bluuebunny/crossref_metadata_2025 huggingface dataset:
Coexistence of Mutated and Unmutated IgVH Forms in Patients with Chronic Lymphocytic Leukemia.
Authors: Sheikholeslami M. , Ma W. , Uyeji J. , Tornyos K. , Cone L. , Mamus S. , Keating M. , Albitar M.
Published: April 2020

The mutation status of the immunoglobulin heavy chain variable region (IgVH) gene is an independent prognostic indicator in patients with chronic lymphocytic leukemia (CLL). Unmutated IgVH is associated with rapid disease progression and shorter survival. The assay for determining IgVH mutation status depends on specific amplification of the mRNA of the expressed clonal IgVH. The presence of more than 1 clone in patients with CLL has been speculated and can be documented when the 2 clones express different light chains. Determining the IgVH gene family that is expressed in subclones also allows the confirmation of the presence of two clones in some cases. We describe 3 patients with two clones of CLL discovered in the process of determining the IgVH mutation status. PCR amplification of the expressed IgVH mRNA yielded 2 distinct bands. Sequencing each band separately revealed 2 different clones in each patient. Interestingly, each clone had a different mutation profile. In 2 of the 3 patients, the 2 clones were from completely different IgVH gene families (VH1-2 with VH2-5 and VH4-34 with VH5-51); both of these patients showed kappa light chain restriction. In the third patient the expressed IgVH gene family was VH3-21 in both clones, but 1 was mutated (6.8%) and the second was unmutated. Two of the 3 patients had evidence of aggressive disease with hepatosplenomegaly and lymphadenopathy; 1 of these patients expressed the VH3-21 gene family, which is known to be associated with aggressive disease irrespective of its mutation status. The other patient had a 9-year history of indolent disease without therapy. The intensity of the expressed IgVH clones as determined by PCR indicated that the mutated clones were dominant in 2 patients (approximately 80%). In the patient with VH3-21 expression, the unmutated clone accounted for 80% of total expressed IgVH. These data suggest that the presence of more than 1 clone should be considered when testing for IgVH mutation status and, more importantly, that evolution of a more aggressive second clone should be suspected. The presence of 2 clones—1 arising in naïve cells and the second in memory cells—suggests the possibility that the first hit occurred very early in the ontogeny of lymphocytes and that a second hit occurred at a later stage. However, the presence of naïve and memory clones in the same family raises the possibility that the naïve leukemic cells might still go through the germinal center programming process.
The VH3-21 Gene Status Correlates with Elevated β2-Microglobulin Serum Levels and Shorter Overall Survival of Patients with Chronic Lymphocytic Leukemia.
Authors: Lech-Maranda Ewa , Robak Tadeusz , Szmigielska-Kaplon Anna , Makuch-Lasica Hanna , Solarska Iwona , Jamroziak Krzysztof , Balcerczak Ewa , Mirowski Marek , Majewski Miroslaw P. , Warzocha Krzysztof
Published: April 2020

Introduction: The prognostic role of immunoglobulin variable heavy chain (IgVH) region somatic hypermutation status in B-cell chronic lymphocytic leukemia (B-CLL) is now well established. However, an overrepresentation of the VH3-21 gene segment has been recently reported to influence clinical course of B-CLL regardless of the IgVH mutational status in different geographic populations. It might suggest the role of a common antigen epitope in the pathogenesis of some B-CLL cases.

Aims: We therefore investigated the frequency of the VH3-21 gene usage in IgVH rearrangements in Polish population of B-CLL patients, and assessed whether it might influence B-CLL clinical characteristics and prognosis.

Patients and Methods: The study comprised of 97 B-CLL patients treated at the Department of Hematology, Medical University of Lodz in Poland. Peripheral blood mononuclear cells were separated on Ficoll and DNA was isolated using column method. The monoclonal population of lymphocytes with VH3-21-JH rearrangement was tested using framework 3 region primer and consensus JH primer described by Davies et al. (Leukemia 2004, 18: 872). The PCR reactions were followed with heteroduplex analysis of PCR products and separation in polyacrylamide gel. The positive VH3-21-JH samples were also confirmed by sequencing.

Results: The frequency of the VH3-21 gene segment in VH-JH rearangements in B-CLL patients reached 9% in Polish population. Beyond an elevated β2-microglobulin serum levels (p=0.03, Mann-Whitney test), no association were found between the VH3-21 gene status and clinical characteristics including CD38 expression at diagnosis The mortality rate was significantly higher in patients carrying VH3-21 (p=0.03, χ2 test), and majority of deaths were related to CLL. With a median follow-up of the surviving patients of 55,5 months (range 2–209 months), the subgroup of patients with the VH3-21 had significantly shorter overall survival (p=0.025, log-rank test). However, this genetic marker was not associated with freedom from progression survival.

Conclusions: We conclude that the frequency of the VH3-21 gene segment in VH-JH rearrangements in Polish population is similar to those previously published for British and Belgian populations and seems to be lower than in Mediterranean area populations (Blood2005, 15: 1678). The presence of VH3-21 gene segment correlates with increased β2-microglobulin levels at presentation and predicts shorter overall survival of B-CLL patients.
Complex Karyotype in Patients with Chronic Lymphocytic Leukemia.
Authors: Yañez Lucrecia , Cuadrado Maria Angeles , Insunza Andres , Mesones Belen Gonzalez , Bermudez Arancha , Conde Eulogio , Bureo Encarna , Iriondo Arturo
Published: April 2020

Chronic Lymphocytic Leukemia (CLL) is heterogeneous and there are two clinical forms with different outcome, in which initial stage and parameters of tumor burden, expression of CD38 and /or ZAP 70, chromosome abnormalities such as 11q23 and 17p13 are associated with a more aggressive course of disease.

The aim of this study is to analyse clinical and biological characteristics and outcome of patients with complex karyotype, and to asses if there are differences with other genomic aberrations.

