50%. Patients were stratified into two groups based on the surveillance strategy employed. Baseline patient characteristics, prognostic features, treatment type, and outcomes were compared.

Results

391 patients with DLBCL treated with R-CHOP or similar regimens who obtained CR1 were analyzed. There were 129 patients in the CS group and 262 in the RSI group. Patient characteristics (age, gender, stage, and IPI) were similar in the two groups. The median follow up is 5 years (range 1 – 12). Relapse after CR1 was detected in 26 (20%) of patients in the CS group and 46 (18%) of the RSI group. The median number of images in the CS group was 0 (range 0-14) and 4 (range 1-27) in the RSI group, p<0.0001. The median average number of images per year of follow up in the CS group was 0 (range 0-6) and 1 (range 1-13) in the RSI group, p<0.0001. Relapses were detected through clinical manifestations in 100 % of CS cases versus 43% in RSI cases, p=0.01. The 5 year progression-free survival (PFS) was 76% in the CS group and 82 % in the RSI group (p = 0.31). The 5 year overall survival (OS) was 87% in the CS group and 92 % in the RSI group (p=0.15). The table shows an analysis of OS by IPI and type of surveillance.

Conclusions

The majority of relapses in patients with DLBCL after achieving CR1 to an R-CHOP or similar regimen occur when signs or symptoms of the disease lead to evaluation and are not detected by RSI. Although asymptomatic relapses are occasionally detected by RSI, in this large cohort of patients neither a PFS nor OS benefit could be demonstrated in favor of RSI. Given the additional cost, radiation exposure and risk of additional procedures, we conclude that the use of RSI in patients with DLBCL in CR1 has limited clinical utility.

Disclosures:

No relevant conflicts of interest to declare.
The Role Of Modified Glasgow Prognostic Score As Predictor In Diffuse Large B Cell Lymphoma Treated With The R-CHOP Regimen
Authors: Kim Yundeok , Kim Yu Ri , Kim Soo Jeong , Jieun Jang , Shin-Young Hyun , Dohyu Hwang , Kim Jin Seok , Hong Min Yoo , Cheong June-Won , Hyun Lee Jung
Published: October 2019

Background

The modified Glasgow Prognostic Score (mGPS), an inflammation-based prognostic score consisting of serum C-reactive protein (CRP) levels and serum albumin levels, shows significant prognostic value in several types of tumors. However, the prognostic value of GPS in lymphoma remains unclear. In this study, we evaluated the prognostic significance of mGPS in patients with diffuse large B cell lymphoma (DLBCL).

Methods

The medical records of 285 DLBCL patients (156 males, 128 females) who were treated with R-CHOP chemotherapy at Yonsei University Severance Hospital were retrospectively reviewed. Patients with an elevated CRP level (³10 mg/L) and hypoalbuminemia (<3.5 g/dL) were given a score of 2, patients with CRP elevated were assigned a score of 1, and patients with neither of these abnormalities were scored 0.

Results

The study population included 285 patients with a median age of 58 years (range, 21-87 years). According to mGPS classification, 204 patients (71.5%) had an mGPS of 0, 57 patients (20%) had an mGPS of 1, and 24 patients (8.5%) had an mGPS of 2. Our study found that high mGPS were associated with poor prognostic factors including older age (>60 years), extranodal (EN) involvement, advanced disease stage, unfavorable IPI scores, and the presence of B symptoms. After 3 cycles of R-CHOP chemotherapy, 144 of the 285 treated patients (50.5%) achieved complete response (CR) or CR unconfirmed (CRu). The CR rate after 3 cycles of R-CHOP chemotherapy was significantly higher in patients with mGPS of 0 (53.8%) compared to those with mGPS of 1 (33.3%) or 2 (25.0%) (P=0.001). Fifty-four (30.2%) patients eventually died. The estimated 5-year progression-free survival (PFS) and overall survival (OS) rates for 285 patients were 77.2% and 72.8%, respectively. Patients with mGPS of 0 had significantly better OS than those with scores of 1 (5-year PFS, vs. 74.1%) or 2 (80.9% vs. 54.7; P=0.036). Patients with mGPS of 0 also displayed significantly better OS than patients with mGPS of 2 (P<0.001). The 5-year PFS rate was 76.1% in patients with mGPS scores of 0, 62.3% in patients with scores of 1, and 66.4% in those with scores of 2. Patients with lower mGPS scores tended to have longer PFS compared to patients with higher mGPS, although it was statistically insignificant (P=0.112). Multivariate analyses revealed that the GPS score was a prognostic factor for the CR rate of 3 cycle R-CHOP therapy (P=0.044) as well as OS (P=0.037).

