bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of Preleukemic Clones at Birth in the Majority of Children with B-Lineage Acute Lymphoblastic Leukemia.
Authors: Gruhn Bernd , Pfaffendorf Nadine , Wittig Susan , Zell Roland , Häfer Ralf , Steinbach Daniel , Herrmann Falko H. , Zintl Felix
Published: October 2019

The proof for the prenatal origin of childhood acute lymphoblastic leukemia (ALL) comes from the detection of concordant leukemia in monozygotic twins and the identification of translocation breakpoint genomic sequences at birth in a limited number of ALL patients with t(4;11) or t(12;21) chromosomal translocation. However, most patients with childhood ALL lack leukemia-specific fusion gene sequences. Therefore, we have used the rearranged immunoglobulin heavy chain (IgH) genes as a marker for the detection of preleukemic clones at birth. Guthrie card blood spots of 32 children with B-lineage ALL treated at our institution were available for this retrospective study. The ALL patients had a median age of 5 years (range, 15 months to 14 years) and had median presenting white blood cell (WBC) counts of 10150/μl (range, 800 to 103800/μl). In all patients a monoclonal IgH gene rearrangement was obtained from diagnostic bone marrow and sequenced. Clone-specific primers were designed using the specific D-N-J and N-D-N sequences. A two-stage polymerase chain reaction (PCR) using a semi-nested approach was developed to improve sensitivity and specificity of amplification. In all 32 patients, one leukemic cell could be detected in a background of 105 normal blood mononuclear cells. Nineteen of the 32 patients (59%) had detectable IgH gene rearrangements at birth using the sensitive semi-nested PCR. Sequencing of the PCR products obtained from Guthrie card blood spots revealed the identical sequences identified from diagnostic leukemic cells. The fetal characteristics of the leukemic cells were indicated by the small numbers of nucleotides inserted into the N region and the shortened D germ line segments. Interestingly, five of the six children (83%) with hyperdiploid ALL had detectable preleukemic clones at birth. Four of the five children (80%) with pro-B ALL, 13 of the 21 children (62%) with cALL and only two of the six children (33%) with pre-B ALL had preleukemic clones on their cards. We did not observe any differences in age at diagnosis or presenting WBC count between the 19 patients with preleukemic clones at birth and the 13 patients whose Guthrie cards were tested negative. Our results suggest that the majority of children with B-lineage ALL has preleukemic clones already at birth indicating a prenatal origin of leukemia. In addition, postnatal factors are important in leukemogenesis as well because of the long latency periods until clinical diagnosis of leukemia.
Proteomic Discovery of Max as a Novel Interacting Partner of C/EBPa: A Myc/Max/Mad Link.
Authors: Zada A. Peer , Pulikkan J. , Bararia D. , Geletu M. , Trivedi A. , Balkhi M. , Behre G.
Published: October 2019

In the present study, we sought to identify novel C/EBPa interacting proteins in vivo through immunoprecipitation using mass spectrometry-based proteomic techniques. We identified Max, a heterodimeric partner of Myc, as one of the interacting proteins of C/EBPa in our screen. We confirmed the in vivo interaction of C/EBPa with Max and showed that this interaction involves the basic region of C/EBPa. Endogenous C/EBPa and Max, but not Myc and Max co-localize in intranuclear structures during granulocytic differentiation of myeloid U937 cells. Max enhanced the transactivation capacity of C/EBPa on a minimal promoter. A chromatin-immunoprecipitation assay revealed occupancy of the human C/EBPa promoter in vivo by Max under cellular settings and by C/EBPa and Max under retinoic acid induced granulocytic differentiation. Interestingly, enforced expression of Max and C/EBPa, results in granulocytic differentiation of the human hematopoietic CD34+ cells as evidenced by CD11b and CD15 expression and by real-time PCR for various genes. Silencing of Max by short hairpin RNA in CD34+ and U937 cells strongly reduced the differentiation-inducing potential of C/EBPa, indicating the importance of C/EBPa-Max in myeloid progenitor differentiation. Taken together, our data reveal Max as a novel co-activator of C/EBPa functions thereby suggesting a possible link between CEBPa and Myc-Max-Mad network.
Discovery, Engineering and Pre-Clinical Evaluation of an Anti-vWF A1 Domain Aptamer.
Authors: Diener John L. , Lagasse H.A. Daniel , Gilbert Madaline , Merlino Patricia , Killough Jason , Marsh H. Nicholas , Boomer Ryan M. , Fraone Joseph M. , McCauley Thomas , Makim Ankit , Lewis Scott D. , Cai Larry , Hutabarat Renta , Rottman James B. , Benedict Claude , Fontana David
Published: October 2019

Following coronary artery damage, von Willebrand Factor (vWF) multimers adhere to exposed collagen via the vWF A3 domain. Platelets passing through damaged vessels under the conditions of high shear force associated with stenosed arteries will adhere to the vWF A1 domain via GPIb receptors. This initial platelet binding event triggers the formation of a thrombus that can limit blood flow to the myocardium and produce the symptoms of acute coronary syndrome (ACS). Through in vitro selection (SELEX) and subsequent lead optimization, we have generated a nuclease-resistant DNA aptamer that binds to the vWF A1 domain with high affinity. The anti-vWF aptamer inhibits both botrocetin-induced platelet aggregation of platelet rich plasma and shear force-induced platelet aggregation in citrated whole blood. Continuous intravenous infusion of the aptamer in cynomolgus macaques is able to effectively inhibit thrombus formation induced by electrical injury to the carotid artery. Modest effects on template bleeding are observed with the vWF-specific aptamer relative to anti-GPIIb/IIIa therapy. Based upon these results, this aptamer is an excellent candidate for treatment of ACS.
Tumor-Endothelial MMP1/PAR1 Crosstalk Promotes Formation of UL-VWF-Strings on Human Endothelium.
Authors: Goerge Tobias , Barg Alexej , Schnaeker Eva-Maria , Luger Thomas A. , Steinhoff Martin , Schneider Stefan W.
Published: October 2019