Fifty patients diagnosed of CLL between 1980 and 1994 were studied (median age: 71 yrs; female/male: 18/32; median follow up 6.6 yrs.). Biological studies with flow cytometry and fluorescence in situ hybridization were done on cryopreserved blood cells. Clinical stages at diagnosis were: Rai stage 0: 56%; I: 20%; II: 16%; III: 6%; IV: 2%. Binet A: 72%; B: 20%; C: 8%. At the time of diagnosis 37% of patients needed treatment and 61% presented disease progression. At the time of this study, 10% of patients are alive with a median follow up of 15.3 yrs.

Seven patients (28%) presented complex karyotype. At the time of diagnosis 43% patients needed treatment and clinical stages were: Rai stage O: 29%, I: 14%, II: 43%, III: 14%, IV: 0%. Binet A: 43%; B: 43%; C: 14%. Median overall median survival was 72 months.

The most frequent genomic aberrations associated with complex karyotype were: del (17p13) and trisomy 12 (60 %). Cellular expression of CD38 and ZAP 70 was not able to separate this group.

Compared with other chromosome abnormalities, there were not important differences in clinical stage at diagnosis or expression of CD38 or ZAP 70, but overall median survival could distinguish three groups: del 11q22-23 or del 17p13 (34 months, p < 0,05) complex karyotype (72 months), and trisomy 12 or del 13q14 (79 months, p > 0,05).

In conclusion, complex karyotype is frequently formed by del 17p13 and trisomy 12 and identifies a subgroup of patients with better prognosis compared with isolated p53 deletion. More studies have to be realized to determine if trisomy 12 is a cell defence mechanism.
Involvement of PKC-δ in the Regulation of Notch2 in B-CLL.
Authors: Hubmann Rainer , Duechler Markus , Hilgarth Martin , Schnabl Susanne , Schwarzmeier Josef D. , Jaeger Ulrich , Shehata Medhat
Published: April 2020

We have recently shown that NOTCH2 signaling is involved in the overexpression of CD23 in B-cell chronic lymphocytic leukemia (B-CLL) cells (Hubmann et al., BLOOD 2002). There is an increasing evidence that NOTCH2 plays a determining role in the development/homeostasis of self-reactive CD5+ B-cells, suggesting a potential function of NOTCH2 in B-cell leukemogenesis. Here we study the regulation of NOTCH2 signaling in B-CLL cells and its possible function in B-lymphocytes using NOTCH2 transduced BL41 cells as a model system. Cultured B-CLL samples (n=30) lose their nuclear NOTCH2 (N2IC) activity within one day as demonstrated by electrophoretic mobility shift assays (EMSA). However, DNA-bound NOTCH2 complexes could be maintained in culture by exposure to the phorbolester TPA and is accompained by increased cell viability. The effect of TPA is prevented by the PKC-δ inhibitor Rottlerin indicating that PKC-δ is involved in the regulation of NOTCH2 signaling in B-CLL cells. The activity of N2IC in the leukemic cells appeared to be resistant to the γ-secretase inhibitors DAPT and compound E, two substances known to block ligand mediated release of the NOTCH2 intracellular domain (N2IC). Since B-CLL cells are locked in an anergic state, we next asked whether NOTCH2 modulates B-cell receptor (BCR) signaling and found that retrovirally transduced N2IC rescues the B-cell line BL41 from surface immunoglobulin M (sIgM) mediated apoptosis, a mechanism proposed to prevent the uncontrolled expansion of self-reactive CD5+ B-cells. In summary, our data suggest that B-CLL cells express an activated form of NOTCH2 which might be involved in the protection of the malignant clone from peripheral negative selection.
Interphase Fluorescence In Situ Hybridization Detection of Cytogenetic Abnormalities in B-Cell Chronic Lymphocytic Leukemia.
Authors: Xu Wei , Li Jianyong , Pan Jinlan , Li Li , Qiu Hairong , Xue Yongquan , Ruan Changgeng
Published: April 2020

The most frequent chromosomal abnormalities in B-cell chronic lymphocytic leukaemia (B-CLL) are deletions on 13q14 and 17p13, trisomy 12 and 14q32 rearrangement. Conventional metaphase cytogenetic analysis underestimates the frequency of specific chromosome aberrations in B-CLL due to the low rate of spontaneous mitoses and poor response to mitogen stimulation. The aim of this study was to investigate the incidence of chromosomal changes in bone marrow or peripheral blood cells (or both) of B-CLL patients using a molecular cytogenetic method, interphase fluorescence in situ hybridization (I-FISH). Probes for 13q14 (D13S319), 17p13 (P53 gene), the centromere of chromosome 12 (D12Z3) and 14q32 (Ig10 and Y6) were applied to detect chromosomal aberrations on bone marrow and peripheral blood smears from 83 B-CLL patients (60 male, 23 female,). Molecular cytogenetic aberrations were found in 60 (72.3%) cases, and 8 (9.6%) patients showed two kinds of abnormalities. The most frequent abnormalities detected in our patients was deletions of 13q14 in 34 cases (41.0%), followed by trisomy of chromosome 12 in 16 patients (19.3%), deletions of 17p13 in 10 patients (12%) and 14q32 rearrangement in 8 patients (9.6%). Statistical analyses were performed to correlate the molecular cytogenetic findings with Binet stages. No apparent differences in distribution were noted for anomalies del(13q14), del(17p13), +12 or 14q32 rearrangement among patients with various Binet stages. FISH was found to be a more rapid, exact and sensitive technique for the analysis of chromosome aberrations in CLL. FISH could provide accurate information of molecular cytogenetics for CLL.
Quantitative RT-PCR Assessment of Cannabinoid Receptor CB2 and Fatty Acid Amine Hydrolase in Neoplastic Cells of Patients with B-Cell Chronic Lymphocytic Leukaemia.
Authors: Piszcz Jaroslaw A. , Kloczko Janusz S. , Ziarko Slawomir , Radziwon Piotr , Mazgajska-Barczyk Katarzyna Z.
Published: April 2020

Background:

Chronic lymphocytic leukaemia (CLL) is one of the diseases in which processes of proliferation and apoptosis are seriously altered. Endocannabinoid system can modulate these processes. Cannabinoid receptor CB2 mainly expressed on lymphocytes and fatty acid amine hydrolase (FAAH) constitute a part of endocannabinoid system responsible respectively for interaction and degradation of cannabinoids. Both the receptor and enzyme are membrane proteins that can influence the lymphocyte fate. It has recently been suggested that this system may be a new potential target of anticancer therapy.

