bluuebunny/crossref_metadata_2025 huggingface dataset:
High Resolution Amplicon Melting Curve Analysis: A Sensitive and Rapid Method To Detect the Janus Kinase 2 (JAK2) Gene Mutation.
Authors: Park Sunita I. , Winton Elliot F. , Mann Karen P. , Hill Charles E.
Published: April 2020

Chronic myeloproliferative disorders (CMPDs) are clonal hematopoetic stem cell disorders resulting in the increased production of cells of the myeloid lineage. The recently described G to T point mutation at nucleotide 1849 in the JAK2 gene may be a specific molecular marker to aid in the diagnosis of CMPDs, including polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF). Previously described methods of detection for this point mutation include sequencing, allele-specific PCR, and restriction digestion. We report here the development of a novel method to detect the JAK2 point mutation using high resolution amplicon melting curve analysis. Genomic DNA was obtained from peripheral blood samples from patients with PV, ET, CIMF, and suspected CMPD using the Qiagen DNA Blood kit (Valencia, CA). DNA was quantified using PicoGreen (Invitrogen) fluorescent dye followed by spectrophotometry and samples were normalized to a standard concentration. Real time PCR was performed on the Roche Light Cycler in the presence of the double stranded DNA binding dye LCGreen1 using primers that flank the point mutation. High Resolution Melting curve analysis was then performed on the HR-1 instrument (Idaho Technologies), and mutation status assigned by comparing the melting curve position and shape to controls of known mutation status. For comparison, the samples were also restriction digested using BsaX I and analyzed by agarose gel electrophoresis. Mutation status was confirmed by sequencing of the PCR amplicons (Lark Technologies, Houston, TX) for a subset of specimens. Twenty six patients with known CMPD as defined by the WHO criteria (2001) were included in the study. In addition, seven patients with suspected CMPD were studied. The point mutation in JAK2 was identified in 9 (100%) of 9 PV patients, 7 (58%) of 12 ET patients, 1 (50%) of 2 CIMF patients, 3 (100%) of 3 CMPD NOS patients, and 2 (29%) of 7 patients with suspected CMPD. Patients testing homozygous for the JAK2 mutation included 6 patients with PV, 1 patient with ET, and 1 patient with CMPD, NOS; all other positive patients were heterozygous for the mutation. We observed excellent correlation between the melting curve analysis and restriction digest results. High resolution melting curve analysis is a rapid, sensitive, low reagent cost method to assess for the JAK2 point mutation in cases of suspected or known CMPDs. The advantages of this technique include the ability to discriminate heterozygous from homozygous mutations, increased sensitivity as compared to sequencing, rapid turn around time, no post-amplification modification, and the ability to screen for other mutations or polymorphisms within exon 12 of the JAK2 gene.
Differences between Peripheral Blood, Lymph Nodes and Bone Marrow in Expression of Integrins, ICAMs, CD38 and CD31 Correlate with Clinical Characteristics in B-CLL.
Authors: Jaksic Branimir , Jaksic Ozren , Kardum-Paro Mirjana M. , Kardum-Skelin Ika
Published: April 2020

B-CLL patients have variable tumor mass distribution within major cell compartments, and as a consequence different clinical presentation. Adhesion molecules (AM) expression phenotype of integrins (CD11a, CD11b, CD11c, CD18, CD29, CD49c, CD49d and CD61), ICAMs (CD54, CD102 and CD50), CD38 and CD31 was determined in samples taken from peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) by two/three color flow cytometry in 33 B-CLL patients. Differences for particular AM expression between compartments were quantified to express respective intensity and direction (gradients). We found following significant differences: stronger expression for CD11a and CD102 in PB than in BM, while CD54 was stronger in BM than in PB; CD11b and CD102 were stronger in PB than in LN, opposite to CD54 and CD38; CD102 was stronger in BM than in LN, while CD18, CD11a, CD11c and CD38 were stronger in LN then in BM. Observed gradients were compared with clinical and laboratory parameters: PB lymphocytosis, LN size, Spleen size, Total Tumor Mass score (TTM), TTM distribution (TD), BM failure (BMF), Rai and Binet stages. Peripheral blood lymphocytosis positively correlated with LN to PB gradient for CD11a, CD11b, CD18 and CD31 (p<0.05). Lymph node size negatively correlated with LN to PB gradient for CD11b and with BM to PB gradient for CD54 and CD61 (p<0.05). Spleen size negatively correlated with BM to PB gradient for CD11c and CD102 (p<0.05). TTM negatively correlated with BM to PB gradient for CD11c, CD61 and CD102 (p<0.05). TD positively correlated with LN to PB gradient for CD11b and CD102, and with BM to PB gradient for CD11c and CD102 (p<0.05). Isolated BMF irrespective of tumor load, as well as clinical stages that incorporate BMF (Rai and Binet) were associated with significant PB to BM gradient for CD18, CD11a, CD11c, CD49d, CD54 and CD102, and with PB to LN gradient for CD102 (p<0.05). Cluster analysis corroborated these findings. This study shows that expression of selected integrins, ICAMs, CD38 and CD31on B-CLL cells is significantly different among lymphoid compartments suggesting possible role in resulting clinical presentation. Taking together, this investigation disclosed yet unexplained interesting interactions and warrants further studies of AM role in B-CLL.
X-Linked Gray Platelet Syndrome Due to a GATA1 Arg216Gln Mutation.
Authors: Tubman Venée N. , Levine Jason E. , Campagna Dean R. , Fleming Mark D. , Neufeld Ellis J.
Published: April 2020

