bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive Oxygen Species Regulate Spontaneous and Fas-Mediated Apoptosis in SBDS-Deficient Cells.
Authors: Ambekar Chhaya , Das Bikul , Yeger Herman , Dror Yigal
Published: April 2020

Background and hypotheses: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, pancreatic insufficiency, and a marked propensity for myelodysplastic syndrome and leukemia. Approximately 90% of the patients have mutations in the SBDS gene, but the function of the gene is unknown. We previously showed that marrow cells from SDS patients and SBDS-deficient HeLa Cells are characterized by accelerated apoptosis, overexpression of Fas and hypersensitive to Fas stimulation. Involvement of reactive oxygen species (ROS; oxidative stress) have been shown to be related to Fas hypersensitivity and overexpression in a variety of cell types. Therefore, we hypothesized that functional deficiency in SBDS in cells that express Fas could lead to impaired ROS generation and a subsequent increase in spontaneous and Fas-mediated apoptosis and decrease in cell growth.

Methods: We used shRNA-mediated SBDS-knockdown HeLa cells as a model. We investigated whether SBDS-deficiency increases ROS levels and if antioxidants can rescue the cell growth and apoptosis phenotype. To measure ROS formation cells were incubated with DCFH-DA and fluorescence measured in Gemini Spectra MAX microplate reader. Staining with annexin V and propidium iodide was done to determine apoptosis and necrotic cell death. MTT assay was used to measure cell viability.

Results: ROS levels in SBDS knockdown cells were significantly increased compared to control. Apoptosis analysis by annexin V and propidium iodide showed a marked decrease in cell viability in the SBDS-knockdown cells. NAC treatment decreased ROS levels, enhanced ERK phosphorylation (pERK), improved cell viability, and decreased apoptotic and necrotic cell death. Stimulation of the Fas signaling pathway by CH-11 (activating anti-Fas antibody) and Fas ligand showed increased ROS production in SBDS Knockdown cells. CH-11 treatment showed a marked increase in apoptotic and necrotic cell death after 24 and 48hrs incubation. Cell viability decreased by 40% and 80% after 24 and 48hrs incubation with CH-11. Treatment with NAC lowered ROS levels, enhanced pERK expression, protected the cells from Fas-mediated early apoptosis and improved cell survival.

Conclusion: We have demonstrated that stable loss of SBDS results in increased ROS levels, leading to apoptotic and necrotic cell death. Thus, increased baseline and Fas-stimulated ROS could result in increased sensitivity to apoptosis and necrotic cell death. NAC appeared to reverse the ROS-mediated decrease in cell survival and apoptotic cell death. Our data support the novel concept that SBDS may be a homeostatic regulator of oxidative stress
Recurrent Chromosomal Intermingling Interactions at the BCL2 Locus in T(14;18) +Ve and −Ve Cell Lines.
Authors: Yang Youwen , Wrench David J , Lister T. Andrew , Fitzgibbon Jude
Published: April 2020

Introduction Chromosomes locate non-randomly within the nucleus where they physically associate via intermingling. This physical association leads to co-ordinated regulation of gene expression but also makes these regions prone to translocations. The loci involved in many of the common translocations in lymphoma co-localise together and include IGH and BCL2 which are juxtaposed by t(14,18)(q32q21) in ~90% of Follicular Lymphoma (FL). This frequency suggests the BCL2 locus may be prone to intermingling, with the major breakpoint region (MBR) representing a potential interactive hotspot as it flanks a matrix attachment region (MAR) which tethers chromatin.

Methods-Results To investigate this possibility, intermingling associations with the BCL2 locus were determined using the Chromosome Conformation Capture technique (3C) in B-lympho-blastoid (HRC57 and NcNc), t(14;18) +ve (RL, SuDHL4, SuDHL6, DoHH2) and -ve (HT, SuDHL8, SuDHL5, RAJI) B-NHL cell lines. The 3C technique allows cross-linking of associating chromosomal segments, generating a library of DNADNA hybrid molecules. Intermingling profiles were established for the following three BCL2 fragments contained within this library:

a 10kb section containing the BCL2 promoter region including exons 1 and 2; a 7kb segment within intron 2 containing a highly Conserved Non-coding Sequence (CNS) (associated with gene regulation) and overlapping MAR; and a 6kb section containing part of exon 3 and the MBR with flanking MAR.