Aims:

The aim of our study was the assessment of receptor CB2 and FAAH mRNA expression in the lymphocytes B derived from patients with newly diagnosed chronic lymphocytic leukaemia.

Material and methods:

We assessed 15 peripheral blood samples from the CLL patients and 5 samples from healthy donors as a control group. The expression of CB2 receptor and FAAH proteins were measured indirectly by determination of their specific mRNA. The isolation of mononuclear cells from peripheral blood was carried out with the use of density centrifugation method. The resolution of mononuclear cell population to lymphocytes B subpopulation was done with negative isolation method utilising magnetic microballs. Total RNA was isolated from B-lymphatic cells of which 10ng was used in each reaction. RT-PCR was carried out with the use of Light Cycler (Roche) and respective commercial kits. The nucleotides sequence and parameters of the amplification process were based on method previously described by Matias et al (2002).

Results:

We found significantly higher mRNA levels of CB2 receptor in the study group in comparison to the control group. The levels of FAAH mRNA were also higher in the study group in comparison to the controls. Furthermore the number of mRNA copies was higher in case of CB2 receptor than FAAH in the CLL patients. Those numbers were comparable in the healthy controls. The results are presented as a mean number of copies in figure below.

Conclusions:

The results of our study show that during the leukaemic transformation there might be an alteration in the expression of some constituents of the endocannabinoid system. Those findings suggest the potential role of the endocannabinoid system in the neoplastic processes. Thus the assessment of the CB2 and FAAH mRNA might help to understand the biological reactions underlying the complex etiology of neoplastic disorders. Furthermore the explanation of all aspects of the role of endocannabinoid system in lymphoid malignancies should help to make it a new target of antileukaemic treatment.

figure1 figure1.
Comparison of IgH Gene Rearrangement Configuration between Hairy Cell Leukemia (HCL) and Hairy Cell Leukemia Variant (HCL-v).
Authors: Gonzalez David , Morilla Ricardo , Morilla Alison , Else Monica , Osuji Nnenna , Catovsky Daniel , Dearden Claire , Morgan Gareth J. , Matutes Estella
Published: April 2020

Hairy cell leukemia (HCL) is a rare B-cell disorder, with a variant form, which differs from the former in clinical behaviour, morphology and immunophenotype. The resemblance of these malignancies to splenic marginal zone lymphoma (SMZL), has led to the suggestion that they may share a common clonal progenitor that homes within the marginal zone of the splenic germinal centres. The immunophenotype and recent global gene expression results offer some support for this view, with a similar profile seen in HCL, and a subset of memory B-cells that are localized to the marginal zone. However, studies of the mutational status of the Ig genes are controversial. The majority of HCL cases are mutated, whereas up to 30% of SMZL cases are unmutated. Further investigation to understand these differences and to identify the cell of origin in these disorders is needed. In addition, there are no data on gene expression or VH mutation in HCL-variant (HCL-v), an entity with a poorer outcome and for which there is as yet no effective therapy.

We have analysed a series of 13 HCL and 23 HCL-v, for the configuration of the Ig heavy chain rearrangements. VDJH rearrangements were PCR-amplified and sequenced using an automated 3130xL sequencer (Applied Biosystems, Warrington, UK). The sequences obtained were compared to the closest germline segments using V-base (CRI, Cambridge, UK) in order to study the level of somatic hypermutation as well as gene segment usage in IgH rearrangements.

The majority of the HCL (92%) showed more than 2% deviation from the closest germline VH gene segment, compared to only 17/23 (74%) in the HCL-v cases. In the mutated cases, the percentage of mutation was similar for HCL and HCL-v (mean 5.8% and 5.9%, respectively), and none of the cases appeared to show ongoing hypermutation. Within the HCL-v group, 4 out of the 6 unmutated cases used VH4-34 segment, and the remaining 2 used VH1-03. Both genes are known to be associated with autoimmune diseases, and they are excluded from the normal and malignant memory plasma cell repertoires. Also, JH5 segment was found in 47% of the HCL-v, while it was absent in the HCL repertoire. This finding is particularly interesting, as JH5 segment is normally under-represented in most normal and malignant B-cells, except for a characteristic group of IgG-expressing chronic lymphocytic leukemias. Our data suggest that HCL-v is, unlike HCL, heterogeneous regarding IgVH mutations, but has restricted IgH repertoire that, at least in some cases, might be derived from activated, self-reactive B-lymphocytes that have not experienced the germinal-centre reaction.
Symptom Assessment in Patients with Chronic Lymphocytic Leukemia: Validation and Application of the M.D. Anderson Symptom Inventory.
Authors: Wang Xin Shelley , Ferrajoli Alessandra , Broadway Jackie B. , Mobley Gary M. , Mendoza Tito R. , Keating Michael J. , Cleeland Charles
Published: April 2020