Extravasation of circulating tumor cells relies on efficient tumor-endothelial crosstalk. Only recently, we were able to demonstrate, that tumor-derived MMP-1 is capable of activating endothelial expressed PAR1 in the macro- and microvasculature (Cancer Res, in press). In the present study we further explore the implications of endothelial PAR1 activation on VWF release under shear flow. For this purpose, we designed a nano-fluidic assay, that allows high-resolution analysis of human platelet-endothelial interactions under shear flow. By immunofluorescence studies and in the absence of ADAMTS13, we are capable of visualizing the activation of human endothelial cells by tumor-derived supernatant, displayed by the formation of UL-VWF platelet strings. Using MMP-1 and PAR1 antagonists, we can significantly inhibit this process. Our findings provide a molecular mechanism of communication between tumor and endothelial cells. Tumor-induced UL-VWF formation contributes to the understanding of thrombus formation as seen in cancer patients.
Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome in Pregnancy: A Report of Four Case.
Authors: He Yang , Master Jianmin Hu , Yang Weiwen , Yan Jun
Published: October 2019

Objctive: Little information exists regarding thrombotic thrombocytopenic purpura(TTP) and hemolytic uremic syndrome(HUS) during pregnancy. We report 4 cases of TTP and HUS complicating pregnancy, with emphasis on diagnosis and management of these rare disorder.

Methods: From Sep. 1988 to May. 2006, case history of four cases with either TTP(n=2) or HUS(n=2) were analyzed. Clinical and laboratory findings and maternal and fetal conditions were recorded from the medical records.

Results: Of 4 cases,3 were nulliparous, one multipara. All women were in good health before pregnancy. Three in the third trimester (34w to37w), had eclampsia, 2 of whom complicated abruptio placenta. The diagnosis was made at admission as HELLP syndrome with eclampsia in 3 cases and one sespected as TTP. The laboratory analyses showed that all 4 cases were anemic(range 29.0 t0 66.0g/L) and 3 had abnormal fragmented red cells on peripheral blood smear. The platelet count ranged from 25 to 40 ×109/L The mean peak serum LDH value was 2616.75U/L(range 914 to >4000U/L),7-to 11 fold the peak serum AST and ALT values. The mental status abnormalities was in 3 cases. All cases had oliguric,2 of whom had acute onset of anuria after delivery. The mean peak serum BUN and creatinine values were 26.14±12.96mmol/L(13.1 to 37.56 mmol/L) and 647.85±347.4μmol/L(184.4 to 1027μmol/L), respectively. The renal biopsy performed on day 8 in one case with anuria, revealed findings consistent with HUS with glomerular crescents. vWF-cleaving protease was measured after plasmapheresis in case of TTP, in whom the report was normal. Treatment included fresh-frozen plasma in 3 cases except one who died before treatment. Of the 3 surviving cases, one received CRRT and plasma exchange and 2 received CRRT and hemodialysis. Of 4 cases,2 delivered vaginally, one ceasarean section, one spontaneous abortion. The course of illness was prolonged, but uneventful 24 to 25 days after treatment and discharge. All fetal death occurred before delivery. During the follow up 3 months to 16 years after discharge, one of 3 cases had another pregnancy complicating preeclamsia at 36 weeks without any other complication and the other two were all normal in renal function and BP.

Conclusion: TTP and HUS complicating pregnancy is associated with high maternal mortality and morbility. The endothelial dysfunction appears to be an important factor in TTP-HUS. With the intense concern about the distinction of preeclampsia/eclampsia/HELLP syndrome from TTP/HUS, the physician must remain alert for the possibility. Improved survival after this disorder had been attributed to treatment with plasma transfusion or plasmapheresis.
Prognostic Indicators in Severely ADAMTS13 Deficient TMA Patients.
Authors: Erickson Yasuko O. , Samia Noelle I. , Bedell Bruce , Raife Thomas J.
Published: October 2019