This selection contained 81% of the BCL2 coding sequence. The intermingling partners of these BCL2 fragments were amplified using either BCL2- “unknown DNA walking primer” or BCL2-IGH enhancer primer combinations and their identity determined by direct sequencing. Nine intermingling partners were identified from the 3C libraries of at least two cell lines. Of these, IGH enhancer–BCL2 promoter associations were identified in RL, SuDHL4 and DoHH2 and appear restricted to t(14;18)+ve cell lines. MYCBP2 (chr13q22), which functions as a transcriptional activator of MYC, associated with the CNS+MAR sequence within intron 2 and this association was detected solely in malignant cell lines. In comparison, OPCML (chr11q25), a tumour-related gene, associated with the BCL2 MBR+MAR region in all cell lines examined.

Conclusions These preliminary studies suggest that the BCL2 locus has multiple recurring intermingling partners. These may be specific to BCL2, as demonstrated for OPCML which was a consistent partner in all lines investigated, or may be restricted to cells containing the translocated allele as shown for the IGH enhancer.
High-Resolution Array-Based Comparative Genome Hybridization (CGH) Identifies Novel and Recurrent Regions in CLL.
Authors: Grubor Vladimir , Krasnitz Alex , Troge Jennifer E. , Meth Jennifer L. , Lakshmi B. , Kendall Jude T. , Yamrom Boris , Alex Garrick , Pai Deepa , Navin Nicholas , Hufnagel Lisa A. , Lee Yoon-ha , Cook Kerry , Allen Steven L. , Rai Kanti R. , Damle Rajendra , Calissano Carlo , Chiorazzi Nicholas , Wigler Michael H , Esposito Diane
Published: April 2020

Although we have some understanding of the genetic abnormalities occurring in B-cell chronic lymphocytic leukemia (CLL) and their association with clinical outcomes, there is an incomplete comprehension of all of the mutations contributing to disease development and progression. In most abnormalities previously observed, it has been difficult to pinpoint specific candidate genes, reflecting the inadequacy of present tools for assessing chromosomal damage.

We examined copy number changes in the genomes of B cells from 58 patients with CLL using a 390,000-probe microarray, enabling us to perform comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least one genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported were also observed, most of which were highly recurrent. We observed the boundaries of known events with greater clarity and identified lesions previously not described, some of which were also recurrent.

A newly identified 3.6 Mb deletion at 8p21.2-p12 includes the gene TRIM 35. A second novel deletion at 2q37.1 (587kb) encompasses the gene SP100/110/140. Of the refined regions, a 249kb region at 9p21.3 spanning the CDKN2A (p16-INK4) and a 156kb region at 18q23 containing NFATC1 are particularly interesting. In the case of NFATC1, the minimal region of overlap spans that single gene.

Furthermore, we tilized our arrays to examine the clonal heterogeneity of CLL within the same patient from mixed sub-populations. The presence of greater than 30% of B-cells with the CD38 cell surface marker has been associated with poor outcome in CLL. It is an open question whether this reflects genetic heterogeneity and possibly clonal evolution. To investigate this possibility, we analyzed CD38+ and CD38− fractions from individual patients and demonstrated that three out of the four patients examined had undergone clonal diversification leading to new subclones of appreciable size.

Additionally, we have utilized a 2.1 million probe ultra-high density (HD2) array from NimbleGen (Madison, WI), providing us with the capability to scan the human genome for copy number changes at a resolution of a probe every 1500 bp. Utilizing the HD2 array, we are presently re-analyzing all the CLL samples in our study, and in the process are discovering many more previously unpublished lesions, several of which pinpoint single genes. We will present the results of our findings and discuss the potential applications of high resolution CGH analysis in a clinical setting.
A High Number of Losses in 13q14 Chromosome Is Associated with a Worse Outcome and Biological Differences in Patients with B Chronic Lymphoid Leukemia.
Authors: Hernández José-Ángel , Rodríguez Ana-Eugenia , González Marcos , Benito Rocío , Fontanillo Celia , Sandoval Virgilio , Romero Mercedes , Martín-Núñez Guillermo , de Coca Alfonso García , Fisac Rosa , Galende Josefina , Recio Isabel , Ortuño Francisco , García Juan-Luis , de las Rivas Javier , Gutiérrez Norma-Carmen , Hernández Jesús-Maria , Miguel J.F. San
Published: April 2020

Background: Cytogenetic abnormalities in B chronic lymphoid leukemia (B-CLL) define groups of patients with different prognosis. Thus, patients with B-CLL and 13q14 deletion display a favourable outcome. However, whether the percentage of cells showing 13q-, as it has been demonstrated in other genetic aberrations such as loss of P53, could influence the prognosis or the biological characteristics of the disease remains unknown.