Gray Platelet Syndrome (GPS) is characterized by a variable, mild bleeding diathesis, associated with thrombocytopenia and large, agranular, functionally abnormal platelets. While the disorder has been well-described both biochemically and pathologically, to our knowledge, no genetic mutation has been associated with the disease. Most cases are sporadic, with a few sibships and apparent autosomal kindreds reported. In our investigation, the proband is a healthy 1 year-old female whose father and uncle had recently been diagnosed with GPS. The child had a platelet count of 382 x103 platelets/μl, comprised of a dimorphic population of normal and large, agranular forms. Her CBC and peripheral blood smear were otherwise unremarkable. We subsequently analyzed four generations of this family. In addition to the proband’s father and uncle, several other members had known bleeding tendencies. The symptomatic individuals were invariably males, consistent with a sex-linked pattern of inheritance. Several symptomatic and asymptomatic family members were evaluated by CBC and blood smear. Large, agranular platelets were found in symptomatic males, while obligate carrier females exhibited both normal and abnormal forms. Using a set of fifteen microsatellite markers spanning the X chromosome, a common haplotype was identified in all affected men, their mothers, and their daughters. Aided by the published sequence of the X chromosome, we examined this region for candidate genes. Although the common haplotype between markers GATA144D04 and DXS6797 (Xp11.3-Xq22.3) contains hundreds of genes, only two, GATA1 and WAS, have known associations with thrombocytopenia. PCR amplification and sequencing of a 3′ segment of the GATA-1 promoter and the five coding exons of the GATA-1 gene revealed an G759A missense mutation resulting in an Arg216Gln substitution in exon 4 (NCBI RefSeq: NM_002049) that segregated with the phenotype and was present in all obligate carrier females. This mutation has been previously associated with X-linked thrombocytopenia and beta-thalassemia (XLTT), a syndrome characterized by splenomegaly, thrombocytopenia, and imbalanced globin chain synthesis (Balduini et al, Thromb Haemost. 2004; 91:129). Comparison of the ultrastructural characteristics of the platelets in both disorders and a review of literature on both diseases suggests that XLTT, and the associated mutation in GATA1, could represent one genetic origin for GPS. As carrier females generally display a less severe phenotype than affected males, without thrombocytopenia and with subtle dimorphism on smears, it is possible that this mutation may account for some previously reported “sporadic” cases of GPS.
Combined Therapy with Alemtuzumab and Pentostatin Is Feasible and Effective in T- Lymphoprolifertive Disorders.
Authors: Ravandi-Kashani Farhad , Kantarjian Hagop , Verstovsek Srdan , Ferrajoli Alessandra , Wierda William , Odinga Shirley , Huang Xueling , Pierce Sherry , Faderl Stefan , Giles Francis , O’Brien Susan , Keating Michael
Published: April 2020

Mature T-cell lymphoid neoplasms are clonal proliferation of post-thymic T-cells, which are generally refractory to traditional chemotherapy regimens. The prognosis for most patients with these disorders, particularly those with relapsed disease, is poor. New treatment strategies are urgently needed. Alemtuzumab and pentostatin have been used as single agents in various T-cell neoplasms with response rates of up to 75% and 50%, respectively. Immunosuppression and opportunistic infections are the principal overlapping toxicities. We have treated 9 patients with T-lymphoid malignancies (5 T-PLL, 1 ATL, 1 γδ-T-cell hepatosplenic lymphoma, 2 T-LGL) on a phase II study of alemtuzumab 30 mg IV three times weekly for up to 3 months with concomitant pentostatin 4 mg/m2 weekly x 4 followed by alternate weekly administration for up to 6 months. Prophylactic antibiotics including valcyclovir, trimethoprim/sulfamethoxazole, and fluconazole were administered during therapy and for 2 months after completion. Median age of patients was 57 years (range 25–80 years), median WBC was 78.5 x 109/L (range 0.7 – 279.5 x109/L), median serum β2M was 4.1 mg/L (2.7 – 10.8 mg/L). Six patients had splenomegaly (1 – 6 cm), and 3 lymphadenoapthy. Six had received prior therapy (median 5, range 1 to 6 regimens). To date, 6 of 8 evaluable patients have responded (5 CR, 1PR). One patient who had failed both single agent pentostatin and single agent alemtuzumab achieved CR with the combination. Monoclonal T-cell receptor gamma chain gene rearrangements were detected by PCR in all patients and became negative in 3 of 5 evaluable responding patients. Median response duration is 5+ months (range 2 – 6+ months). Two patients proceeded to an allogeneic stem cell transplant, 2 (1 with ATL and 1 with T-LGL) have died from disease progression after a response, 2 are alive and disease-free, and 2 were refractory to therapy. Opportunistic infections have included reactivation of CMV and HSV in one patient, recurrence of pre-existing Serratia pneumonia in 1 patient and Aspergillus pneumonia in 1 patient. We conclude that the combination of alemtuzumab and pentostatin is effective in T-cell neoplasms; infections can be prevented with prophylactic antibiotic therapy and close monitoring of patients.
The Treatment of Complicated and High Risk Chronic Lymphocytic Leukemia (CLL) with Fludarabine, Cyclophosphamide and Rituximab (FCR).
Authors: Donthireddy Vijaya , Shurafa Muhammad S. , Saleh Murad , Kamboj Ginny , Wang Ding , Janakiraman Nalini
Published: April 2020