Conclusion

Similar to several other cancers, GPS can be an independent predictor of survival outcomes in DLBCL patients treated with R-CHOP therapy.

Disclosures:

No relevant conflicts of interest to declare.
Interactions Between PD-1 and PD-L1 and PD-L2 Promote Malignant B-Cell Growth in Waldenstrom Macroglobulinemia
Authors: Grote Deanna M. , Ziesmer Steven C. , Hodge Lucy S. , Price-Troska Tammy L. , Novak Anne J. , Yang Zhi-Zhang , Ansell Stephen M
Published: October 2019

Programmed death 1 (PD-1) is a T cell co-inhibitory receptor with two ligands, PD-L1 and PD-L2. In B-cell malignancies, this pathway plays a major role in immune resistance in the tumor environment by promoting the differentiation of regulatory T-cells and inducing T-cell exhaustion. Recent data have shown that blockade of this pathway can enhance antitumor immune responses. While interactions between PD-1 and its ligands have been studied in various lymphoid tissues, little data exist regarding these interactions in the bone marrow, particularly in patients with Waldenstrom macroglobulinemia (WM). The goal of this study was therefore to determine the expression of PD-1 and its ligands in the bone marrow of patients with WM and to assess the functional role of PD-1 signaling in this disease.

Using immunohistochemistry, the expression of PD-1, PD-L1 and PD-L2 in bone marrow (BM) specimens from patients with WM (n=6) was compared to normal control BM. PD-1 expression was modest and the pattern of staining in WM was similar to normal BM. In contrast however, intense staining for PD-L1 and PD-L2 was seen in WM compared to controls. When analyzed by flow cytometry, PD-L1 and PD-L2 expression was seen on malignant B-cells and also on dendritic cells, monocytes and macrophages isolated from the bone marrow. To determine the functional effect of PD-1 signaling in WM, we stably transfected PD-L1 and PD-L2 into the HEK-293 cell line. The WM cell lines, MWCL-1, BCWM.1 and RPCI-WM1 as well as patient specimens, were then cocultured with either the PD-L1 or PD-L2 expressing HEK-293 cells. When compared to cells expressing an empty vector control, proliferation and viability of WM cells was increased when they were cocultured with cells expressing PD-L1 or PD-L2. Addition of blocking antibodies abrogated the increase in proliferation.

These results show that PD-L1 and PD-L2 are highly expressed on both malignant B-cells and non-malignant cells within the BM microenvironment in WM and promote malignant B-cell growth. Inhibition of PD-1 signaling may therefore be a promising therapeutic strategy in patients with WM.

Disclosures:

No relevant conflicts of interest to declare.
Oral Arsenic Trioxide Based Regimen As Salvage Treatment For Relapsed Or Refractory Mantle Cell Lymphoma
Authors: Gill Harinder , Au Wing-Yan , Cheung Winnie W.W. , Kwong Yok Lam
Published: October 2019

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoproliferative disorder, and relapsed/refractory disease has a poor prognosis. In patients with relapsed / refractory MCL, optimal treatment strategy remains undefined. Oral arsenic trioxide (As2O3) was initially developed for the treatment of relapsed acute promyelocytic leukemia (APL). As2O3 inhibits neoplastic cellular proliferation by a wide array of mechanisms, including induction of apoptosis, targeting of signaling pathways, and down-regulation of BCL-2. Evidence in vitro also suggested that As2O3 might be effective in lymphoma, but clinical data are hitherto not available. In this study, we investigated the use of an oral-As2O3-based regimen for the treatment of patients with relapsed / refractory MCL. Thirty-nine patients (men=34, women=5) at 64 (41–82) years of age with relapsed/refractory MCL, who had received 2 (1–5) prior regimens and were ineligible for high-dose chemotherapy, were treated with a continuous oral regimen, comprising oral arsenic trioxide (oral-As2O3), chlorambucil and ascorbic acid. The overall response rate was 49% (complete response: 28%; partial response: 21%). Only grade 1/2 toxicities were observed (hematologic: 56%, hepatic: 8%). Independent prognostic factors for response were increased lactate dehydrogenase (P=0.04) and unfavorable MCL international prognostic index (P=0.04). At a median follow up of 21(range:1-118) months, the median progression-free-survival (PFS) was 16 months, and overall-survival (OS) 38 months. The 2-year and 5-year PFS were 41% and 29% respectively. The 2-year and 5-year OS were 56% and 43% respectively. Independent prognostic factors for PFS were female gender (P=0.002), Eastern Cooperative Oncology Group (ECOG) performance score of 2 (P=0.009), and non-response to treatment (P<0.001). Independent prognostic factors for OS were female gender (P<0.001), ECOG performance score of 2 (P=0.03), non-response to treatment (P<0.001), and disease progression while on treatment (P=0.05). These findings showed that an oral regimen of oral-As2O3, chlorambucil and ascorbic acid was an active regimen with minimal toxicity in relapsed/refractory MCL, achieving durable responses in some cases.