Aim: To analyze the clinical and biological characteristics of B-CLL displaying, at diagnosis, loss of 13q as the sole cytogenetic aberration.

Patients, material and methods: A total of 350 patients diagnosed of B-CLL were included. All the clinical data as well as FISH information (13q14, 12, 11q21, 14q32 and 17p13 probes) and molecular studies were carried out. In addition, a gene expression profile from a microarray-based analysis was also performed.

Results: One hundred and sixty-two patients (46.3 %) had 13q14 deletion. In 109 out of the 350 cases (31.1 %) loss of 13q was the sole cytogenetic aberration at diagnosis. Interestingly, in the subgroup of patients with ³ 80 % of cells with loss of 13q (18 cases, 15.5%), the OS was 143 months (95 % CI: 104 m-154 m), while in the group with < 80 % of losses in 13q, the OS has not been reached (95 % CI: 175 m-253 m). In the multivariate analysis for OS in patients with 13q-, the variables included in the final model were the percentage of losses of 13q14 (P = 0.001) and the presence of B symptoms (P = 0.007). Regarding the time to the first therapy (TFT), in the group with more than 80 % of losses in 13q the median TFT was 134 months while in the group of patients with < 80 % of losses it was 38 months (P = 0.05). In the multivariate analysis for TFT in patients with 13q- the variables selected were unmutated status of IgVH (P = 0.001) and a high level of b2microglobulin (P = 0.003). In addition, the gene expression profile showed that patients with a high number of losses in 13q14 had a high proliferation rate, downregulation of genes apoptosis-related, and deregulation of genes related to mitochondrial functions compared to B-CLL patients with low numbers of 13q- cells.

Conclusion: B-CLL patients with a high number of losses in 13q14 as the sole cytogenetic aberration display different clinical and biological features: short OS and TFT as well as more proliferation and less apoptosis. Therefore, a quantification of the number of cells showing a genetic abnormality should be included in the study of the prognostic factors of B-CLL.
Invariant Deletion of One Mir-34b/C Locus in CLL with del11q.
Authors: Malek Sami , Ouillette Peter
Published: April 2020

CLL with del11q constitutes a clinical syndrome characterized by a relatively aggressive disease course, short response duration to therapies and compromised overall survival. CLL with del11q carries mutations in ATM in about one third of cases, which may further negatively affect prognosis. Little is known about additional molecular changes within or due to del11q in CLL and it remains unclear what the selective advantage of acquisition of del11q is for CLL cells.

To gain insights into the physiology of CLL with del11q, we have mapped this lesion using the Affymetrix 50K SNP array platform in 178 CLL cases using DNA from sorted CD19+ cells versus paired buccal DNA. Focusing on the region that spans ATM, a minimal deleted region (MDR) of approximately 8 Mb was identified, spanning approximately 106.6–114.6 Mb physical position.

Scanning the MDR for genes with the potential to affect the physiology of cancerous cells uncovered invariant monoallelic deletion of the miR-34b/c gene, the only known human microRNA gene within the del11q MDR. miR-34a-c have been implicated as direct p53 target genes by a number of groups and these microRNAs have been shown to be direct regulators of pro-proliferative and anti-apoptotic genes, including E2F3, Cyclin E2, Cdk6 and Bcl2. Given that ATM acts upstream of p53 in the ds-DNA damage response pathway, it appears that del11q lesions in CLL affect p53 apoptotic effector functions through two independent processes (ATM loss or mutation and miR-34b/c loss).

Work is in progress measuring miR-34 b/c expression levels in a large CLL cohort and correlating miR-34b/c levels with expression levels of critical miR-34 targets in CLL. In summary, our data implicate miR-34b/c loss as a potential contributor to the physiology of del11q in CLL and should stimulate investigations into miR-34 function as a response predictor of therapy for CLL.
Heat Shock Protein Localisation in Chronic Lymphatic Leukaemia.
Authors: Dempsey Nina C , Leoni Francesca , Williams John H.H. , Hoyle Christine
Published: April 2020

Heat Shock Proteins (Hsps) are a highly conserved group of molecular chaperones, the over-expression of which has been well documented in many cancer types. However, the location of these proteins within the cancer cell appears to be crucial in terms of tumour progression. Intracellular Hsp70 expression in particular appears to confer resistance to apoptosis, while surface Hsp70 expression has been shown to aid immune recognition of the cancer cell by NK cells. High intracellular levels of Hsp90 and Hsp27 also contribute to cell survival, while surface Hsp90 and Hsp27 have been shown to be required for tissue invasion and metastasis. However, the importance of these stress proteins in CLL progression is still poorly understood.