Between November 2000 and November 2004, we treated 11 patients with the FCR regimen for complicated CLL. The median age was 64 years (51–80 years) and the median number of prior regimens received was 1(0–4). Median number of FCR cycles delivered was 4 (2–6). Four patients were treated due to active autoimmune hemolytic anemia (AIHA), three patients due to massive lymphadenopathy, two patients for short lymphocyte doubling time, one patient for active AIHA and immune thrombocytopenia and one patient for progressive splenomegaly. Two patients received only 2 cycles of FCR due to prolonged pancytopenia secondary to hypoplastic bone marrow. Four of the patients with AIHA had prior splenectomies. Overall response rate, according to NCIWG criteria, was 100% including the patients who had received only 2 cycles. One patient achieved a complete remission, seven patients achieved nodular partial remissions and three achieved partial remissions. Among the 7 patients with nodular PR’s, 4 had no evidence of residual CLL by flow cytometry and are continuing to be in remission at a median follow up of 53 months. The other 3 patients with nPR who had evidence of disease by flow cytometry relapsed at a median of 23 months (16–33 months). The patient who had a CR by NCIWG criteria, still had evidence of disease by flow cytometry and relapsed at 35 months. All patients with AIHA and immune thrombocytopenia achieved a remission of their autoimmune processes, including those who failed splenectomy. Grade 3–4 neutropenia, thrombocytopenia and anemia occurred in 8, 2 and 2 patients respectively. The median progression free interval was 33 months (9+ to 54 m) and the overall survival was 56 months (9+ to 56m). Four patients had expired at the time of this report due to progression of CLL(1), sepsis while still in remission(1), high grade transformation and subsequent sepsis(1) and complications of allogeneic transplant(1). Cytogenetic remissions were achieved in 7/9 patients. Three of them continue to be in remission at the time of this report with a median follow up of 53 months since initiation of therapy.

Conclusion: FCR is an effective treatment regimen for CLL associated AIHA, even after failing splenectomy and can result in high response rates and prolonged remissions. Flow cytometry remission seems to correlate with prolonged disease free survival in patients achieving nodular partial remissions according to the NCIWG criteria. We suggest treatment with FCR until elimination of minimal residual disease by flow cytometry irrespective of histopathological remission.
Induction of Chronic Lymphocytic Leukemia (CLL) Apoptosis by Nitric Oxide Synthase (NOS) Inhibitors: Drug Efficacy Correlates with Lipid Solubility and NOS1 Dissociation Constant.
Authors: Levesque Marc C. , Ghosh Dipak K. , Beasley Bethany E. , Chen Youwei , Volkheimer Alicia D. , O’Loughlin Charles W. , Gockerman Jon P. , Moore Joseph O. , Weinberg J. Brice
Published: April 2020

The viability of CLL cells may be dependent on the autocrine production of nitric oxide because nitric oxide synthase (NOS) inhibitors induce CLL cell apoptosis and CLL cells express inducible NOS (NOS2). Our previous study indicated that the non-specific NOS inhibitor NMMA induced CLL cell apoptosis but only at high concentrations (> 1 mM) (Levesque et al., Leukemia17:442, 2003). Therefore, we performed the current study to identify NOS inhibitors that induce CLL cell apoptosis at lower concentrations and to understand factors that promote NOS inhibitor-induced CLL cell toxicity. We isolated and enriched CLL cells from the blood of CLL patients and cultured the CLL cells in media containing various concentrations of 21 different NOS inhibitors. We determined CLL cell viability following culture with each NOS inhibitor. We found that NOS inhibitors with specificity for neuronal NOS (NOS1) induced CLL cell death at concentrations lower than non-specific NOS inhibitors and lower than inducible NOS (NOS2) specific inhibitors. There was a weak correlation (r2 = 0.29, p = 0.1608) of the NOS1 (but not NOS2) half-maximal inhibitory concentration (IC50) of each NOS inhibitor for purified recombinant NOS and its ability to induce CLL cell death. We confirmed the specificity of the NOS inhibitors by inhibition of purified recombinant NOS1 and NOS2 enzyme activity, and we confirmed that NOS1 specific inhibitors induced CLL cell death by apoptosis. Because there was only a weak correlation of the NOS1 IC50 with NOS inhibitor induced CLL cell death, we considered whether other factors such as the Kd and hydrophobicity of each compound correlated with CLL cell death. We found that there was a direct correlation between the NOS1 (but not NOS2) dissociation constant (Kd) of NOS inhibitors and CLL cell death (r2 = 0.77, p = 0.0041) and a direct correlation of the partitioning coefficient (a measure of hydrophobicity) of each NOS inhibitor and its ability to induce CLL cell death (r2 = 0.68, p < 0.0001). Therefore, NOS inhibitors that bound tightly to NOS1 and were hydrophobic induced CLL cell death at lower concentrations. There was variable expression of CLL cell NOS1 mRNA (6 of 28 samples positive) and we were unable to demonstrate CLL cell expression of NOS1 protein by immunoblotting. This suggests that if NOS1 is present in CLL cells, it exists at very low levels. Taken together, we believe that low level NO production promotes CLL cell viability and that inhibition of CLL NOS induces CLL cell apoptosis. Importantly, our studies provide direction for the rational design and selection of NOS inhibitors that may be useful as CLL therapeutics.
Fludarabine and Alemtuzumab Versus Fludarabine and Rituximab in the Treatment of Relapsed B-Cell Chronic Lymphocytic Leukemia: Results from a Phase II Study.
Authors: Gribben John , Stephans Katherine , Marshal Blossom
Published: April 2020