Previous studies have shown that severe ADAMTS13 deficiency in TMA is associated with lower platelet counts, a greater number of plasma exchange procedures, and a higher rate of relapse compared to non-severely-deficient ADAMTS13. To further explore prognostic factors in TMA, we retrospectively analyzed associations between several pre-TPE laboratory analytes and the number of TPE procedures in a large cohort of surviving TMA patients. We evaluated TMA patients with severely deficient (<15%) pre-TPE ADAMTS13 activity levels (ADAMTS13-D patients) separately from TMA patients with non-severely deficient ADAMTS13 activity (ADAMTS13-N patients). In 37 ADAMTS13-D patients and 40 ADAMTS13-N patients we compared pre-TPE platelet counts and hematocrits (Hcts) with number of TPE procedures. Because TMA is associated with inflammatory conditions, in subsets of 23 ADAMTS13-N and 30 ADAMTS13-N patients we measured C-reactive protein (CRP) and the sepsis marker procalcitonin (PCT), and compared them to number of TPE. The median number of TPE procedures among ADAMTS-D patients with platelet counts below the median (13,000/μL) was 4-fold greater (36 TPE) than in patients with platelet counts above the median (9 TPE) (p=0.006, rank sum). Similarly, the median number of TPE in ADAMTS13-D patients whose Hcts were below the median (24%) was half (12 TPE) that of the cohort with Hcts above the median (26 TPE) (p=0.044). In ADAMTS13-D patients, CRP ≤1.0 ng/dL was associated with >2-fold more TPE procedures (median 6.5 TPE) than CRP>1.0 ng/dL (median 15 TPE) (p=0.0026). By contrast, there was no relationship between platelet count, Hct or CRP level and the number of TPE in ADAMTS13-N patients. However, evidence of sepsis (elevation of both PCT and CRP) was present in 40% of ADAMTS13-N patients, but was not associated with number of TPE procedures. No ADAMTS13-D patient had elevated PCT. In conclusion, lower pre-TPE platelet counts, higher Hcts and higher CRP levels were associated with significantly greater numbers of TPE in ADAMTS13-D TMA. By contrast, no laboratory parameter was associated with number of TPE in ADAMTS13-N patients. ADAMTS13 levels combined with other laboratory parameters may therefore be helpful in the prognosis, resource management, and guidance of adjunctive therapy in TMA. In addition, rapid assays such as PCT may allow early identification of sepsis in TMA patients, which may not have prognostic significance, but could prompt antibiotic treatment that may enhance outcome. The distinct difference in prognostic factors between ADAMT13-D and ADAMTS13-N patient subsets further supports the pathogenic importance of ADAMTS13 in TMA.
Cryosupernatant and Solvent Detergent Fresh Frozen Plasma (Octaplas, Octapharma) Usage in Acute Thrombotic Thrombocytopenic Purpura-Review of 50 Episodes.
Authors: Scully Marie , Flynn Michael , Berryman Jenny , Machin Samuel J.
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is an acute, life threatening disorder. The mainstay of treatment is plasma exchange (PEX) as a source of ADAMTS 13. In the UK, 20–25% of all plasma consumed is in patients with TTP. In our protocol (up until 31st December 2005) apheresis was initially with cryosupernatant (National Blood Service, UK) unless patients had a previous severe allergic reaction or refractory disease. Apheresis therefore continued with Solvent-Detergent Fresh Frozen Plasma (S/D FFP) Octaplas, (Octapharma, Vienna Austria) virally inactivated plasma, available throughout Europe. We reviewed 50 acute TTP episodes involving 32 patients. Thirteen episodes used cryosupernatant only and in 15 episodes, treatment started with cryosupernatant and changed to Octaplas. The reasons for changing were refractory disease in 2 episodes and major or recurrent allergic reactions to cryosupernatant in 13 cases. Once Octaplas had been used, it was continued on further admissions. In 22 episodes, Octaplas was used exclusively; in 4 cases as physicians choice and in the remaining due to previous reactions to cryosupernatant. The total volume of cryosupernatant used was 508250mls, 27.6% of all plasma; total volume of Octaplas was 1327600mls, 72.4% of all plasma. Citrate mediated reactions associated with symptomatic hypocalcaemia during apheresis were present in 11% of Octaplas and 20% of cryosupernatant. Acute or delayed urticarial or allergic reactions were noted in 5% of Octaplas and 10% cryosupernatant procedures. A particular complication of apheresis is central line infection. There were 21 line infections and in 43% of cases the infection was associated with a reduction in platelet count < 150 × 109/L. In all 50 episodes, the only documented thrombosis was a superficial non central vein in a patient who had received Octaplas. Prevention of venous thrombosis is by use of thromboembolic stockings, low dose aspirin and low molecular weight heparin in patients when platelet counts >50 × 109/L. In episodes receiving only cryosupernatant or Octaplas, there was no significant difference in the median number of PEX to remission, 7(3–14) and 8.5 (5–30) respectively. Baseline viral screen in all episodes was negative after discharge following an acute TTP episode. In conclusion: cryosupernatant and S/D FFP (Octaplas) appear equally efficacious. However, the risk of allergic/urticarial reactions was twice as common with cryosupernatant, as were citrate reactions. Milder allergic reactions to cryosupernatant are possibly higher, but may have been treated with antihistamines and data not recorded. There was no documented viral transmission with either product.
A Study on the Induction of Mesoderm Differentiation of Mouse Embryonic Stem Cell in Serum Free Culture.
Authors: Suzuki Takao , Takahashi Masatomo , Miyagi Tsukasa , Irei Michiko , Shibuya Yasushi , Kato Masayuki , Inoue Yasuyuki , Koike Mitsuru , Miura Ikuo
Published: October 2019

(Introduction) Several products which can maintain the growth of ES (Embryonic stem) cell with no feeder cells or fetal calf serum (FCS) is now commercially available. Culti Cell ®(CC) can be a substitute for FCS and has ability of mainitaining mouse ES cells without feeder cells. Previously, it was reported that ES cell differentiate into FLk (fetal liver kinase) positive cells derived from mesoderm in methylcellulose and FCS. We investigated whether ES cell is able to differentiate into Flk positive cell in CC and methylcellulose without FCS.