CLL is the most prevalent form of adult leukemia. The life expectancy of patients affected by CLL varies significantly, with a large fraction of patients with survival expectation of 10 years or longer. Furthermore a number of patients are observed without treatment intervention. These patients have been labeled as “asymptomatic” and very little attention has been placed to the symptom burden that accompanies CLL. In order to measure the quality of life of patients with CLL, we explored the reliability and validity of the M. D. Anderson Symptom Inventory (MDASI). We enrolled 99 consecutive patients followed at our outpatient center. To assess and construct validity, we performed principal axis factor analysis with oblimin rotation. To assess reliability, we calculated separate coefficient alphas for the severity and interference subscale items. Patients were 63% male with a mean age of 59 years (range 29–79), with 43% age 60 years or older. 94% were White Non-Hispanic patients. 61% were employed. 83% had higher than high school education. 75% had Rai staging 0–1, and 53% had received prior treatment. Factor analysis of the MDASI items resulted in a two-factor solution (symptom severity and interferences) that satisfied criteria for interpretability and model fit in a confirmatory setting. Cronbach coefficient alphas for the MDASI were 0.91 for severity and 0.96 for interference. We also examined the criterion validity of the two MDASI subscales by investigating their correlation to similar subscales of the SF-12. MDASI “worst pain” was highly correlated with the “body pain” subscale of the SF-12 (r=−.84, p=.000). MDASI “fatigue” was significantly correlated with the SF-12 “energy” item (r=.79, p=.000). The patient self reported most severe symptoms were fatigue, sleeping disturbance, drowsiness, distress, and shortness of breath. There were no significant differences of symptom severity by age group (more or less than 60 years old) and by staging (Rai 0–1, 2–3). However, fatigue severity was significantly higher in patients who had more than 2 therapies than patients without prior therapy (4.26 vs 2.07 on 0 to 10 fatigue scale on MDASI, P=.003). The most burdensome symptoms were interference of working, activity and enjoyment of life. Moderate to severe symptoms that would be the targeted area of symptom control in CLL patients were fatigue (25%), drowsness, (13%), shortness of breath (11%), and sleeping disturbance (12%). In conclusion this preliminary data analysis showed that the MDASI is a reliable and valid tool for assessing symptom severity and interference with daily functioning in CLL patients. This versatile assessment tool can be used to measure CLL-related symptom burden in the clinical practice of symptom management as well as to measure changes in symptom burden in patients enrolled in therapeutic clinical trials.
Mutually Exclusive Cdk4-Cyclin D1 and Cdk6-Cyclin D2 Pairing Inactivates Rb and Promotes Cell Cycle Dysregulation in Multiple Myeloma.
Authors: Ely Scott , Di Liberto Maurizio , Niesvizky Ruben , Hatada Eunice , Cho Hearn , Knowles Daniel M. , Lane Joseph , Chen-Kiang Selina
Published: April 2020

Multiple myeloma (MM), the second most common hematopoietic cancer, ultimately becomes refractory to treatment when self-renewing MM cells begin unrestrained proliferation by unknown mechanisms. Cell cycle reentry and progression is modulated by the balance between positive cell cycle regulators [cyclins and cyclin-dependent kinases (Cdk)] on the one hand and Cdk inhibitors (CKI) on the other. Here we show that one, but not more than one, of the three early G1 D cyclins is elevated in each case of MM. However, cyclin D1 or D3 overexpression rarely leads to phosphorylation of the retinoblastoma protein Rb and G1 cell cycle progression, unless cyclin-dependent kinase 4 (Cdk4) is correspondingly elevated in the absence of Cdk6. By contrast, cyclin D2 and Cdk6 are coordinately increased, thereby overriding inhibition by Cdk inhibitors, p18INK4c and p27Kip1, and phosphorylating Rb in conjunction with Cdk4. Thus, Cdk4 pairs exclusively with cyclin D1 whereas Cdk6 pairs exclusively with cyclin D2, although Cdk4 can also pair with cyclin D2 in MM cells. The basis for the exclusive Cdk4-cyclin D1 and Cdk6-cyclin D2 pairing is coordinated transcriptional activation. Cyclin D1 or D3 expression is scattered or uniform among bone marrow MM cells and at a constant frequency in the clinical course. By contrast, phosphorylation of Rb by Cdk6-cyclin D2 occurs in discrete foci of bone marrow MM cells early in the clinical course before cell proliferation, then is heightened with proliferation and disease progression. Mutually exclusive Cdk4-cyclin D1 and Cdk4/6-cyclin D2 pairing, therefore, critically controls cell cycle re-entry and G1 progression, which underlies the expansion of self-renewing MM cells in MM progression.
Pax-5 Demonstrates Residual B-Cells in Bone Marrow Trephines Post Anti-CD20 Immunotherapy.
Authors: D’Souza Alwyn , Juneja Surender , Trivett Melanie , Carney Dennis , Westerman David
Published: April 2020

Background: Immunohistochemical stains for CD20 are reliable in both formalin and mercuric-based fixed tissues. Anti-CD20 (rituximab (R)) immunotherapy for B- cell malignancies has negated the usefulness of CD20 immunohistochemistry post-therapy. Many other routine B-cell immunohistochemical stains are unreliable in mercuric-based fixatives. Thus, a reliable B-cell stain is needed post-R treatment.

Pax-5 is a transcription factor specific for B-cells that is required for normal B-cell development. It is expressed from the Pro-B stage through to mature B-cells and is expressed on both normal and malignant B-cells. The Pax-5 immunohistochemical stain has proven to be reliable in formalin-fixed tissue. Its role in mercuric-fixed tissue has yet to be determined.

Aim: To demonstrate residual CD20-negative B-cells, using the Pax-5 immunohistochemical stain, on mercuric-fixed bone marrow trephines following R treatment.

Methods: We performed CD20 and Pax-5 immunostains on bone marrow trephines in patients who had received combined chemoimmunotherapy with R. Patients underwent restaging bone marrows midway through treatment (range 2–4 cycles) and at the end of treatment (range 6–8 cycles). We divided analysis into two groups; 1. Patients with overt residual disease on H&E stains. 2. Patients with morphologic complete remission on trephine.