Disclosures:

No relevant conflicts of interest to declare.
Real World Data On Primary Treatment For Mantle Cell Lymphoma 2000-2011 – a Nordic Lymphoma Group Observational Study
Authors: Abrahamsson Anna , Lindblad Alexandra Albertsson , Brown Peter de Nully , Wennerholm Stefanie Baumgartner , Pedersen Lars Moller , d'Amore Francesco , Nilsson-Ehle Herman , Jensen Paw , Pedersen Michael , Geisler Christian H. , Jerkeman Mats
Published: October 2019

Background

There is a consensus that younger patients with mantle cell lymphoma (MCL) should receive intensive immunochemotherapy regimens including high-dose cytarabine, rituximab and high-dose chemotherapy with stem cell support, but the optimization of treatment for elderly or unfit patients, as well as patients with localized or indolent disease, remains a challenge.

Methods

Our study is based on data from the Swedish and Danish Lymphoma Registries from the period of 2000-2011, in Sweden supplemented by review of patients´ records. The Lymphoma Registries comprise >95% of all diagnosed lymphoma patients in Sweden and Denmark.

Results

1389 patients were diagnosed with MCL, confirmed by positive cyclin-D1 and CD5 staining, in Sweden and Denmark between 2000 and 2011. In this population based cohort, we could confirm the prognostic impact of MIPI, but in addition, male sex was associated with inferior overall survival (OS) in multivariate analysis (HR 1.4, p<0.001). Treatment with the Nordic MCL2 regimen (n=324) was associated with superior outcome compared to the majority of other regimens (3-year OS: 80%). In patients >65 years, chlorambucil (n=132) was superior to CVP (n=35) (HR 1.8, p=0.003), when adjusted for MIPI and rituximab, but there was no significant difference between CHOP (n=311) and CHOP+Cytarabine (n=84). Rituximab (HR 1.5, p<0.001) and autologous stem cell transplantation (HR 1.9, p<0.001 ) per se were both associated with prolonged OS in multivariate analysis. Forty-three (3.1%) patients with stage I-II disease received radiotherapy as primary treatment with curative intent showing an estimated 3-year OS of 93%. A small proportion, 29 patients (2.1%), were followed without treatment. Estimated 3-year OS for this group of patients was 79%.

Conclusion

By a population-based approach, we were able to provide novel data on prognostic factors and primary treatment of MCL, applicable to routine clinical practice.

Disclosures:

No relevant conflicts of interest to declare.
Network Analysis Of Single Cells Identifies DDIT3 As a Nucleator Of Erythroid Signatures In Early Erythro-Myelopoiesis
Authors: Pina Cristina , Teles Jose , Fugazza Cristina , Guo Yanping , May Gillian , Peterson Carsten , Enver Tariq
Published: October 2019

Analysis of individual cells within phenotypically-uniform stem and progenitor populations reveals heterogeneity of cell states and affords exploration of their differential contribution to fate transitions. Herein, we investigate the transcriptional composition of lineage commitment transitions in the mouse hematopoietic system, obtained either spontaneously or driven by individual transcription factors (TF), to define alternative molecular states at the emergence of erythroid and myeloid lineage identity.