(Materials and Methods) Thawed ES cells were cultured with methylcellulose and CC in the absence of FCS and feeder cells. EB (Embryoid body) formation was completed after 7 days. The separated cell from EB was adjusted to 1×104/ml. These ES cells were cultured with 15% CC and 0.8% methylcellulose for 7 days at 37°C in 5% CO2. The expression of Flk and mesoderm differentiation was analyzed with flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) at day 1, 2, 3, 5 and 7. Oligonucleotide primers for Flk1 and brachyury were used for RT-PCR. In addition, the expression of CD31 was also examined with flow cytometry.

(Results) No Flk positive cells were detected in these culture periods. The expression of brachyury, which is a marker of mesoderm differentiation, was consistently observed throughout culture days. The expression of CD31 gradually diminished as the culture period increased.

(Conclusion) These results suggest that ES cell is maintained and differentiate into mesoderm in suspension culture with CC and methylcellulose, but diferentiation to Flk positive cell can not be induced in this culture condition. Further examination is necessary to clarify unidentified factors for differentiation to Flk positive cell in serum free culture.
Negative Regulation of Primitive Hematopoiesis by the FGF Signaling Pathway.
Authors: Nakazawa Fumie , Nagai Hiroki , Shin Masahiro , Sheng Guojun
Published: October 2019

Hematopoiesis is controlled by multiple signaling molecules during embryonic and postnatal development. The function of FGF pathway in this process is unclear. Here we show that FGF plays a key role in the regulation of primitive hematopoiesis. Using hemoglobin mRNA expression as a sensitive marker, we demonstrate that timing of blood differentiation can be separated from that of initial mesoderm patterning and subsequent migration. High FGF activity inhibits primitive blood differentiation and promotes endothelial cell fate. Conversely, inhibition of FGFR activity leads to ectopic blood formation and down-regulation of endothelial markers. Expression and functional analyses indicate that FGFR2 is the key receptor mediating these effects. The FGF pathway acts through crosstalk with VEGFR signaling and regulates primitive hematopoiesis by modulating gata1 expression level and activity. We propose that the FGF pathway mediates repression of globin gene expression and that its removal is essential before terminal differentiation can occur.
Differences in Human Growth Factors Induced by Non-Human Cell Expression.
Authors: Pilkington Glenn , Marks Denese , Crofts Linda , Domagala Teresa , Liddell Kate , Russell-Jones Greg
Published: October 2019

Expression of recombinant human proteins in cells from different species, including bacteria, yeast, insect, mouse, hamster or primates, imparts species-specific changes post-translation. The impact of these species-specific differences in post-translational modifications (PTMs) can be far-reaching, resulting in effects ranging from inaccurate quantitation of proteins in serum to in-vivo immunogenicity (Porter, J Pharm Sci 90: 1–11, 2001) or even autoimmunity to the patient’s own native protein (Ryan et al., Int Immunopharmacol 4:647–55, 2006). Currently, most human proteins are still produced from bacteria, including several of the World Health Organization international growth factor standards. For example, present-day clinical grade recombinant human growth factors such as G-CSF, IL-4 and GM-CSF are manufactured from bacteria. The expression of human proteins that naturally exhibit a high degree of glycosylation, such as GM-CSF, in bacterial cells has major implications for immunoassays and therapeutics. Since bacterial cells do not glycosylate human proteins, exposure of cryptic or masked epitopes, normally buried on the native protein, can result in generation of antibodies that bind falsely in immunoassays and can cause reduced potency or adverse reactions in patients. The expression of recombinant human proteins in non-human mammalian cells can result in non-natural epitopes due to the addition of non-human sugar structures. We have expressed several human growth factors in human cells and have demonstrated differences in their immunoreactivity patterns when compared to the same recombinant proteins expressed in non-human cells. These changes in the biological properties of the proteins are likely related to differences in glycosylation and other PTMs unique to proteins expressed in human cells. We have demonstrated that expression of human growth factors in human cells may have significant benefits for the accuracy of native human protein assays and the therapeutic efficacy of human growth factors.
Chronic Refractory Anemia with Sideroblastic Bone Marrow: A Study of Four Cases
Authors: BJÖRKMAN SVEN ERIK
Published: October 2019

Four cases of severe refractory anemia with an enormous increase of demonstrable free iron in the normoblasts are reported. Three cases were of unknown etiology and of benign course but in the fourth the anemia was probably related to rheumatoid arthritis or to the onset of myeloblastic leukemia. The significance of the sideroblastic bone marrow in these cases is discussed and the opinion expressed in the literature—that it indicates a defect in hemoglobin synthesis is subscribed to. Thus, it is believed that the anemia in these cases is due to degenerative changes in the bone marrow which also have some morphologic manifestations. The disturbance of marrow activity is probably best reflected in the reticulocyte counts.
Homozygous Hemoglobin C Disease in Siblings: Further Comment on Intraerythrocytic Crystals
Authors: WHEBY MUNSEY S. , THORUP OSCAR A. , LEAVELL BYRD S.
Published: October 2019

1. Two Negro brothers with homozygous hemoglobin C disease are reported. In one splenectomy was performed.

2. Intraerythrocytic crystals in smears of peripheral blood were demonstrated only in the patient who had splenectomy. The crystals could not be found in re-examination of bone marrow preparations of this patient that were made prior to splenectomy.

3. Absence of the spleen may be a necessary factor for the formation of these crystals.

4. The presence of homozygous C disease is compatible with a normal life span.
Atypical Familial Hemolytic Anemia
Authors: SMILEY R. K. , DEMPSEY H. , VILLENEUVE P. , CAMPBELL J. S. , BEST BARBARA
Published: October 2019

1. The genetic, clinical and hematologic features of an atypical chronic hemolytic anemia in two siblings of a French Canadian family have been described.