Results: Patient diagnoses included: CLL/SLL n=20; Follicular lymphoma n=2; Mantle cell lymphoma n=1; Waldenstrom macroglobulinaemia n=3. Patients’ morphologic responses were: complete remission (CR) n=5; partial remission (PR) n=9; nodular partial remission (nPR) n=10; no response (NR) n=2. In the non-CR group (PR, nPR, NR) 4 patients were positive for CD20 and all 21 patients were positive for Pax-5. In the CR group 0 patients were positive for CD20 and 5 patients were positive for Pax-5.

Conclusion: Pax-5 is a robust and reliable immunohistochemical stain that demonstrates B-cells post anti-CD20 immunotherapy. While it stains both normal and malignant B-cells, it may prove to be a useful tool in assessing minimal residual disease following immunotherapy.
IL-21 and CpG ODN Are Synergistic in Their Ability To Induce Apoptosis of Chronic Lymphocytic Leukemia (CLL) Cells and Benign B1 Cells.
Authors: Jahrsdoerfer Bernd , Blackwell Sue E. , Wooldridge James E. , Andreski Melinda W. , Jacobus Laura S. , Taylor Christiana M. , Weiner George J.
Published: April 2020

We previously reported the TLR9 ligand CpG ODN can induce apoptosis of CLL cells. Interleukin 21 (IL-21), a recently discovered cytokine with structural homology to IL-2, IL-4 and IL-15, has pleiotropic effects on lymphocyte populations including NK, T and B cells. IL-21 is known to co-stimulate normal human B cell proliferation induced by CD40 ligation, and to inhibit B cell proliferation induced by anti-IgM antibodies and IL-4. We explored the impact of these two agents on CLL because of the overlapping nature of their signaling pathways. Gene chip array studies demonstrated CLL cells express the receptor for IL-21, and expression of this receptor is upregulated by CpG ODN. IL-21 as a single agent induced apoptosis of CLL cells in most samples studied. The combination of IL-21 and CpG ODN induced a greater degree of apoptosis than either agent alone.

Figure Figure

Dosing studies demonstrated synergy between these two agents in their ability to induce apoptosis of CLL cells.

Figure Figure

The effect of IL-21 and CpG ODN was also evaluated in peripheral blood from normal donors, and in umbilical cord blood. In both types of samples, therapy preferentially eliminated CD5(+) B-cells. Monoclonal antibody against CD5, but not control antibody, was able to inhibit the pro-apoptotic effect of IL-21. Our results suggest treatment with IL-21 and CpG ODN preferentially eliminates CD5(+) B cells in both malignant and benign samples, and may be a useful combination for the treatment of CLL or autoimmune disorders dependent elevated B1 cell levels. Ongoing studies are exploring the underlying signaling pathways which might be responsible for these effects.
Subcutaneous Administration of Alemtuzumab: A Single Institution Experience.
Authors: Bubis Jeffrey A. , Schaal Anna D. , Kimtis Elizbeth A. , Gautier Marc
Published: April 2020

Alemtuzumab is an active drug in the treatment of a number of lymphoid malignancies and is currently indicated for intravenous administration to patients with B-cell chronic lymphocytic leukemia (CLL) who have failed therapy with fludarabine. Unfortunately, despite premedication, grade 3/4 infusion-related reactions (IRR) are common during alemtuzumab IV administration (fever = 19%, rigors = 16%, and hypotension = 5%, amongst others). If grades 1/2 IRR are included, these numbers increase to 86%, 85%, and 32%, respectively. Nausea also occurs in 54% of patients. In addition to complicating therapy, these reactions sometimes make it necessary to prematurely discontinue therapy. These toxicities may be avoided by administering alemtuzumab subcutaneously (SC); a promising modality, but one with limited supporting data. We report the results of our institutional experience with SC alemtuzumab administration. We performed a retrospective chart review examining data points related to demographics, safety, and efficacy. This IRB - approved review looked at 20 patients who were treated with SC alemtuzumab (13 = CLL, 1 = CLL/AML, 3 = CTCL, 3= PTCL); 2 of these were retreated subcutaneously after receiving IV therapy. Treatment was attempted with a dose-escalation scheme of 3mg-10mg-30mg; with the goal of a 12-week regimen. The average age of the patients was 61 (median 62, range 45–76). 13 were male and 7 were female. The average number of weeks of subcutaneous therapy received was 7 (median 7, range 1–12 weeks). The average number of prior treatment regimens was 3 (median 2, range 1–7). ORR to therapy with alemtuzumab= 60%. (CR = 4, PR = 8). 9 patients had refractory or progressive disease. Patients tolerated the therapy well. Toxicities were all grades 1/2 and usually resolved with supportive therapy and continued alemtuzumab administration; these included hemolytic anemia (n=1), mild pruritis (n=1), anemia (n=1), neutropenia (n=2), fever (n=3), rigors (n=2) mild skin reactions (n=2). Infectious complications included bacterial pneumonia (n=1), herpes zoster (3 months after the completion of therapy, n=1), CMV reactivation (antiviral therapy with alemtuzumab held for two weeks, n=1). No patients discontinued therapy due to grade 3/4 IRR. Of particular interest are the two patients retreated with SC alemtuzumab after previously receiving the drug IV. (1 = PR after first and second course, 1 = PR after first course and no response to second course.) These patients tolerated the drug well with minimal toxicity. One of the retreated patients who had experienced bronchospasm with IV therapy, had no respiratory issues with subcutaneous administration. Based on this data, we conclude that SC alemtuzumab is a safe, well-tolerated, and effective alternative to IV administration. Final data will be available at presentation.
Treatment Outcomes in Patients (Pts) with Transformed Chronic Lymphocytic Leukemia (CLL) in One Institution.
Authors: Acs Peter , Mavromatis Blanche , Ozdemirli Metin , Hartman Daniel , Cheson Bruce
Published: April 2020

CLL is usually an indolent lymphoproliferative disease; however pts are at risk over time (3-5% yr) of transformation to a more aggressive histology, e.g., prolymphocytic leukemia (PLL), or Richter’s transformation (RT). The outcome of these pts is poor with median overall responses of 5–43%, CR 5–43%, and overall survival of 5–8 months regardless of the chemotherapeutic approach.