To this end, we transcriptionally profile hundreds of individual cells across a spectrum of lineage states by quantitative RT-PCR on a Fluidigm platform. We explore the dual binary (on-off) vs. continuous (expression level) nature of PCR data and respectively calculate odds ratio, and Spearman correlation on co-expressing cells, for any pair of genes, in order to infer the circuitry underlying alternative routes to lineage commitment. This approach identifies Ddit3 as a specific target of GATA-driven networks that antagonizes Cebpa transcriptionally at the emergence of myeloid fate.

Knockdown and knockout approaches indicate a requirement for Ddit3 in early erythroid decisions from stem cells. Enforced expression of Ddit3 in granulocytic-monocytic progenitors (GMP) results in down-regulation of individual Cebpfamily members and their downstream transcriptional programmes, including myelo-monocytic cytokine receptors. Notably, the resulting cells have robust self-renewal potential and express an early erythroid signature that is augmented in the presence of interleukin-3 and erythropoietin.

Taken together, the data suggest that Ddit3 can nucleate incipient erythroid-affiliated programmes in early erythro-myelopoiesis, and may contribute to fate choice through antagonism with Cebp networks. Interestingly, ex-vivo culture and expression profiling of individual wild-type and DDIT3-enforced GMP suggest that the erythroid-primed state captured through Ddit3 expression may be found in a small proportion of native GMP, suggesting some fluidity of lineage identity in early progenitors.

Disclosures:

No relevant conflicts of interest to declare.
Patterns Of Care and Outcomes Of Anthracyclines In Grade 3 Follicular Lymphoma (FL)
Authors: Nastoupil Loretta J. , Rai Ashish , Lipscomb Joseph , Williams Jessica N , Ward Kevin , Howard David H , Flowers Christopher R.
Published: October 2019

Background

The role of anthracyclines in the management of Grade 3 (G3) FL is unclear. Furthermore, the patterns of care and outcomes of first-line treatment strategies for G3 FL in older adults are not clearly established. We describe the patterns of use, determinants of treatment, and survival outcomes of first-line management strategies for G3 FL with emphasis on four common first-line regimens: cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), rituximab (R) plus CHOP (R-CHOP), cyclophosphamide, vincristine, and prednisone (CVP), and R plus CVP (R-CVP).

Methods

We used the linked Surveillance, Epidemiology, and End Results -Medicare database to identify 1,308 G3 FL patients (pts) diagnosed between 1995 and 2009 and focused on pts diagnosed between 1999 and 2009 when claims with R appear. We ascertained first-line management strategies from Medicare claims made within 90 days of diagnosis. We used multiple variable logistic regression models to evaluate the relationship between pt characteristics and the use of first-line R-CHOP. We used Kaplan-Meier estimators stratified by FL stage to evaluate survival functions for first-line management strategies and performed multiple variable Cox proportional hazards regressions adjusted for pt demographics, comorbidity index, disease characteristics, and year of diagnosis to compare the impact of first-line management strategies on survival.

Results

Of the 1,308 G3 FL pts, 59% were female, 91% were Caucasian, 3% were African American, 44% had stage III/IV disease, 6% had B-symptoms, and 36% had extranodal involvement. The median age at diagnosis was 75 (interquartile range 70-80). Common first-line management strategies were: observation (obs), 29%; R-CHOP, 26%; CHOP, 9%; radiotherapy alone (XRT), 8%; R alone, 8%; R-CVP, 7%; and CVP, 5%. The use of R-CVP and R-CHOP increased over time (Figure 1).

In the cohort of pts diagnosed between 1999 and 2009 the use of R-CHOP was less commonly associated with age >80 years (ref. age 66-70 years; OR 0.19; 95% CI 0.11-0.31), comorbidity index ≥ 2 (ref. index =0; OR 0.54; 95% CI 0.31-0.92), and more commonly associated with stage III/IV FL (OR 1.46; 95% CI 1.05-2.02), and year of diagnosis (ref. years 1999-2001; OR for 2002 3.68; 95% CI 1.77-7.64; steady increase thereafter).

The table displays median survival and hazard ratios (HRs) for first-line management strategies. The most favorable outcomes were associated with first-line R-chemotherapy (R-Chemo). Among first-line R-chemo regimens, R-CHOP was associated with the most favorable outcomes (Figure 2).

