bluuebunny/crossref_metadata_2025_split huggingface dataset:
B-Cell Receptor Configuration and Adverse Cytogenetics Are Associated with Autoimmune Hemolytic Anemia in Chronic Lymphocytic Leukemia
Authors: Maura Francesco , Visco Carlo , Falisi Erika , Gianluigi Reda , Fabris Sonia , Agnelli Luca , Tuana Giacomo , Lionetti Marta , Guercini Nicola , Novella Elisabetta , Nichele Ilaria , Montaldi Anna , Autore Francesco , Gregorini Anna Ines , Barcellini Wilma , Callea Vincenzo , Mauro Francesca Romana , Laurenti Luca , Foa Robin , Neri Antonino , Rodeghiero Francesco , Cortelezzi Agostino 
Published: October 2019

Abstract
Abstract 1780

Background:
Biological features related to the development of autoimmune hemolytic anemia (AHIA) in patients with chronic lymphocytic leukemia (CLL) are crucial insights in the understanding of the pathogenesis of autoimmune phenomena in the course of the disease.

Design and Methods:
We retrospectively analyzed 585 CLL patients with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration (HCDR3). Of them, 73 developed AIHA. The clinical characteristics at CLL diagnosis and follow-up were available in all patients, while cytogenetic analysis at the time of diagnosis was available in 409 patients.

Results:
Occurrence of AIHA was significantly associated with an IGHV unmutated (UM) status (p<0.0001) and unfavorable cytogenetic lesions [del(17)(p13) and del(11)(q23)] (p<0.0001). Stereotyped HCDR3 sequences were identified in 173 of 585 patients (29.6%) and were similarly represented among patients developing AIHA (28,7%) or not (29.6%). Of the stereotyped subsets, subset #3 was associated with a significantly higher risk of AIHA occurrence than the other HCDR3 configurations (p=0.004). Restricting the analysis to UM patients, a strong association was found between AIHA and “truly” UM patients, defined as patients carrying a 100% identity with the germ line configuration. Multivariate analysis showed that “truly” UM IGHV, del(17)(p13) and del(11)(q23) were the strongest independent variables associated with risk of developing AIHA (p=0.02, p=0.0002 and p=0.01, respectively).
Based on the results of the multivariate analysis, we constructed a risk score of developing AIHA during time, according to the presence of none (low risk = favorable cytogenetics and mutated (M) IGHV), one (intermediated risk = unfavorable cytogenetic or UM), or two (high risk = unfavorable cytogenetic and UM) risk factors. This scoring system allowed a significant patient risk stratification (Figure 1).

Conclusions:
Taken together, our data indicate that an UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped BCR subsets in a proportion of cases.

Disclosures:
No relevant conflicts of interest to declare.
Stromal Tumor Microenvironment in CLL: Regulation of Leukemic Progression
Authors: Chaturvedi Nagendra K , Ahrens Adam K , Shukla Ashima , Gilling Christine E , Mittal Amit K. , Bierman Philip , Weisenburger Dennis D , Lu Runqing , Joshi Shantaram S 
Published: October 2019

Abstract
Abstract 1781
Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patientsâ€™ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression.
Figure: Association of MYLK expression with time to first treatment Figure:. Association of MYLK expression with time to first treatment

Disclosures:
No relevant conflicts of interest to declare.
Characterization of Peripheral B Cells Expressing CLL-Associated Receptor Tyrosine Kinase ROR1 in Healthy Donors
Authors: Milleck Julia , Kreuzer Karl-Anton , Assenmacher Mario , Schmitz Jürgen , Jähn Peter S 
Published: October 2019