Although single-agent fludarabine is associated with high response rates (60%–80%) in patients with previously untreated B-cell chronic lymphocytic leukemia (B-CLL), this therapy is not curative and patients will relapse from persistence of minimal residual disease. Response rates to subsequent lines of therapy drop dramatically, as does survival. Antibodies including alemtuzumab and rituximab act in synergy with fludarabine and improve responses in salvage CLL therapy. In an effort to identify an effective chemoimmunotherapy regimen for patients with relapsed CLL, a phase II, multicenter, open-label, randomized trial was initiated. B-CLL patients who had failed prior therapy were randomized to treatment with either fludarabine combined with alemtuzumab or fludarabine combined with rituximab. Four patients randomized to the cohort received fludarabine 25 mg/m2 IV and alemtuzumab 30 mg SC, on Days 1–5. Eight patients received fludarabine 25 mg/m2 IV on Days 1–5, and rituximab 375 mg/m2 IV on Days 1 and 4 of the first cycle, followed by fludarabine 25 mg/m2 IV on Days 1–5, and rituximab 375 mg/m2 IV on Day 1 in subsequent cycles. Patients were assessed monthly for response while on therapy, and interim restaging occurred at cycle 4. Those who achieved a CR received no further therapy, whereas those who achieved a PR or SD received 2 additional cycles. 12 patients (7 male and 5 female) participated in this trial and the median age was 67 years. Nine patients had Rai III/IV (2 patients in alemtuzumab arm and 7 patients in rituximab arm). All patients had failed 1 course of therapy; 9 had failed treatment with a fludarabine-based regimen, and 3 had failed treatment with alkylating agents. In the alemtuzumab arm, 2 patients developed a CMV reactivation, one of whom developed CMV viremia, which was successfully treated with gancyclovir. Of the 8 patients in the rituximab plus fludarabine arm, 6 withdrew due to adverse events and 2 patients died while on study. In the alemtuzumab plus fludarabine arm, 1 person withdrew due to adverse events and 1 patient died after the trial was closed. Overall, 3 of 4 patients in the alemtuzumab arm responded (2 complete response [CR], 1 partial response [PR]), and 3 of 7 patients in the rituximab arm (1 CR, 2 PR). Recently published data has prompted a shift in CLL therapy. Increased numbers of patients are receiving chemoimmunotherapy combinations earlier in treatment. An increased use of FR and FCR in the first-line setting made further recruitment difficult. The potential of randomizing patients to the FR arm of the study prompted low accrual and the subsequent closing of the study.
A Novel Anti CD23 Fully Human Monoclonal Antibody Potentially Useful for B-CLL Therapy.
Authors: Delcommenne Marc , Klingemann Hans-Georg , Gregory Stephanie A.
Published: April 2020

B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematological malignancies and is, in most cases, characterized by an increased expression of CD23 on the cell surface. Since cross-linking CD23 induces B-CLL apoptosis, it is an attractive target for B-CLL antibody-based immunotherapy. In this study we show that an anti-CD23 human IgG1 monoclonal antibody, C6F5, may be useful in treating B-CLL. This antibody is derived from the human single chain antibody (scFv) C6F5 that was originally raised against the RPMI-8226 multiple myeloma cell line using the antibody phage display technique. While the C6F5 scFv did not bind to other myeloma cell lines, it was able to bind weakly to normal peripheral blood B lymphocytes and strongly to EBV transformed B cells and B-CLL cells. The antigen recognized by C6F5 was also upregulated on B lymphocytes that had been stimulated by CD40 ligand. Immunoprecipitations by the scFv C6F5 identified a protein of 45 kDa which co-migrated with CD23. Furthermore, this protein was recognized by an anti-CD23 mouse mAb in Western blot analyses. Immunofluorescence staining with the C6F5 scFv was inhibited if cells were preincubated with an anti-CD23 polyclonal antiserum. Taken together, these results verify that C6F5 recognizes CD23. The VH and VL regions of C6F5 antibody were then cloned into a baculovirus transfer vector encoding the human IgG1 heavy and light chains so that fully human C6F5 IgG1 antibody could be produced in baculovirus infected SF9 cells. Since C6F5 binding specificity was preserved in the IgG1 format, this antibody is ready to be tested in in vitro cytotoxic assays against B-CLL cells.
Characterization of IMiDs (Immunomodulating Agents) Induced “Flare Reaction” in Patients with Chronic Lymphocytic Leukemia (CLL) and Correlation with Changes in Serum Cytokine Levels.
Authors: DiMiceli Laurie , Miller Kena C. , Rickert Michael , Wallace Paul , Marshall Patricia , DePaolo Dawn , Padmanabhan Swaminathan , Landrigan Beverly , Chanan-Khan Asher A.
Published: April 2020

Introduction: Several cytokines (including TNF-alpha, IL-6 and VEGF) are reported to play a critical role in the pathogenesis of CLL cells. Targeting the tumor microenvironment through modulation of the cytokine milieu thus is a potential way to treat CLL. In 2 separate clinical studies we investigated the role of IMiDs (thalidomide and lenalidomide) either in combination with standard chemotherapy (thalidomide + fludarabine, FT) or single agent (lenalidomide, L) in pts with previously untreated or relapsed/refractory CLL, respectively. In these studies we observed tumor flare reaction not previously reported. In this report we describe the clinical characteristics of this side effect, its management and correlation with changes in serum cytokine levels. Flare Reaction (FR): CLL pts treated with IMiDs were noted to have tender increase in lymph nodes size starting within 24–48 hours of initiating therapy with or without erythema and rash. Some patients also increased their total white cell as well as absolute lymphocyte count (ALC).