2. The anemia is normocytic, normochromic and not associated with any characteristic morphologic abnormality of the red cells.

3. Slight increases in osmotic and incubated mechanical fragility, as well as a more definite increase in aumtohemolysis were found which could not be demonstrated after splenectomy.

4. The survival time of normal red cells was shortened before splenectomy in one patient. Normal red cell survival was demonstrated in both patients after splenectomy.

5. The features which differentiate this hemolytic anemia from hereditary spherocytosis are discussed.

6. French or French Canadian ancestry has been noted in some of the reported patients most similar to our own.

7. The association of this type of hemolytic anemia with blood group A has been confirmed in our patients.

8. Splenectomy decreased the severity of the hemolytic process in both patients. This benefit may have resulted from removal of an extracorpuscuar hemolytic mechanism.
Multiple Marrow Aspiration in Man from the Posterior Ilium
Authors: BIERMAN HOWARD H. , KELLY KEITH H.
Published: October 2019

A procedure for aspirating large volumes of bone marrow with minimal admixture of blood is described.
Sickle-Cell Anemia in South Turkey. A Study of Fifteen Cases in Twelve White Families
Authors: AKSOY MUZAFFER
Published: October 2019

1. Fifteen cases of sickle-cell anemia in twelve white families in southern Turkey are reported.

2. In all these cases of sickle-cell anemia and their families, there was no suggestion of anything Negroid in their features or coloring.

3. The hereditary trait of these patients has been investigated. In 53 healthy members of twelve families 37 persons had sickle-cell traits.

4. With the exception of case 12, both parents of all cases had sickle-cell trait.

5. The possible explanations of the frequent occurrence of sickle-cell anemia and the fairly high incidence of sickle-cell trait in this white community have also been considered.
Detection of p57KIP2 Gene Methylation Is Useful Marker for Minimal Residual Disease in Diffuse Large B Cell Lymphoma.
Authors: Hagiwara Kazumi , Nagai Hirokazu , Yokozawa Toshiya , Kato CHiaki , Hamaguchi Motohiro , Hotta Tomomitsu , Horibe Keizo
Published: October 2019

Background: The t(14;18)(q32;q21) translocation, which causes a BCL2/IgH gene rearrangement, is a characteristic chromosomal abnormality found mainly in B cell lymphomas, and its detection and quantification is useful for monitoring of minimal residual disease (MRD) in follicular lymphoma (FL). However, in diffuse large B cell lymphoma (DLBCL), the frequency of this rearrangement is relatively low. We revealed that the promoter region of p57KIP2 gene is frequently methylated in B cell lymphomas. Aberrant DNA methylation in the promoter regions of genes is known as the major mechanism for inactivation of tumor suppressor genes, so the detection of the methylation is expected to be used as a promising tumor specific marker. Therefore, we investigated the possibility of p57KIP2 gene promoter methylation as a biomarker for MRD detection in B cell lymphoma, especially in DLBCL.

Methods: We analyzed lymphoid cell line DNA, tumor DNA from 64 patients with DLBCL, and 4 patients with FL. The bisulfite-modified DNA was used as a template for conventional methylation-specific PCR (MSP) and real-time quantitative MSP (Q-MSP).

Results: In clinical samples (tumor DNA), p57KIP2 gene methylation was detected by Q-MSP in 73% (47/64) DLBCL, and 50% (2/4) FL. Using cell line DNA, which was fully methylated in promoter region of p57KIP2 gene, we determined the detection limit of Q-MSP assay. The methylated DNA could be detected in the presence of a 1000-fold excess of unmethylated DNA by Q-MSP. We could validate the level of methylated DNA in each sample by Q-MSP evaluating the p57KIP2/b-actin ratio. The sensitivity to detect MRD in bone marrow by this method was found to be equivalent to real time quantitative PCR for BCL2/IgH major breakpoint region.

Conclusion: The methylation of p57KIP2 gene is detected at high frequency in DLBCL. This biomarker is thought to be conventional and widely applicable to the detection of MRD in DLBCL.
Bone Marrow Biopsy for the Staging of Diffuse Large B-Cell Lymphoma: Is It Necessary Bilateral Trephine Biopsy?.
Authors: Bellesso Marcelo , Velasques Rodrigo D. , Pracchia Luis F. , Beitler Beatriz , Aldred Vera L. , Chamone Dalton , Kutner Jose M. , Pereira Juliana
Published: October 2019

In this retrospective analysis we study the usefulness of bilateral rather than unilateral iliac trephine biopsies in demonstrating Diffuse Large B-Cell Lymphoma (DLBCL) in the bone marrow (BM). A total of 28 DLBCL cases with BM infiltration were investigated with bilateral iliac trephine biopsy during staging. Our propose was to evaluate the incidence of unilateral BM involvement, and we compare the BM biopsy size with negative and positive BM infiltration. In 28 cases studied it was analyzed 70 BM fragments. These fragments were reviewed separately by pathologist about size and infiltration. In this group, 6 cases (21.4%) were unilaterally positive. The median number of fragment per case was 2.5; the median size of BM fragment was 11.01 mm (± 5.12) and BM fragment per case was 27.53mm. It was not found difference between size BM fragment in negative or positive fragments 11,57mm (±5.2) versus 10.95mm (±5.1), p>0.05, respectively. In 24 cases it was possible to compare negative and positive BM infiltration with age, sex, LDH and computerized tomography (CT) staging. In addition, it was not found difference between unilateral and bilateral BM infiltration about LDH and age, and CT. (Table 1).