Abstract
Abstract 1787
Recent studies have described an extensive expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1) on the surface of malignant B cells in patients suffering from chronic lymphoid leukemia (CLL). ROR1 expression has also been detected in ovarian cancer, renal cancer, melanoma, and lung adenocarcinoma, suggesting a general role of ROR1 in cancer genesis and/or maintenance. However, low levels of ROR1 mRNA or protein expression have also been found on undifferentiated embryonic stem cells, adipose tissue and on early stage of B-cells in bone marrow, but not in major adult tissues and peripheral B cells.
In contrast, our results describe for the fist time expression of ROR1 on rare B cells in peripheral blood of healthy donors. Using magnetic cell sorting techniques we were able to enrich ROR1 positive B cells from PBMCs of healthy donors. The average frequency of ROR-1+ B cells in PBMCs ranged from 1×10−3 to 1×10−4. Interestingly, ROR1+ B cells revealed a heterogeneous phenotype and could further be classified into a CD19+/CD27+/CD38− and a CD19+/CD27−/CD38+ subpopulation as assessed by flow cytometry. Remarkably, in contrast to the CD27+ subpopulation the CD38+ subpopulation showed higher levels of CD5 and CD23 expression, which are both markers known to be upregulated on B cells of CLL patients. Both populations do not show elevated expression of transcription factor ZAP70 and Bcl-2 but expressed higher level of Bcl-6 compared to CD19 B cells. Bcl-6 is known to be required for pre-B cell self-renewal but has also been described to play an essential role in resistance of leukemic cells to therapeutic tyrosine kinase inhibitors. Moreover ROR1+ B cells revealed an immature and non-activated sate as evidenced by the expression of IgD and IgM, and the lack of IgG and CD69. Further functional examination and immunophenotyping of these rare cells are under investigation.
We have identified rare B cells expressing the tumor-associated receptor tyrosine kinase ROR1, which are heterogenic in CD27 and CD38 expression and show an immature phenotype. We are currently assessing a potential relationsphip ROR1+ peripheral B cells and ROR1 expressing B cells in malignancies like CLL, mantel cell (MCL) and marginal zone lymphoma (MZL).

Disclosures:
Milleck: Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Jähn:Miltenyi Biotec GmbH: Employment.
Minimal Residual Disease Stratification in Fludarabine Ccyclophosphamide Rituximab (FCR) Therapy in Chronic Lymphocytic Leukaemia (CLL
Authors: Hayat Amjad , O'Brien David , Quinn Fiona , Keane Fionnuala , Parker Imelda , Kelly Johanna , Wieczorkowska Marzena , Crotty Gerard , Leahy Maeve , Enright Helen , Hennessy Brian , Cahill Mary , Vandenberghe Elisabeth A. 
Published: October 2019

Abstract
Abstract 1791
Six courses of FCR is considered standard therapy for fit, non-(17p) deleted patients with Chronic Lymphocytic Leukaemia (CLL), however the optimal dose of Rituximab in combined chemotherapy for CLL or the number of FCR courses required has never been formally assessed. Minimal residual disease (MRD) eradication (assessed by 6 colour flow-cytometry(FC) is associated with an increased disease free interval and is a logical endpoint in determining length/efficacy of treatment. Serial MRD testing following therapy provides an objective test of disease re-emergence.
A multi-centre prospective phase II study in treatment-naïve CLL patients with modified FCR (Rituximab 375mg/m2) using MRD to determine treatment length/efficacy and identify disease emergence recruited from 2009–2012. Standard pre-treatment assessment plus C-T scan, FISH and Ig somatic hypermutation (SHM) analyses were performed. Patient in radiological and MRD-ve remission after 4 courses of FCR stopped therapy and the remainder received 6 courses. All were followed by 6 monthly MRD assessment.
Fifty-two patients were included (35M/17F), mean age 52 years (range 37–72), mean WCC 51 × 109/L (range 8–386), elevated LDH 23 of 45 (51%), lymphadenopathy 43 (83%) and hepatosplenomegaly in 37 (71%). Abnormal FISH results were, del(11q) in 15, +12 in 5, del(13q) in 18; SHM 15(29%) mutated and 37(71%) unmutated or with V3–21 gene usage. Post-treatment MRD is available in 43 patients; 36 (70%) were MRD –ve including 18 (42%) after 4 courses. Nine patients did not complete treatment (toxicity −7, progression-1, non-compliance-1). With a mean follow-up of 20.7 months (range 4.5–38), 6 patients have reverted to MRD +ve at a mean of 13 months (range 10–19) from therapy; only the patient with primary treatment failure has required further chemotherapy. Myelotoxoicty resulted in 23 NCI grade 3 episodes and 7 treatment delays of a mean duration of 25 days (range15–32). One further grade 3 toxicity of pneumonia was identified.
Modified FCR was effective in this patient cohort with high risk features (38% unfavourable FISH, 71% unfavourable SHM), with 70% patients achieving MRD-ve status on completion of therapy. 42% of patients became MRD-ve after 4 courses of FCR, suggesting that some patients may not require 6 courses of therapy. Myelotoxicity remains an issue with 7 patients not completing therapy. Longer follow-up will clarify whether shortened therapy will have an impact on MRD+ve reversion, time to re-treatment and survival.