Results: A total of 48 pts (20 on FT and 28 on lenalidomide) are collectively treated on 2 clinical trials. Median age was------, -----had limited stage (0-II) and ----- has advance Rai stage (III, IV). ------(%) on the FT and 22 (78.5%) on the L developed a FR, which was noted as early as 24 hours after initiation of therapy (median 48 hours). Most pts resolved FR by day 14. A comparison of serum cytokine level at day 0 and 7 was done in 10 pts (9 with FR) treated with L. An increase in TNF-a (n=4), MIG (n=6), IP-10 (n=8), IL-8 (n=7) and IL-10 (n=6) was noted at day 7.

Management: FR was treated with ibuprofen 400 mg q 6 hr with addition of oral morphine (or agents of equivalent potency) for improved pain control.

Conclusion: FR is a new side effect noted in CLL pts treated with IMiDs that is fairly well managed by anti-inflammatory agent along with aggressive pain control. Higher incidence is noted with L. There seems to be no correlation of FR with clinical response to therapy. Identification of the FR is important to avoid confusion with disease progression. An increase in levels of inflammatory cytokine was noted in all pts treated with L which may explain the underlying mechanism of this phenomenon.
Multiple Myeloma Presenting as a Clivus and Brain Stem Plasmacytoma.
Authors: Rezazadeh Arash , Kloecker Goetz H. , Laber Damian A.
Published: April 2020

Multiple myeloma is the most common hematological malignancy. MM can affect many organ systems; therefore, it may mimic different clinical syndromes at presentation. We report an extremely rare presentation of MM.

CASE REPORT: A 62-year-old man presented with progressive cervical pain for 2 months and double vision for 2 weeks. Physical exam demonstrated a cranial nerve 6 palsy, nuchal rigidity and no other abnormalities. A magnetic resonance image (MRI) of the brain revealed a 5 cm enhancing mass in the clivus which extended into the brain stem, left internal auditory and carotid canal, nasopharynx, sphenoid and cavernous sinuses, without midline shift or hydrocephalus. MRI of the spine showed 2 small enhancing lesions in thoracic vertebrae 4 and 11. Computer tomography of the chest abdomen and pelvis demonstrated a lesion in the sternum and no other abnormalities. Technetium-99 bone scan showed uptake in the skull base mass only. Complete blood count and metabolic profile were normal except for an albumin of 3.2 g/dL. Serum protein electrophoresis with immunofixation revealed hypogammaglobulinemia without a monoclonal protein. 24 hour urine collection demonstrated monoclonal free kappa light chains. Transsphenoidal stereotactic biopsy of the mass showed an atypical plasma cell proliferation with multi-nucleation and mitotic activity, positive for kappa light chain. Bone marrow aspirate revealed 17% of the nucleated cell to be abnormal plasma cells, some with plasmablastic morphology. He improved rapidly with dexamethasone and was referred for radiotherapy.

DISCUSSION: The most common neurologic complications of MM are spinal cord compression due to epidural plasmacytomas or vertebral fractures, and peripheral polyneuropathy due to the presence of antibodies directed against myelin structures or due to amyloid deposits. Brain involvement in MM is uncommon. Furthermore, neurologic symptoms due to plasmacytomas located either in the clivus and brain stem are extremely rare. A MEDLINE search from 1950 until July 2005 revealed only 9 cases of MM or plasmacytoma involving the brain stem. Since there is no published autopsy series on MM patients describing the percentage of patients with brain involvement at death, the denominator is difficult to assess. Clinically, extramedullary involvement from MM bears a poor prognosis, and has been described in cases of plasma cell leukemia. We speculate that the aggressive behavior of the MM in our patient might be consistent with the atypical phenotype of the plasma cells.

CONCLUSION: The unusual presentation of this patient adds to the medical knowledge about the clinical spectrum of multiple myeloma.
Bone Marrow Microcirculation Parameter Amplitude A of Contrast-Enhanced Dynamic MRI Is a Prognostic Factor for Progressive Multiple Myeloma for Patients with Low Beta2-Microglobulin.
Authors: Hillengass Jens , Wasser Klaus , Benner Axel , Delorme Stefan , Zechmann Christian , Ho Anthony D. , Goldschmidt Hartmut , Moehler Thomas
Published: April 2020

Introduction: We have previously shown that contrast-enhanced dynamic magnetic resonance imaging (dMRI) microcirculation parameters Amplitude A and distribution rate constant kep are significantly increased in patients with multiple myeloma (MM) compared to healthy controls and correlate with osteolytic bone involvement. Furthermore an elevated Amplitude A is associated with a high plasmacell-infiltration and increased microvessel density in bone marrow. We now evaluated the prognostic value of changes in microcirculation of bone marrow as detected by dMRI for overall (OAS) and event free survival (EFS) in patients with MM.