Conclusion: Although in unilateral infiltration cases did not change risk factor in International Prognostic Index (stage III–IV) by CT, we conclude that bilateral trephine biopsy is superior to unilateral, because bilateral biopsy could increase by 21,4% the detection of BM involvement by DLBCL, without difference between the size in negative versus positive fragments.

Table 1. Distribution of Unilateral and Bilateral Bone Marrow infiltration by categories Unilateral infiltration (%) Bilateral infiltration (%) Age < 60 21% 50% Age ≥ 60 4% 25% Sex Male 16% 29% Sex Female 8% 47% CT staging I–II - 35% CT staging III–IV 26% 39% LDH Normal 8% 34% LDH Elevated 16% 42%
A Comparative Study of Diffuse Large B-Cell Lymphoma (DLBCL) between African Americans and Caucasians: Single-Center Experience at the University of Alabama at Birmingham (UAB).
Authors: The Angelina , Li Yufeng , Reddy Vishnu , Davis Randall , Baird Melissa , Foran James
Published: October 2019

Background: SEER data indicate that African Americans (AA) have a lower incidence of DLBCL but a higher mortality rate than Caucasians (C). To investigate this, we conducted a single-center analysis of AA and C patients with DLBCL at UAB. UAB is a primary care facility and the main tertiary referral center in Alabama. It is located in Jefferson County where the average African American population of 39% is nearly triple the national average.

Methods: After IRB approval, patients diagnosed with DLBCL from 1995 to 2007 were identified from pathology, referral, and UAB tumor registry databases. Baseline demographic data including race, age at diagnosis, stage, treatment administered, response to treatment, and survival were extracted. Serum LDH levels and performance status were not consistently available in medical records and therefore not included in the final analysis. Patients with monomorphic post-transplant lymphoproliferative disease and primary CNS lymphoma were excluded. Associations between race and stage, treatment (first-line rituximab, anthracycline, or combination therapy), response, and outcome were analyzed using Chi-squared or Cochran Mantel-Haenszel statistical analysis.

Results: A total of n=309 (n=32 AA and n=277 C) patients were identified. AA patients were diagnosed at a significantly younger age than C patients [median age 49 (range: 22–90) vs. 61 (range: 16–91), respectively; p=0.0131]. AA patients also presented with advanced stage disease (Ann Arbor stage III–IV) more frequently (69% vs. 52%, p=0.046). No difference in extranodal disease presentation or in complete response rate to first-line therapy was noted. As expected, those with early stage disease, age <60, and those given first-line rituximab +/− anthracycline therapy had superior survival regardless of race. No difference in overall survival was seen between AA and C patients (p=0.1306). However, AA patients had an inferior survival to C patients when the analysis was restricted to patients who received either first-line treatment with rituximab (p=0.0658), anthracycline (p=0.0188), or combination (p=0.0196).

Figure Figure

Conclusions: Compared to C patients, AA patients were diagnosed with DLBCL at a significantly younger age, more advanced stage, and had worse survival despite first-line rituximab +/− anthracycline therapy. This single-center experience confirms SEER data and suggests a possible difference in tumor biology or treatment effect, although potential socioeconomic factors can not be excluded.
Marqibo (Vincristine Sulfate Liposomes Injection (OPTISOME™) Demonstrates Activity in Patients with Relapsed and Refractory Peripheral T-Cell Lymphoma.
Authors: Rodriguez M. Alma , Winter Jane N. , Lu Biao , Huang Gary , Choy Gavin S. , Deitcher Steven R.
Published: October 2019

Introduction: New and more effective agents are needed to improve treatment outcomes for patients with relapsed and refractory peripheral T-cell lymphoma (PTCL). Marqibo is a novel formulation of vincristine (VCR) encapsulated in sphingomyelin/cholesterol liposome called OPTISOME. In preclinical studies, OPTISOME technology has been shown to provide targeted, increased, and sustained delivery of VCR to tumor cells compared to non-encapsulated VCR or VCR encapsulated in conventional liposomes.

Methods: PTCL patients from two multi-center phase 2 studies of Marqibo in relapsed and refractory lymphoid malignancies are integrated for safety and efficacy evaluation. In both studies, Marqibo (2.0 mg/m2 without dose capping IV over 60 minutes) was administered every 14 days for up to 12 cycles or until toxicity or progressive disease was observed. The primary efficacy endpoint of the 2 studies was the objective response rate (ORR) defined as the percentage of patients whose best response was complete response (CR), complete response unconfirmed, or partial response (PR). Best response was determined according to the International Workshop Response Criteria. Secondary endpoints included adverse event evaluation, time to progression (TTP) and overall survival (OS).

Results: Six (3M/3F) patients diagnosed with PTCL received at least one dose of Marqibo. At baseline, the median age was 59 years (range, 34–86). All six patients had aggressive disease and most were heavily pretreated. The median number of prior lines of chemotherapy and immunotherapy regimens was 3 (range, 1–6) and four patients (66.7%) had received prior autologous bone marrow transplant. All patients had prior exposure to neurotoxic therapy: platinum compounds (33.3%), taxanes (16.7%) and VCR (100%). All patients had achieved a CR or PR to their frontline therapy and 50% (3/6) had achieved a CR or PR to their last line of therapy prior to receiving Marqibo. Patients received a median of 4 cycles of Marqibo (range, 1–9). Median cumulative dose of Marqibo was 7 mg/m2 (range, 2–17.4). The most commonly reported adverse event was peripheral sensory neuropathy (3 of 6 patients) and all of Grade 1 severity. Three patients had PR, 1 patient had stable disease, 1 patient experienced disease progression, and 1 patient was unevaluable. Median OS and TTP were 194 and 70 days, respectively.