Disclosures:
No relevant conflicts of interest to declare.
A 3-Dimensional Co-Culture Model to Investigate Adhesion-Mediated Drug Resistance in Multiple Myeloma
Authors: Aljitawi Omar S. , Xiao Yinghua , Ramachandran Karthik , Lipe Brea C. , Garimella Rama , Krishnan Amrita , Stehno-Bittel Lisa , Li Linheng 
Published: October 2019

Abstract
Abstract 1826

Background:
In multiple myeloma (MM), evidence suggests that the adhesion of malignant plasma cells to bone marrow microenvironment elements is central to MM growth and drug resistance, otherwise referred to as adhesion–mediated drug resistance. As other investigators have shown, the MM-stroma interaction is in part mediated by adhesion molecules like N-cadherin.

Goal:
To develop a 3-dimensional MM in vitro co-culture model where MM cells interact with stromal elements mimicking the bone marrow microenvironment and use the model to investigate N-cadherin mediated drug resistance.

Methods:
We co-cultured U266 cells, a MM cell line, with mesenchymal stromal cells (MSCs) isolated from Wharton's jelly (WJ) in a synthetic copolymer (polyglycolic acid/ poly L-lactic acid 90/10 copolymer) (3-dimensional culture conditions). For controls, we cultured U266 cells over a monolayer of WJMCS (2-dimensional conditions) or U266 cells in suspension. Cell proliferation was measured by Ki-67 immunohistochemistry (IHC) stain and lambda light chain expression was measure by enzyme-linked immunosorbent assay (ELISA) and IHC stains. Bortezomib in 1, 5, and 10 nM concentrations was used to treat U266 cells for 24 hours under the three culture conditions. Cytotoxicity was measured by modified Alamar Blue assay.

Results:
U266 cells in our 3-D model adhered to stromal cells and formed clusters close to matrix material. U266 cells proliferated as evidenced by positive Ki-67 stain, and expressed lambda light chain by IHC and ELISA. Lambda light expression measured by ELISA peaked at 1 week of co-culture in the 3-D model. Though N-cadherin was expressed in both stromal cells and U266 MM cells in 3-D, it was expressed only in stromal cells in 2-D culture conditions. Finally, pre-incubating U266 cells with N-cadherin blocking antibody resulted in less population of the 3-D model by U266 MM cells. Treatment of U266 with bortezomib resulted in less cell cytotoxicity in 3-D and 2-D co-culture conditions compared to U266 cells treated in suspension.

Conclusions:
Using a 3-D in vitro co-culture model, we demonstrated that only 3-D co-culture conditions resulted in N-cadherin expression in U266 MM cell line. We also demonstrated that U266 population of the 3-D model was successfully blocked using an N-cadherin blocking antibody. We propose N-cadherin mediated adhesion as a mechanism to explain reduced cytotoxicity to bortezomib in 3-D. We propose this model to be used to investigate adhesion-mediated drug resistance.