Methods: Between 1999 and 2001 62 patients with progressive MM requiring chemotherapy according to international standards (6 newly diagnosed patients, 56 patients with relapse or refractory disease) were investigated with dMRI of the lumbar spine. OAS and EFS were updated for all patients in February 2005. The estimated median follow up was 4.5 years. All patients underwent standardized dMRI with high temporal resolution (T1w-turboFLASH) before start of therapy. The contrast uptake was quantified using a two compartment model with the output parameters Amplitude A and distribution constant rate kep reflecting bone marrow microcirculation. To examine the prognostic value of the findings of dMRI we used the Proportional Hazards model as proposed by Cox.

Results: We recorded a correlation with borderline significance (p-value 0.1) between Amplitude A and EFS. We did not find a correlation of dMRI parameters with OAS. The multivariable analysis of Beta2-Microglobulin, age, Lactat Dehydrogenase (LDH) and Albumin revealed Beta2-Microglobulin as the only statistically significant prognostic factor for EFS in this group of patients. When patients were classified according to high or low Beta2-Microglobulin, Amplitude A was able to provide significant additional information characterizing EFS. Patients with low Beta2-Microglobulin and increased Amplitude A had significant shorter EFS than patients with low Beta2-Microglobulin and low Amplitude A.

Conclusion: Our investigations indicate that dMRI parameter Amplitude A which reflects the increased bone marrow microcirculation and angiogenesis is a novel prognostic factor for progressive multiple myeloma in patients with low Beta2-Microglobulin. So a more precise differentiation of patients may be possible through dMRI. The prognostic impact of dMRI for newly diagnosed myeloma patients and patients with monoclonal gammopathie with undetermined signifikance will now be evaluated in a prospective study.
Modification of Cytokine Levels and CD34+ Stem Cell Adhesion Molecules during Mobilization in Multiple Myeloma (MM) Patients.
Authors: Zappasodi Patrizia , Corso Alessandro , Rusconi Chiara , De Amici Mara , Consensi Erica , Pascutto Cristiana , Varettoni Marzia , Mangiacavalli Silvia , Pica Gianmatteo , Lazzarino Mario
Published: April 2020

The mechanisms underlying the mobilization of peripheral blood stem cells (PBSC) are still unknown, even though studies on healthy donors and on cancer patients have hypothesised a central role for CD34+ stem cells adhesion molecules and for chemokines serum levels. The type of mobilizing regimen and the pathologic condition can exert different effects on cytokine levels and adhesion molecules expression. To evaluate the modification of these parameters during mobilization we studied 10 MM patients treated with DCEP chemotherapy (Decadron, Cyclophosphamide, Etoposide, cis-Platin) followed by G-CSF (5 mg/kg/day starting 48 hours after chemotherapy until PBSC collection).

The expression of four CD34+ cell adhesion molecules (Thy1, L-selectin, VLA4, CXCR4) was measured in the bone marrow before mobilization therapy and in the leukapheresis product. Serum levels of five cytokines (TGF-β, IL-8, HGF, VEGF-A, sVCAM-1) were assessed, through specific ELISA kits, before therapy, 5 days after the G-CSF stimulation and at stem cells collection. Each parameter was correlated with the number of CD34+ cells collected.

Thy1 significantly decreased (p=0.007) and L-selectin significantly increased (p=0.038) in nearly all cases. By contrast, no statistically significant differences were observed for VLA4 and CXCR4 due to a consistent variability. Serum levels of IL-8 showed an increase at day 5, then a significant decrease at the time of the harvest (p=0.013); VEGF-A constantly increased (p<0.001) and TGF-β decreased (p=0.001). The modifications registered for sVCAM-1 and HGF did not reach statistical significance. The expression of CD34+ adhesion molecules did not correlate with the number of PBSC collected, while among cytokines, high baseline levels of TGF-β and VEGF-A significantly correlated with high number of CD34+ stem cells yielded (p=0.016, p=0.042 respectively).

In conclusion, this study shows that during stem cells mobilization in MM Thy-1 expression significantly decreases while L-selectin increases; the behaviour of VLA-4 and CXCR4 is extremely variable. As regards cytokines, we found a statistically significant increase of VEGF-A level, while TGF-β, which induces the stem cell homing, decreases during the mobilization allowing stem cells to migrate in the peripheral blood. High baseline levels of TGF-β and VEGF-A predict a good stem cells harvest.
Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.
Authors: Molloy Timothy J. , Cindy Baulch-Brown , Deng Yi-Mo , Spencer Andrew , Ma David F.
Published: April 2020

We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.
Cytogenetic Abnormalities in Histologically Confirmed Multiple Myeloma.
Authors: Mohamed Anwar N. , Bentley Gail , Bonnett Michelle L. , Palutke Margareta , Al-Katib Ayad M.
Published: April 2020