Conclusions: These preliminary results suggest encouraging activity and tolerability of Marqibo in heavily pre-treated relapsed and refractory PTCL patients. Given the high proportion of exposure to neurotoxic agents preceding Marqibo, the low grade neurotoxicity noted following Marqibo is a favorable finding.
Rituximab Maintenenance Therapy Prolongs Event Free Survival in Patients with CD20+ B-Cell Non-Hodgkin-Lymphoma.
Authors: Witzens-Harig Mathias , Hensel Manfred , Schmier Johann W. , Neben Kai , Benner Axel , Dreger Peter , Kuhn Christian , Schmidt-Wolf Ingo , Kraemer Alwin , Ho Anthony D.
Published: October 2019

Clinical and pharmacokinetic data suggest that the effect of rituximab could be improved by prolonged exposure to the drug. To test for this hypothesis we performed a prospective randomized trial of rituximab maintenance therapy in patients with CD20+ B-cell Non-Hodgkins-Lymphoma. After completion of standard treatment patients were randomized to either observation or maintenance therapy with rituximab (375 mg/m2) every 3 months for 2 years. Patients after first line therapy as well as relapse patients were included in the study. Patients with aggressive lymphoma were enrolled if they had achieved a complete response (CR) after initial treatment. Patients with aggressive lymphoma with residual tumor mass were examined with positrone emission tomography (PET) and qualified for randomization if PET showed no signs of tumor activity. Patients with indolent lymphoma qualified for the study if at least a partial response (PR) was achieved. After recruitment of 172 patients a planed interim analysis was performed. Complete data sets of 162 patients (pts) with CD20+ B-cell Non-Hodgkins-Lymphoma were evaluable for analysis. Histological subtypes included diffuse large cell lymphoma (69 pts), follicular lymphoma (41 pts), mantle cell lymphoma (18 pts), primary mediastinal lymphoma (15 pts), marginal zone lymphoma (9 pts), Burkitt’s lymphoma (3 pts), immunocytoma (2 pts), primary intestinal lymphoma (1 pt), hairy cell leukemia (1 pt), chronic lymphocytic leukemia (1 pt) and unclassified B-cell lymphoma (2 pts). The interim analysis showed that event free survival was significantly prolonged in the rituximab maintenance group compared to the observation group (p<0.05). However, no difference in overall survival between the two groups was observed so far. Two patients in the treatment group developed WHO grade III adverse events (1 leucopenia, 1 infection). Both pts recovered shortly after appropriate treatment. We conclude that rituximab maintenance therapy is feasable, safe and well tolerated in patients with CD20+ B-cell Non-Hodgkins-Lymphoma and may prolong event free survival in this patient population.
Bortezomib, Pegylated-Lyposomal-Doxorubicin and Dexamethasone Followed by Melphalan 100 mg/m2 in Elderly Newly Diagnosed Patients: An Interim Analysis.
Authors: Palumbo Antonio , Avonto Ilaria , Patriarca Francesca , Crippa Claudia , Petrucci Maria T. , Rossini Fausto , Corradini Paolo , Liberati Anna M. , Giuliani Nicola , Pescosta Norbert , Falco Patrizia , Larocca Alessandra , Omedè Paola , Offidani Massimo , Fanin Renato , Boccadoro Mario
Published: October 2019

Bortezomib has been evaluated as induction regimen to improve cyto-reduction before autologous stem cell transplant. Combinations including bortezomib, doxorubicin and dexamethasone have shown encouraging results. Melphalan at 100 mg/m2 has been suggested as reduced-intensity conditioning regimen for elderly patients. In this prospective multicenter phase II study, bortezomib, pegylated-liposomal-doxorubicin and dexamethasone (PAD) followed by tandem melphalan 100 mg/m2 has been investigated in newly diagnosed patients aged 65–75 years. The induction regimen included four 21-day PAD cycles (bortezomib 1.3 mg/m2 days 1, 4, 8, 11, pegylated-liposomal-doxorubicin 30 mg/m2 day 4 and dexamethasone 40 mg days 1–4, 8–11, and 15–18 for cycle 1 and days 1–4 for cycles 2–4). Cyclophosphamide (3 g/m2) plus G-CSF were used to harvest stem cells. Patients were then conditioned with tandem melphalan 100 mg/m2 followed by stem cell infusion. A single interim analysis has been planned. Sixty-five patients have been enrolled in the study and 37 completed the induction cycles. Median age was 69 years; median β2-microglobulin 3 mg/L; median albumin 4.1 g/L; chromosome 13q deletion was detected by FISH in 35% of patients. After the 4 courses of PAD 97.1% of patients achieved at least a partial response (PR), 50% at least a very good partial response (VGPR), 11.8% an immunofixation negative complete remission (CR). After tandem melphalan 100 mg/m2, all patients achieved at least a PR, 80% at least a VGPR and 30% an immunofixation negative CR. After 4 cycles of PAD, grade 3–4 hematologic events were thrombocytopenia (13.5%) and neutropenia (8.1%); more frequent grade 3–4 non-hematologic toxicities were peripheral neuropathy (21.6%) and infections (10.8%). One early toxic death was reported (central nervous system bleeding) and two patients had to discontinue therapy due to pneumonia and HBV reactivation. Eighty-two percent of patients achieved successful stem cell harvest (>4 CD34+ cells/Kg). PAD is an effective induction approach, it may improve autologous transplant results in selected elderly patients. Updated results of the interim analysis will be presented at the meeting.
Predicting Structural and Serologic Modifications in Blood Group Antigens by Homology Modeling.
Authors: Westhoff Connie M. , Wylie Dwane E.
Published: October 2019