Disclosures:
No relevant conflicts of interest to declare.
The CD317-Targeted Immunotoxin HM1.24-ETA′ Efficiently Eliminates Malignant Plasma Cells in Vitro and in Vivo
Authors: Staudinger Matthias , Glorius Pia , Kellner Christian , Guenther Andreas , Repp Roland , Burger Renate , Gramatzki Martin , Peipp Matthias 
Published: October 2019

Abstract
Abstract 1838
Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma still needs improvement. Recently antibody-based targeted therapies with a toxic payload have documented impressing activity. HM1.24 (CD317), a surface molecule overexpressed on malignant plasma cells, is efficiently internalized and may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the generation and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described.
HM1.24-ETA′ was generated by genetic fusion of a CD317-specific single-chain Fv antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and the protein was purified to homogeneity by affinity chromatography. HM1.24-ETA′ efficiently inhibited growth of myeloma cell lines (INA-6, RPMI8226, U266) analyzed in MTT assays. Half maximal growth inhibition was observed at low nanomolar concentrations. Target cell killing occurred via induction of apoptosis as indicated by annexin V / propidium iodide staining and analysis of PARP cleavage. The delivery of HM1.24-ETA′ to target cells is antigen-specific, because excess of unconjugated parental antibody completely blocked the cytotoxic effect. The proliferation of IL-6 dependent INA-6 was efficiently reduced by HM1.24-ETA′ even in co-culture experiments with bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA′ strongly triggered apoptosis (up to 80% annexin V-positive cells) of freshly isolated tumor cells from 5 of 5 myeloma patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA′ immunotoxin (p < 0.04).
Thus, HM1.24-ETA′ immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of multiple myeloma using immunoconjugates with toxic payloads.

Disclosures:
No relevant conflicts of interest to declare.
LONG-TERM Risk of Late Effects After Treatment for EARLY-STAGE HODGKIN'S Lymphoma (HL).
Authors: Viviani Simonetta , Russo Anna DI , Bonfante Valeria , Valagussa Pinuccia , Bonadonna Gianni , Gianni Alessandro M. 
Published: October 2019

Abstract
Abstract 2732

Background:
Currently the vast majority of patients (pts) with early-stage HL can be cured. However they still face the lifetime risks of late secondary events, mainly secondary malignancies (SM) and cardiovascular complications (CV). To assess the impact of late events on Overall Survival (OS), we evaluated the incidence of SM and CV in pts with early-stage HL (I A, B, II A), enrolled in a prospective randomized trial comparing 4 cycles of ABVD followed by subtotal nodal irradiation (STNI, arm A) with 4 cycles of ABVD followed by involved field irradiation (IFRT, arm B).

Material and Methods:
From 1990 to 1995, pts with early stage, defined as stage I A and B and II A, ± bulky disease were randomized. After 4 cycles ABVD, RT was STNI (36 Gy on involved sites and 30 Gy on uninvolved sites) were delivered in arm A and IFRT (36 Gy) in arm B. As previously reported (Bonadonna G, J Clin Oncol 2004;15:2835), no significant survival difference was documented between the two arms. We extended follow-up to evaluate the incidence of late secondary events.

Results:
140 pts were randomized, 68 to arm A and 72 to arm B. Median follow-up is 210 months (range:1–264). At 20 years, freedom from progression (FFP) is 95% for arm A and 90% for arm B (p=ns). Eight deaths occurred in arm A and 6 in arm B for a 20-yr OS of 84% for ABVD-STNI and 92% for ABVD-IFRT (p=ns).Cutaneous basal cell carcinoma excluded, a total of 13 SM were documented in arm A (1 MDS after 18 mos from treatment start, and 13 solid tumors after a median of 172 mos (range, 22–237): 7 breast, 2 prostate, 1 ovarian, 1 sigma, 1 cutaneous squamous cell) whereas only 3 SM (3 breast) occurred in arm B after a median fu of 170 mos (range, 160–198). The cumulative incidence of SM at 20 yr is 38% in arm A and 7% in arm B. Overall fatal myocardial infarction was documented in one pt in arm A and in two in arm B; fatal stroke occurred in 1 pt in arm A. Thirty-seven pregnancies occurred in 29 out of 70 premenopausal females (41%) after a mendian of 74 mos from treatment start; 35 males consented to have semen analysis: 63% were normospermic and 37% had some semen abnormality (oligo- ± astheno-± teratospermia) after a median of 42 mos; overall 10 pts fathered.