In multiple myeloma, conventional cytogenetics is limited by low proliferation activity of plasma cells in culture. Despite that, nonrandom chromosomal abnormalities have been reported in one third of cases. Recently, the karyotype has emerged as an important prognostic factor in multiple myeloma. In this study, we identified 104 histologically confirmed multiple myeloma cases with satisfactory cytogenetic evaluation and abnormal karyotype. FISH analysis was used to evaluate certain chromosome abnormalities or to monitor treatment response. Hyperdiploid karyotype was found in 67 cases (64%), hypodiploid in 25 cases (24%), and the remaining 12 cases (12%) had a pseudodiploid karyotype. The most common numerical abnormalities were gains of chromosomes 9, 15, 3 followed by chromosomes 19, 21, 11, 7, and 5. Whole chromosome losses were also frequent involving primarily chromosomes 13, 8, 14, 22, and 16. In the hypodiploid cases, loss of chromosome 13 was more evident, seen in 19/25 (76%) cases. Most cases showed also structural rearrangements leading to del(1p), dup(1q), del(6q), del(9p), del(13q), del(17p), del(8p), 3p/3q abnormalities, and 11p/11q abnormalities. Translocations affecting 14q32/IGH region was the most frequent seen 36 times in 34/104 (33%) cases; t(11;14)(q13;q32) in 13 cases, t(1;14), t(6;14), and t(8;14) in 2 cases each, and the remaining cases had various t(V;14) partners or of an undetermined origin. The 14q32/IGH translocations were less frequent in the hyperdiploid karyotypes than the hypodiploid and pseudodiploid karyotypes (18% vs 60%). Nine cases showed break at 8q24/CMYC site; six of those had Burkitt’s-type translocations. Other non-random translocation was t(1;16) seen in three cases. Coventional cytogenetic remains an important tool in elucidating the complex and diverse genetic anomalies of multiple myeloma. Acccordingly well defined cytogenetic subgroups can be identified. At the present time, FISH and other molecular genetic techniques are important adjuncts, but should not be a substitute for conventional karyotyping. The clinical and hematological correlates of the karyotype in our series is being evaluated.
Prognostic Relevance of CD56 Expression in Patients with Multiple Myeloma Treated with High-Dose Melphalan-Based Chemotherapy and Autologous Stem Cell Transplant.
Authors: Yeung Joanna , Samiee Sara , Yi Qi Long , Pantalony Dominic , Chang Hong
Published: April 2020

CD56 is a neural adhesion molecule and expressed in 70–80% cases of multiple myeloma (MM). Lack of CD56 expression has shown to be a poor prognosis in MM patients treated with conventional chemotherapy, but its prognostic relevance in MM treated with high dose chemotherapy and autologous stem cell transplant (ASCT) is not known. CD56 expression was evaluated by immunohistochemistry on bone marrow paraffin embed specimens from 107 MM cases undergoing Melphalan-based high dose therapy and ASCT. The 66 men and 41 women had a median age of 54 years. 53 patients had an IgG paraprotein, 30 IgA, 4 IgD, 15 light chain disease and 5 were nonsecretory. The median post-transplant follow-up was 20 months. Of 107 MM patients, 76 (71%) were CD56 positive and 31 negative. 27 died during the follow-up period including 7(23%) in CD56 negative and 20 (26%) in CD56 positive group; 50 were in relapse including 11 (35%) in CD56-negative, and 39 (51%) in CD56 positive group. The median overall survival for the CD56 positive patients was 48.1 months and for the CD56 negative patients, 44.8 months (p=0.67). The median progression free survival of the CD56 positive patients was 25.8 months and for the CD56 negative patients, 33.1 months (p=0.28). CD56 negative myeloma was associated with bone lesions (69% for CD56 positive vs 90% for CD56 negative group, p=0.032). However, CD56 was not associated with other biological factors including age, sex, hemoglobin, calcium, albumin, c-reactive protein, beta-2 microglobulin, immunoglobulin isotype, stage of the disease or bone marrow plasmacytosis. Furthermore, there was no correlation between CD56 expression and genetic aberrations including deletions of 13q14, 17p13.1 (p53), translocations t(11;14) or t(4;14) as evaluated by fluorescence in situ hybridization (FISH). In contrast to reports of CD56 in myeloma treated with conventional chemotherapy, we found CD56 negativity does not confer a poor prognosis in our patients, suggesting Melphalan-based high-dose chemotherapy and ASCT may overcome the adverse influence of CD56 negative myeloma.
Differential Expression of Genes in Multiple Myeloma (MM) and Identification of Candidates to Targeted Therapy.
Authors: Felix Roberta S. , Vettore André L. , Yamamoto Mihoko , Almeida Manuella S.S. , Silva Wilson A. , Zago Marco A. , Colleoni Gisele W.B.
Published: April 2020

Considering the heterogeneity of genetic aspects related with MM pathogenesis, recent studies have been focused on prognostic factors that could define the best therapeutic approach for each case or to contribute for the development of new therapies. In this way, gene expression profiling analysis can be very helpful to define such prognostic factors. Serial analysis of gene expression (SAGE) allows not only the identification of genes expressed in a given tissue, but also their expression levels. Until now, there was no data of gene expression in normal or neoplastic plasmacytes in public SAGE databases.

Purpose: The aim of this study is to create two SAGE libraries using normal and neoplastic plasmacytes and to identify genes differentially expressed in MM.

Methods: Normal plasmacytes were obtained from palatine tonsils of children who underwent tonsillectomy. MM library was obtained from bone marrow plasmacytes of two IgGk newly diagnosed MM patients. Purified normal and neoplastic plasmacytes were obtained after magnetic sorting of CD-138-positive cells (MACS - Magnetic Cell Sorting of Human Cells, Miltenyi Biotec). Both SAGE libraries were obtained using the kit I-SAGE (Invitrogen), according to manufacture’s instructions.