We report on 15 pts with CLL transformed to PLL or RT treated at our institution over the last 4 years. The median pt age at transformation was 64 (51–79). 9 had PLL, 4 had RT, one had an anaplastic histology, and another with atypical transformation. 7/14 pts were positive for ZAP-70 expression. 10/14 presented with CLL prior to the transformation with a median time of 7 yrs (range 3–27), and had undergone prior therapy with either fludarabine or chlorambucil. Pts were treated with mainly rituximab chemotherapy combinations. One pt who achieved a CR, was consolidated with a matched related donor transplantation and remains disease free for 3 yrs.

Conclusion. CR and ORR were higher than reported in the literature. Also, pts treated with rituximab-based combinations achieved longer overall survivals. Rituximab based-combinations should be considered in treating such pts. Whether they would benefit from consolidation therapy needs further investigation.

Treatment Based Response and Overall Survival in Patients with Transformed Chronic Lymphocytic Leukemia Treatment # Pts Prev. Tx # Pts CR (%) PR (%) OS after Transformation (Months) Pts, patients; Prev., previously; Tx, treated; CR, complete response; PR, partial response; OS, overall survival; R, rituximab; Flu, fludarabine; Cy, cyclophosphamide PLL R/Flu +/− Cy 2/6 6 67 12 36+, 36+, 36, 24+, 4, 12+ RCHOP or R-EPOCH 2/2 2 0 50 8, 3+ Alemtuzumab 1/1 1 0 100 12+ RT RCHOP or REPOCH 1/4 4 75 25 36+, 30+, 3, 9 Anaplastic RCHOP 1/1 1 100 0 24+ Atypical R/Flu/Cy 1/1 1 100 0 24+ Overall 60 27
Maintenance Alemtuzumab in Refractory B-Cell Chronic Lymphocytic Leukemia Patients: Updated Data.
Authors: Thieblemont Catherine , Houot Roch , Toure Boubacari Ali , Traulle Catherine , Michallet Anne-Sophie , Bouafia Fadhela , Espinouse Daniel , Salles Gilles , Coiffier Bertrand
Published: April 2020

Alemtuzumab has an impressive clinical activity in B-cell chronic lymphocytic leukemia. The standard regimen (30 mg three times weekly for 12 weeks) results in high rate of early responses but frequent early progression. To prolong TTP, we realized a phase II study with an identical initial scheme: 3, 10, 30 mg of Campath-1H on sequential days, followed by 30 mg three times weekly until partial response, and maintenance therapy with 30 mg every one or 2 weeks, then, 30 mg monthly until progression or toxicity. Between January 2001 and August 2005, 16 B-CLL patients have been treated with this regimen and were retrospectively analyzed on an intent-to-treat basis. Eighty-one percent (13/16) of the patients had received 3 or more prior chemotherapy regimens. Response was evaluated at 12 weeks (W12) after the beginning of the treatment by the CLL WG criteria. Survivals were defined from the first day of alemtuzumab. With a median follow-up for alive patients at 26 months, median duration of treatment was 8.5 months (range: 1.5–23.5) corresponding to a median number of 18 injections of alemtuzumab (range 6–36) realized. Nine (56%) patients received more than 6 months of monthly injections. Among them, 3 patients received one injection every 2 or 3 months after these 6 months. Two patients progressed in blood during maintenance. Modulation of the doses was then proposed with more frequent injections during a time period allowing to re-obtain a CR. Maintenance was stopped because of nodal progression in 6 patients, opportunistic infections in 2 patients (1HSV and 1 pulmonary infection), persistent CR in 3 patients, and persistant neutropenia in 1 patient. Objective response at W12 was reached in 13 (81%) of the patients, including 5 (31%) CR and 8 (50%) PR. One patient had a stable disease (disappearance of blood infiltration but stability of lymph nodes involvement) and 2 a progressive disease. Median duration of response was 11.7 months (range: 5–30). Median of TTP and overall survival were 12 months and 38 months, respectively. In this series, patients did not experience more hematological toxicities or opportunistic infections than reported with the standard regimen. Of the 13 patients that progressed at the time of analysis, 7 were re-treated by alemtuzumab (n=4) or alemtuzumab + chemotherapy (n=3). Objective responses were obtained in 6 patients during a median time at 8.7 months. In conclusion, maintenance alemtuzumab with modulation of dose intensity allowed improving time to progression and survival, without adding hematological toxicities and infectious complications.

Figure Figure
Combined Chemotherapy with Fludarabine, Cyclophosphamide and Rituximab (FCR) for Relapsed or Untreated Progressive Chronic Lymphocytic Leukemia (CLL). A Single Center Experience.
Authors: Pavlovsky Santiago , Pavlovsky Carolina , Pardo Laura , Sapia Sandra , Fernandez Isolda I. , Juni Mariana
Published: April 2020

Introduction: During the last decade, strong prognostic factors and new compounds emerged for the treatment of CLL. The initial experience with the combination of FCR at MDACC as salvage therapy and later in untreated patients (pts) promote us to use these regimen since March 2001.

Purpose: To evaluate the efficacy of FCR in improving the complete remission (CR), disease free survival (DFS) and overall survival (OS) rates in pts previously treated with chlorambucil-prednisone as a single therapy and untreated CLL pts.