Homology modeling of blood group proteins offers the possibility of predicting the effect of amino acid changes on serologic phenotype and immunogenicity. The location of an amino acid change within known structural motifs, its phylogenetic conservation, and its proximity to known epitopes give insight into its potential effect on protein structure and, consequently, its clinical significance. We applied this approach to investigate the loss of membrane expression of the Dombrock blood group antigens in a patient with a single amino acid change and to investigate RhD alterations in weak D phenotypes. The Dombrock homology model was derived with rat ART2.2 crystal structure as template. For the RhD model, the crystal structure of the Rh-like-ammonia transporter from Nitrosomonas europaea was used. Protein alignment was derived with Clustal X, adjusted visually, and submitted to the Swiss Modeling server. Models were viewed with Deep View Swiss Pdb Viewer. The Dombrock null containes a Phe62Ser substitution. This Phe (F) residue is located in an FDDQY motif near the COOH terminus. This region of the protein also contains a HYYLT motif. These two motifs are highly conserved in the ART protein family and contribute several aromatic amino acids to this region of the molecule. Aromatic side chain interactions between these residues could contribute to the stability of the Do protein. In support, the distance in the ART2.2 crystal structure between Phe in FDDQY and His in HYYLT is 3.7 Å, which is the appropriate distance for aromatic side chain interactions. This is also the measured distance between these two residues in the Do model. Thus, protein modeling indicates that the Phe62Ser mutation disrupts important stacking interactions between Phe62 and His160. When amino acid changes causing weak D phenotypes were examined, some of those affecting expression of RhD were located near the vestigial transport channel. These include the Trp220Arg mutation (weak D Type 16). This Trp residue is part of the transport channel in Nitrosomonas and is conserved in Rh proteins of almost all species. Its role in maintaining Rh structure is indicated by the dramatic effect its modification has on protein and epitope expression. Additionally, Arg114Trp change (weak D Type 17), which is also near the channel, reduces D expression to only 66 antigen sites/cell. GlyXXXGly motifs stabilize interactions of adjacent alpha helices in membrane proteins. Evidence for a role in stabilization of RhD is revealed by the Gly282Asp mutation (weak D Type 15) which is part of such a motif. In addition, a D-epitope in loop 3 is near the 282Asp residue. Alteration of helical packing accompanied by epitope conformation could explain production of anti-D in patients with weak D Type 15. Homology modeling is an important tool for understanding the structure and serologic bases of blood group proteins and will continue to give important insight as more protein crystal structures become available.
Prognostic Significance of CD33, CD34 and CD117 Expression on Bone Marrow Blasts of Patients with Myelodysplastic Syndromes.
Authors: Nador Guido , Molteni Alfredo , Cesana Clara , Nichelatti Michele , Scarpati Barbara , Cairoli Roberto , Barbarano Luciana , Nosari Annamaria , Morra Enrica
Published: October 2019

Introduction: Although blast-rich specimens immunophenotype studies in myelodysplastc syndromes (MDS) could associate bone marrow (BM) blast expression of CD7 and/or CD117 antigens with poor outcome (Ogata et al., Blood 2002), the prognostic role of markers of myeloid cell immaturity and committment in not enriched BM samples is largely unexplored.

Patients and Methods: The expression of CD33, CD34 and CD117 antigens in not enriched BM samples of 50 newly diagnosed MDS was compared with both BM blast WHO category and IPSS score. Immunophenotyping was carried out by using the panel of quadruple monoclonal antibodies CD34/CD117/CD45/CD33, conjugated with the fluorochromes FITC, PE, PerCP, APC, respectively. Acquisition of information on 1x105 stained cells corresponding to the whole BM cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Multiple group comparisons were made using non parametric ANOVA for BM blasts; general linear model with Wald’s test and Kruskal-Wallis (KW) test to confirm significance was used for IPSS.

Results: According to IPSS, 5 (10%) low risk, 27 (54%) intermediate risk-1, 14 (28%) intermediate risk −2 and 4 (8%) high risk pts were identified, respectively. The expression of CD33, CD34 and CD117 significantly correlated with both blast WHO category and IPSS, as shown in the Table 1. Interestingly, by analyzing the subset of 30 pts with BM blasts <5%, a correlation was found between IPSS and the expression of both CD33 (p=0.0097) and CD34 (p=0.0299); CD117 expression increased in pts with higher IPSS, without, however, reaching statistical significance.

Conclusions: Both markers of myeloid cell committment (i.e., CD33 and CD34) and markers of myeloid cell immaturity (i.e., CD117) correlate with BM blast WHO category and IPSS score. Expression of CD33 and CD34 may have prognostic significance independently from blast percentage. The evaluation of CD33, CD34 and CD117 expression on not enriched BM samples may represent a simple tool for the prognostic assessment of MDS patients, in addition to previously established methods.

Tab. 1: Correlation of CD33, CD34, CD117 expression with IPSS and percentage of BM blast in 50 MDS pts at diagnosis