Conclusions:
These findings confirm that ABVD given for 4 cycles followed by reduced extent of irradiation (IFRT) resulted in excellent long-term OS and FFP with an important decrease in the incidence of malignant secondary event. This manageable treatment strategy can still be considered standard treatment for early-stage HL. Long-term follow-up of current combined modality therapy, where lower doses of RT and shorter courses of chemotherapy are applied in favorable presentations, and more intensive chemotherapy regimens like BEACOPP in unfavorable presentations, should lead to even less late secondary events and mortality.

Disclosures:
Gianni: Hoffman-La Roche: Consultancy, Honoraria.
Phase I Trial of Combination Therapy with 90y Ibritumomab Tiuxetan and Gemcitabine in Patients with Non-Hodgkin's Lymphoma, Final Report.
Authors: Borghaei Hossein , Schilder Russell J. , Litwin Samuel , Millenson Michael , Cohen Adam D , Thibodeau Linda , Smith Mitchell R 
Published: October 2019

Abstract
Abstract 2753
Y90-ibritumomab tiuxetan radioimmuotherapy (Y90-RIT) is an effective therapy against CD20+ lymphomas approved for use in patients (pts) with relapsed/refractory low grade, follicular, or transformed B-cell non-Hodgkin's lymphoma (NHL), as well as consolidation of first remission low grade NHL. Gemcitabine (Gem) also is active against NHL and is a potent radiation sensitizer. We conducted a phase I trial to assess the safety of concomitant administration of Y90-RIT and G in patients with NHL. Eligible pts had any histologic subtype of recurrent CD20+ NHL (not candidates for potentially curative options) and met standard Y90-RIT criteria: platelets 150,000/ul; < 25% bone marrow involvement by lymphoma; prior radiation to <25% of bone marrow and no prior bone marrow or stem cell transplant. Initially, nine pts in three cohorts were treated with 250 mg/m2 of Gem IV on days 1 and 8 of the Y90-RIT treatment program (rituximab + 111In-ibritumomab day 1 and rituximab + Y90 ibritumomab day 8), with Y90-RIT at 0.2, 0.3 or 0.4 mCi/kg respectively. We confirmed that a standard Y90-RIT dose of 0.4 mg/kg can be safely administered with Gem at 250 mg/m2. In subsequent cohorts, escalating doses of Gem were used according to a Bayesian based system. Response evaluation was by CT scan criteria (IWG JCO 1999). Between 2004–2012, twenty pts were treated (10 follicular (FL), 3 marginal zone (MZL), 7 diffuse large B-cell (DLBCL) lymphomas). Median age is 71.5 (range 55–82). The median number of prior treatments is 3 (range 1–6). Gem doses ranged from 250 mg/m2 to the maximum planned dose of 800 mg/m2 on days 1 and 8. One DLT occurred (thrombocytopenia) and MTD was not reached. Treatment-related toxicities consisted of grades 3 (N=11) and 4 (N = 2) neutropenia, grade 3 (N=11) leukopenia, grades 3 (N=14) and 4 (N=8) thrombocytopenia. One grade 3 infection occurred, unrelated to study drugs. All pts recovered counts to ≤ grade 1 by week 12. The only grade 3 non-hematologic toxicity was elevated bilirubin in 1 and increased GGT in 2 pts. Best responses seen include: 3 CR/CRu, 7 PR, 4 SD, 4 PD and 2 patients still in follow up. Median PFS for all patients is 192 days. Median PFS for all non-DLBCL histologies (10 FL and 3 MALT) is 202 days and for DLBCL is 77 days.

Conclusion:
Standard dose Y90-RIT combined with gemcitabine days 1 and 8 is safe and well-tolerated at doses up to 800 mg/m2 in pts with relapsed/refractory NHL. Further investigation with the established doses in non-DLBCL histologies is warranted.

Disclosures:
Borghaei: Biogen-IDEC: Research Funding. Off Label Use: 90Y Ibritumomab Tiuxetan in relapsed DLBCL. Schilder:Biogen-IDEC: Research Funding. Smith:Biogen-IDEC: Research Funding.