Patients and methods: A total of 32 CLL pts, 16 after relapse and 16 untreated with progressive disease completed treatment and evaluation, 3 pts are still on treatment. FCR consisted of Fludarabine 25 mg/m2 iv on days 1 to 3, Cyclophosphamide 250 mg/m2 iv on day 1 to 3 and Rituximab 375 mg/m2 iv on day 1 given at 4 weeks interval up to 4–6 cycles. Response was assessed 4–8 weeks after treatment. CR was defined by CLL/NCI-WG. Minimal residual disease (MRD) negative was defined as <1% CD19, CD5 positive cells in peripheral blood and bone marrow as assessed by three colour flow cytometry. Median age was 63 years (range 34–80). Binet’s clinical stage were A: 9%, B: 31% and C: 60%. The CD38 expression was positive (>7% of cells) in 53% and negative in 47% of the cases.

Results: The CR was obtained in 66% of the pts, nodular partial remission (NPR) in 19%, PR in 9% and stable disease in 6%.

Characteristics Pts. # % CR % MRD negative % DFS 24 mo P= # OS 24 mo P= Untreated Pts 16 81 100 81 0.64 90 0.81 Treated Pts 16 50 81 76 87 CD38 Positive 17 65 82 65 0.01 80 0.28 CD38 Negative 15 67 87 100 100 Total 32 66 84 78 89

Neutropenia grade 3–4 was observed in 31% of the courses, 5% of the pts required hospitalisation due to infeccious episodes.

Conclusion: In patients with CLL, FCR induce a high CR, MRD negative and DFS rate. Patients with CD38 negative expression correlated with a better DFS.
High Dose of Rituximab in B-Cell Malignancies.
Authors: Hugec Vladimir , Alexandrescu Doru T. , Oe Yukiko , Paietta Elisabeth M. , Wiernik Peter H.
Published: April 2020

Background: Rituximab is currently used as a standard agent in many B cell malignancies. High doses of rituximab may be more efficacious in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma, because CD20 antigenic sites occur in low density per cell, and soluble CD20 in the plasma binds with infused rituximab. We describe our results with escalated high doses of single agent rituximab given as weekly induction doses followed by monthly maintenance doses.

Methods: 18 patients (10 males, 8 females) with median age 58 years (range 42 – 80); 4 patients with MCL and 14 patients with CLL were enrolled. Rai stages were: Stage 0–1, Stage I–2, Stage II–5, Stage III–8, Stage IV–2. 9 patients had at least one prior treatment (range, 1–8 treatments): fludarabine-7 patients, chlorambucil-6 patients, chlorambucil and fludarabine-4 patients. Median duration of disease prior to rituximab administration was 23 months (1–143 months). 9 patients had bulky disease (mass largest diameter > 5 cm). All patients received the standard single agent rituximab dose 375 mg/m2 with weekly escalation up to 2,250 mg/m2, followed by the highest escalated dose of rituximab as a monthly maintenance dose. The median monthly dose 1000 mg/m2 (range 500 – 2,250 mg/m2), and the median number of monthly doses was 18 (4–76 doses).

Results: The overall response rate (CR + PR) was 88% (CR 50%, PR 38%). CR median duration is 17 + months (6 – 46 + months). CD20 intensity of staining (range of mean fluorescense intensity ratio 162–4766) and CD38 expression (range 5–99%) did not correlate with responses. The median time to progression was not reached. 8 patients progressed and their median time to progression is 25 months (14–48). None of the patients with bulky disease reached CR. 2 CR and 4 PR were observed in previous fludarabine failures. 3 of 4 patients with MCL reached CR and one continues in CR 46+ months off treatment. Rituximab was very well tolerated and only 2 episodes of grade 3 infusion related toxicity were observed. Both patients completed infusion with appropriate supportive care.

Conclusion: Single agent rituximab in high doses can induce durable clinical response in non-bulky early stage CLL/MCL. Higher than standard doses improve responses possibly by saturating CD20 binding sites and overcoming the obstacle of circulating CD 20 antigen.
Applying of Fludarabine in One Patient with CLL/PL Prolymphocytic Transformation - One Case Report.
Authors: He XueFeng , Zhang Ri , Ma QinFen , Tao RuiFang , Wu DePei
Published: April 2020

We reported one case of CLL/PL with prolymphocytic transformation and observed the effect of fludarabine on it. The patient was first admitted for nonspecific hematological symptoms. Her bone marrow showed a mixture leukemia cell’s group consisted of 30% prolymphocytes and 50% mature small lymphocytes. Datas of MIC assay were analyzed. Phenotype indicated some prolymphocytic features. The patient was diagnosed as CLL/PL and a traditional chemotherapy regimen mainly consisted of CHOP and leukeran was applied but showed very limited effect. One month later she was admitted once more for aggravating anemia. MIC was examined again and showed a high proportion of 60% prolymphocytes in marrow. Phenotype by FCM indicated a double positive expression of FMC7 and SmIg. The patient was diagnosed as prolymphocytic leukemia transformed from CLL/PL. Chemotherapy based on CVP and leukeran received no good response. So a single-agent regimen of fludarabine (30 mg*m−2*d−1 IV for 5 days as one cycle) was tried. After two cycles’ chemotherapy, bone marrow was reevaluated. Proportion of prolymphocytes descended to a level of 1% and a mild bone marrow suppression was accompanied. Nearly half a year’s follow-up showed a sustained blood tri-linage low level and the patient still led an acceptable quality life with packed red cell support once a month.
Combination of Fludarabine (FAMP) Cyclophosphamide (CTX) and Alemtuzumab for Patients with Relapsed Chronic Lymphocytic Leukemia (CLL).
Authors: Tedeschi Alessandra , Montillo Marco , Miqueleiz Sara , Draisci Michela , Ricci Francesca , Scarpati Barbara , Mura Mariangela , Crugnola Monica , Morra Enrica
Published: April 2020