Conclusion

R-CHOP is the most commonly used first-line regimen in the United States for older adult pts with FL G3. Even after controlling for disease characteristics and comorbidity, R-CHOP was associated with the most favorable survival outcome.

Disclosures:

No relevant conflicts of interest to declare.
Early Ofatumumab Treatment For High-Risk, Treatment-Naive Patients With Chronic Lymphocytic Leukemia
Authors: Jain Nitin , Keating Michael J. , Ferrajoli Alessandra , Konopleva Marina , Thomas Deborah A. , Pemmaraju Naveen , DiNardo Courtney D. , O'Brien Susan , Wierda William G.
Published: October 2019

Background

Ofatumumab is a human IgG1-kappa monoclonal antibody that binds to CD20 on normal B cells and on B chronic lymphocytic leukemia cells, resulting in B cell lysis. Ofatumumab has single-agent activity in patients (pts) with refractory CLL (Wierda, JCO 2010). Pts with CLL who have early stage disease (Rai 0-II) but have high-risk prognostic markers such as deletion 17p or deletion 11q have an increased risk of disease progression. Currently, these pts are offered watch-and-wait approach. The objective of this Phase II study is to evaluate the efficacy of ofatumumab in treating these pts with the goal to delay time to first chemoimmunotherapy treatment.

Methods

Pts with CLL/SLL were eligible provided they had high-risk for progression based on the presence of at least one of the following features: Rai stage II, serum beta-2 microglobulin (β2M) ≥3 mg/L, absolute lymphocyte count ≥25,000/µL, unmutated (≤2%) IGHV gene or mutated IGHV3-21, ZAP70 positive, CD38 positive (≥ 30%), or 11q or 17p deletion by FISH. Pts having a 2008 IWCLL/NCI-WG indication for CLL treatment were not eligible. Pts with Rai stage III-IV CLL were not eligible. Ofatumumab 300 mg dose 1, then 1000 mg weekly for 7 additional weeks (8 doses) was administered. Response assessment, including bone marrow evaluation, was done at least 2 months after last dose of ofatumumab and pts were followed for progression-free survival and time to first chemoimmunotherapy.

Results

Twenty-five pts (9 women, 16 men) were enrolled so far. The median age was 59 yrs (range, 40-81). The baseline characteristics are listed in the Table. Fifty percent of pts had unmutated IGHV gene. Thirty-four percent of pts had high-risk cytogenetic by FISH analysis. The median follow-up on the study is 4.7 months (range, 0.5-26). Grade 3-4 adverse events included infusion reaction in 6 pts. Autoimmune hepatitis with grade 4 ALT, grade 4 AST, and grade 4 alkaline phosphatase elevations was seen in 1 pt. Other grade 3-4 toxicities included rash (1 pt), shingles (1 pt), and tumor lysis (1 pt). Tumor lysis was seen in the pt with the WBC count of 207 K/µL. Eighteen pts (7 too early) are evaluable for response. Responses are as follows: 6 CR, 3 nPR, 3 PR, and 6 with stable disease. Three pts have progressed at 18.8, 14.1 and 3.2 months after starting the study treatment; 2 pts had unmutated IGHV gene (FISH negative) and one pt had trisomy 12 (IGHV mutation status unknown). None of the pts with deletion 17p or deletion 11q have progressed. All pts are alive at this time. The median overall follow up time is 7.6 months (range, 1-28).

Conclusions

Ofatumumab is well tolerated in pts with early stage CLL and may delay time to first chemoimmunotherapy.

Disclosures:

Ferrajoli: GlaxoSmithKline: Research Funding.
Multiple Myeloma Profile In Latin America: Clinical and Epidemiological Observational Study
Authors: Hungria Vania T M , Maiolino Angelo , Martinez Gracia Aparecida , De Souza Carmino A , Bittencourt Rosane , Bruggemann Peters Lygia Goretti , Colleoni Gisele W. B. , Oliveira de Oliveira Luciana Correa , Sousa Lais , Coelho Erika , Pasquini Ricardo , Magalhaes Silvia M. M. , Nunes Renata , Pinto Neto Jorge Vaz , Delboni Faria Rosa Malena , de Souza Mair Pedro , Hamerschlak Nelson , Fantl Dorotea Beatriz , Navarro Juan Ramon , Conte Guillermo , Almaguer David Gomez , Ruiz-Arguelles Guillermo , Durie Brian G M
Published: October 2019

Introduction

Little is known about the incidence and clinical features of Multiple Myeloma (MM) in Latin America. A clinical registry of Latin American (LA) patients with MM represents an opportunity to gain insight into the prevalence of the disease in this region, the patterns of care and the current treatment status in different LA countries.