Results: We generated 4,000 clones from each library and, after automatic sequencing, we got 29,918 tags from normal and 10,340 tags from tumoral libraries (corresponding to 8,241 and 2,359 unique tags, respectively). After comparison of unique tags expression levels, we selected 15 overexpressed (at least 10 times) genes in the MM library (ZFHX1B, XBP1, PIM2, LGALS1, RANBP2, HSPC132, PRDM2, DDX5, ARMCX3, LSM5, JUND, MAPKAPK2, SP140, KLF2 and NTN1). Once these genes are related with transcription, signaling, cell proliferation and/or apoptosis, they can have important role in the tumorigenesis process and may be possible therapeutic targets for MM control
Dual Roles of T/NK Immunomodulatory and Anti-Tumor Properties for Selected Herbal Medicines in Multiple Myeloma Therapy.
Authors: Fan Kei , Li George Q. , Yang Shihong , An Xin , Ma Guoyi , Roufogalis Basil D. , Joshua Douglas E. , Sze Daniel
Published: April 2020

Thalidomide is one of the current therapies for multiple myeloma (MM) which has multiple mechanisms on myeloma cells, bone marrow microenvironment, T cells and NK cells. Chinese herbal medicines have been used in the treatment of cancer and also have multiple pharmacological activities; however, there is a strong need for evidence-based scientific data to understand their anti-cancer mechanisms. The aim of this study is to integrate thalidomide and Chinese herbs to enhance the beneficial effects and minimize the side-effects of thalidomide. The melanins from an anti-cancer Chinese herb Ligustrum lucidum were extracted and tested. The dual roles of the herbal extracts on T/NK immunomodulatory and anti-tumor properties were investigated in the following three aspects:

Proliferation measured by both [3H] thymidine assays and CFSE assays; Cell survival measured by propidium iodide exclusion and trypan blue staining; and NK cytotoxicity using myeloma cell line 8226 as target cell labeled with CalceinAM, and peripheral blood mononuclear cells (PBMC) from healthy subjects as the source of effector cells.

It was found that in the [3H] thymidine assays, Ligustrum lucidum melanin (LLM) extracts exhibited both stimulatory effect on PBMC proliferation and inhibitory effects on myeloma cell line 8226 proliferation at the concentration of 0.02 to 20 μg/ml. The stimulatory effect on PBMC was also observed with CFSE assays when PBMC was incubated with LLM (20 μg/ml) for 2 days with a proliferated population of 33%, compared with thalidomide (5 μg/ml) 57%, and control, 12%. In the cell survival assay tumor cell line NK92-MI was completely inhibited by LLM (20 μg/ml). NK cytotoxicity assay showed the LLM (0.02 to 20 μg/ml) showed dose response increase of cytotoxic effects of NK cells from 50% to 70%, higher than 42% achieved by thalidomide (5 μg/ml). These results indicate that herbal medicine Ligustrum lucidum posses both T cell / NK cell immunomodulatory effects and anti-tumor effects to myeloma cells. Further works will be to determine the structure of the plant constituent and to assess the synergistic combinations of the active extracts and constituents with thalidomide in the cellular assays.
Peripheral Blood Cells from Multiple Myeloma Patients Show Increased Osteoclast and Decreased Osteoblast Gene Expression.
Authors: Gordon Melinda S. , Berenson Ariana M. , Drucker Charles B. , Katz Matthew , Lee Hee Jin , Campbell Richard A. , Chen Haiming , Yeh Howard S. , Bonavida Benjamin , Said Jonathan , Berenson James R.
Published: April 2020

Bone resorption leading to osteolytic bone disease is characteristic of multiple myeloma (MM). Recent studies show the presence of bone-resorbing osteoclasts and bone-forming osteoclasts in the circulation, and these cells may correlate with bone disease and change with anti-bone resorptive therapies. We have investigated whether there is an imbalance in the expression of osteoblast and osteoclast genes in the peripheral blood mononuclear cells (PBMCs) from MM patients relative to normal age-matched controls and the effect of bisphosphonate treatment on the expression of these genes. We analyzed the expression of a panel of osteoblast-related (bone alkaline phosphatase [bone AP], bone morphogenic protein 2 [BMP2], collagen I and osteocalcin) and osteoclast-related (b3 integrin, calcitonin, receptor for activation of nuclear factor kappa B [RANK] and tartrate-resistant alkaline phosphatase [TRAP]) genes by semi-quantitative RT-PCR on total RNA isolated from PBMCs obtained following density gradient separation. We demonstrated that the expression of the osteoblast-related gene BMP2 was reduced in eight of nine MM patients when compared with normal donors. In marked contrast, three osteoclast-related genes, b3 integrin, RANK and TRAP, were more highly expressed in all nine MM patients compared to the normal donors; only calcitonin expression was similar to the control subjects. Interestingly, patients receiving bisphosphonate treatment appeared to show increased osteoblast gene expression with higher amounts of bone AP, BMP2 and osteocalcin RNA compared to the patients not receiving anti-bone resorptive therapy. However, there was no alteration in the level of the RNA in any of the four osteoclast genes compared to patients not receiving anti-bone resorptive therapy. We are extending our analysis to a larger panel of MM patients in order to determine the relationship between these circulating cells and bone disease, overall clinical status and change in their levels with anti-bone resorptive therapy. In addition, we are also investigating whether there exist larger and smaller numbers of circulating osteoclasts and osteoblasts, respectively, in MM patients, or whether these circulating cells show alteration of their expression of these genes. Our semi-quantitative RT-PCR results are being correlated with immunohistochemical staining results from osteoblast and osteoclast markers obtained on PBMCs from MM and normal subjects. These studies provide evidence that the number of circulating osteoblasts and osteoclasts is altered in patients with MM, and also may suggest that bisphosphonate therapy may also be associated with changes in these cell populations.