Previous studies have shown that FAMP in combination with CTX seems to have a significant advantage over single-agent FAMP in patients with relapsed CLL who had previously received alkylators or alkylators plus fludarabine, but are still fludarabine-sensitive. Despite the improved response rates, patients who achieve CR eventually relapse and require alternate therapies. The high rate of relapse in CLL affirms the need for improved treatments, which reduce or eliminate residual disease (MRD) to induce “better quality,” more durable responses. The monoclonal antibody alemtuzumab (Campath®), which is directed towards the CD52 antigen expressed on the surface of B-CLL cells, acts synergistically with fludarabine in vitro and appears to have synergistic activity in vivo. Alemtuzumab is one of a few single agents that has demonstrated the ability to eliminate minimal residual disease in CLL. Therefore, we designed a phase II study to determine the efficacy and safety of a 4-week combination regimen consisting of FAMP, CTX, and alemtuzumab (FCC) in relapsed patients with CLL. Objectives of this study also included the evaluation of the overall response rate (ORR), duration of response (DR), and the presence of MRD following treatment with FCC. Patients received FCC after a short period of alemtuzumab dose escalation, from 1 mg to 3 mg to 10 mg on consecutive days. The FCC regimen consisted of FAMP 40 mg/m2/day PO (Days 1–3), CTX 250 mg/m2/day PO (Days 1–3), followed by alemtuzumab 10 mg SC (Days 1–3). This combination was repeated on Day 29 for up to 6 cycles. MRD was measured by 4-color flow cytometry and consensus primer PCR. Nine patients (7 male and 2 female), have been enrolled in this trial. The median age of patients was 60.3 years (range, 43–78 years); 2 patients (22%) were in Binet stage C, 3 patients were in stage A and 4 patients were in stage B. Median number of prior treatment regimens was 2 (range, 1–3). Currently, 3 patients completed treatment, and 2 patients received 3 courses while the remaining 4 patients completed the first course. Among the 3 patients who completed treatment, 1 achieved a complete response (CR) and 2 patients a partial response (PR). MRD negativity was achieved in the peripheral blood in 1 patient. No CMV reactivation has been observed. No opportunistic infections have been recorded. Hematologic toxicity was manageable: 2 patients required G-CSF support. No nonhematologic toxicity was documented. This preliminary data on safety and efficacy of this regimen are encouraging and warrant further study.
GRN163L Inhibits Telomerase Activity in B-CLL Cells.
Authors: Lin Claudia , Damle Rajendra N. , Chiorazzi Nicholas , Chin Allison C.
Published: April 2020

B-CLL cells, like many solid and hematologic malignancies, are characterized by short telomeres, suggesting that they would be acutely susceptible to telomerase inhibition. We and others have documented that CLL patients in poorer prognosis subsets, i.e., those without IgVH mutations, had shorter mean telomere lengths and higher telomerase levels than patients with IgVH mutations (Damle et al, 2004; Keating et al, 2003; Bechter et al 1998, Hultdin et al, 2003). Treatment with the telomerase inhibitor GRN163L, a lipid-conjugated 13-mer thio-phosphoramidate oligonucleotide (Geron Corporation), inhibits the growth of human hepatoma (Djojosubroto et al, 2005), ovarian carcinoma (Ertem et al, 2004, 2005), and multiple myeloma (CAG, MM.1S) cell lines in vitro and in vivo (AACR 2004 and 2005 Annual Meetings). Although no validated human B-CLL xenograft models exist, preliminary data indicate effective inhibition of telomerase in freshly thawed B-CLL cells upon exposure to GRN163L. We will present data demonstrating robust uptake of GRN163L into primary B-CLL patient cells, along with the subcellular distribution of the oligonucleotide into cytoplasmic and nuclear compartments. Comparison of the effect of GRN163L and a mismatch oligonucleotide control on telomerase inhibition will be described. Geron initiated a Phase I/II trial with GRN163L in chronic lymphocytic leukemia in July 2005.
The Loss of CD19 in Myeloma Cells Contributes to the Escape from FcγRII-Mediated Growth Suppression.
Authors: Zheng Xu , Mahmoud Maged S. , Abroun Saeid , Asaoku Hideki , Kawano Michio M.
Published: April 2020

CD19 expression is specifically lost on human myeloma cells by the loss of Pax-5 gene expression, while normal plasma cells do express CD19. In order to examine the biological meaning of the loss of CD19 expression in human myeloma cells, we have generated CD19 transfectants of human myeloma cell lines (U266 and KMS-5 cells) and of a human non-myeloma erythroleukemia cell line (K562) and compared their proliferation and survival with those of their corresponding parent or only vector-transfected cells in the serum-containing or serum-free medium (supplemented with BSA and transferrin) in vitro. Exogenous introduction of CD19 expression markedly suppressed the proliferation of these cells in the serum-containing medium, but there existed less suppressive effect of CD19 introduction in serum-free culture. Among the serum components, we focused on the IgG and examined the effect of addition of high concentration of IgG or addition of anti-CD32 (FcγRII) antibody in this serum-free medium. Addition of high concentration of IgG induced the marked suppression of CD19 transfectants more than that of parent or mock transfectants, and furthermore, stimulation with anti-CD32 antibody also showed the suppression of CD19 transfectants more than that of the mock cells. We also confirmed that primary myeloma cells as well as myeloma cell lines predominantly expressed FcγRII(CD32), but not FcγRI(CD64) or FcγRIII(CD16), and were found to express FcγRIIB gene by RT-PCR. Furthermore, the treatment with anti-CD32 antibody induced the tyrosine phosphorylation of CD32b in the CD19+ cells more than that in CD19- mock cells, and also the phosphorylation of SHIP, but not SHP-1 or SHP-2 was enhanced in CD19+ cells. Therefore, these data suggest that myeloma cells as well as plasma cells or B cells express FcγRII(CD32), and CD19- myeloma cells are less sensitive to the FcγRII-mediated growth suppression than CD19+ normal plasma cells or B cells.