Objective

To characterize the demographic and clinical features of patients with multiple myeloma from five LA countries (Brazil, Argentina, Chile, Mexico and Peru) and to create a LA database on MM; in addition to investigating the patterns of care for MM patients in Latin America.

Patients and Methods

This is an observational, non-intervention study, with a prospective evaluation of data. Eligible patients were diagnosed with multiple myeloma, between January 1, 2005, and December 31, 2007, at any one of the participating centers, regardless of disease stage or treatment modality. The follow-up period extended to at least 5 years for each patient (December 31, 2012).

Results

Eight hundred and seventy six patients were included. The median age was 60 years old (25-97), 53.4% male and 46.6% female. The median follow-up was 31.4 months, and the median overall survival was 57 months. The median overall survival to patients who received high-dose chemotherapy was 77 months and for patients who received conventional chemotherapy was 48 months (p<0.001). The multivariate prognostic model included patient baseline variables that were associated with mortality in the Kaplan-Meier univariate analyses. Only hypercalcemia, DSS II and III, ISS stage III andnon- high-dose chemotherapy were independent predictors of mortality.

Conclusion

This current study, which is the largest case series of MM patients in Latin America, recognizes the feasibility of large, collaborative, observational studies among various tertiary-care hematology centers in Latin America.

Note

We will present more details related to the demographic and most frequently used treatments in Latin America for newly diagnosed and relapsed patients in these LA countries.

Disclosures:

No relevant conflicts of interest to declare.
Vitro Culture and Regulation Of Osteoblasts and Cellular Immunity Function In Multiple Myeloma patients
Authors: Fu Rong , Gao Shan , Shao Zonghong
Published: October 2019

Objective

To investigate the biological characteristics of osteoblasts（OBs) cultured in vitro from bone marrow（BM) of multiple myeloma(MM) patients and to explore their generation and osteogenic potential. Then the affects by some factors such as bortezomib and MM patient serum on the OBs were observed. To explore the cellular immunity changes and the relationship between OBs and the immunity in MM.

Methods

OBs from BM of MM patients were cultured in vitro. The generation and osteogenic potential of OBs from MM patients and normal subjects were compared. The changes of their osteogenic potential and biological characteristics were observed. The antigens (CD34、CD138、CD45) on OBs and the quantities of T cell subsets，Dendritic cells(DC)，effective T cells、regulatory T cells and helper T cells (Th）were examined with flow cytometry assay. And then correlation was analyzed between OBs and the immunity. The levels of IL-7 were measured with ELISA. The BMP2 mRNAs were measured by RT-PCR.

Results

OBs from BM of MM patients could be cultured in vitro. The quantity of OBs from MM patients was less than normal subjects. There were calcium depositions in both groups after cultured for 4weeks culture. But the depositions of MM patients was less than that of normal controls. The OBs cultured with MM patient serum were less than those without patient serum. Bortezomib increased those from MM patients. CD138, CD45, CD34 were not detected on the cultured cells. The ratio of CD4+/CD8+、DC1/DC2、CD8+CD25+/CD3+T were reduced, that of helper T cell (Th1/Th2) was significantly decreased; the percentages of CD4+CD25+/ CD3+T and CD4+CD25+CD127low/CD4+T were significantly higher. Some of them were correlated with the quantity of OBs. The level of IL-7 in serum of MM patients was higher than that of normal controls. The expression of BMP2 mRNA was seen in the normal OBs and MM patients’ OBs culured with bortezomib, but not be seen in those without bortezomib.

Conclusions

OBs could be cultured in vitro from BM of MM patients. The proliferation and osteogenic potential of OBs from MM patients were decreased. Bortezomib was a positive regulatory of OBs and MM patient serum was a negative one. They both could affect the proliferation and osteogenic potential of OBs. There were down-regulations of quantity and function of T cell subgroup, DC cell, helper T cell, effector T cell while the regulatory T cells relatively raised. Predominantly of them were correlated with the quantity of OBs.

Disclosures:

No relevant conflicts of interest to declare.