Using flow cytotmetry, we show that CLL patients express significantly higher levels of iHsp90 in lymphocytes (mean MFI CLL = 2463, Control = 748) and significantly higher levels of iHsp27 in lymphocytes (mean MFI CLL = 2138, Control = 760) than that expressed by age-matched control patients. The expression of iHsp90, but not Hsp27, in these patients was shown to be related to the stage of disease, and patients in Binet stage A appear to have a significantly higher expression than those in Binet stage C (p<0.001).

Hsp70 expression in CD5+/CD19+ cells, both intracellular and extracellular, showed either a very high level or a very low level of Hsp70 (see figure below). The high expressing groups were found also to be significantly different (p<0.01) from both the non-malignant cells from the same patients, and normal lymphocytes from age-matched controls. This was not the case for the low expressing groups. iHsp70 expression was also seen to be related to stage of disease as patients in Binet stages A and B were found to express significantly lower levels than those patients in Binet stage C.

Hsp expression was shown not to be associated with the apoptotic condition of the cell as analysis of active caspase-3 in CLL patients proved that the majority of the leukocytes were not apoptotic. Furthermore, the mean of fluorescence was significantly less in lymphocytes from CLL patients (mean MFI = 139) than those from control patients (mean MFI = 278).

Taken together these results show that Hsp expression changes with the natural progression of the disease and therefore manipulation of Hsps may be beneficial in the treatment of CLL.

Figure Figure
Bendamustine Versus Chlorambucil as First-Line Treatment in B Cell Chronic Lymphocytic Leukemia: An Updated Analysis from An International Phase III Study.
Authors: Knauf Wolfgang Ulrich , Lissitchkov Toshko , Aldaoud Ali , Liberati Anna , Loscertales Javier , Herbrecht Roul , Juliusson Gunnar , Postner Gerhard , Gercheva Liana , Goranov Stefan , Becker Martin , Hoeffken Klaus , Huguet Francoise , Foa Robert , Merkle Karlheinz , Montillo Marco
Published: April 2020

Introduction: Bendamustine is a purine analog/alkylator hybrid agent with a unique mechanism of action, which has shown good clinical efficacy and acceptable tolerability in various hematological malignancies, including Hodgkin’s disease, non-Hodgkin’s lymphoma, and multiple myeloma.

Patients and Methods: The efficacy and safety of bendamustine and chlorambucil have been compared in a randomized, open-label, multicenter, Phase III trial in patients with previously untreated advanced (Binet stage B/C) B cell chronic lymphocytic leukemia: an updated analysis from this trial is presented here. Patients were randomized to receive bendamustine (100 mg/m2 on days 1 + 2) or chlorambucil (0.8 mg/kg on days 1 and 15) for up to 6 treatment cycles. The median cumulative dose per patient was 1820 mg and 517 mg for bendamustine and chlorambucil, respectively. The primary endpoints were overall remission rate (ORR), which was defined as complete response, nodular partial response or partial response, and progression-free survival (PFS). Secondary endpoints included overall survival (OS) and safety. The response to treatment was evaluated by a blinded Independent Response Assessment Committee.

Results: A total of 319 patients were randomized (162 bendamustine, 157 chlorambucil), of whom all were included in the efficacy analysis and 312 were evaluable for safety. The mean (± SD) number of treatment cycles was 4.8 ± 1.7 in the bendamustine group and 4.6 ± 1.7 in the chlorambucil group; the median duration of follow-up was 29.2 months (29.8 bendamustine, 27.8 chlorambucil). The ORR was significantly higher with bendamustine than with chlorambucil (67% versus 30%, P<0.0001). The median PFS was 21.5 months with bendamustine and 8.3 months with chlorambucil (P<0.0001). No difference in OS was seen between groups. Most doses were given on schedule. The mean overall relative dose intensity was 86% and 96% in the bendamustine and chlorambucil treatment groups, respectively. At least 1 grade 3/4 neutropenia occurred in 43% of bendamustine-treated patients and 21% of those receiving chlorambucil. Grade 3/4 infections were documented in 7% of bendamustine-treated patients and 4% of chlorambucil-treated patients.

Conclusion: This study has shown that bendamustine offers significantly greater efficacy than chlorambucil, with manageable toxicity, and should be considered as first-line chemotherapy for patients with advanced B-CLL.
Treatment with Lenalidomide Has a Positive Immunomodulatory Effect in Patients with Chronic Lymphocytic Leukemia.
Authors: Gao Hui , Ferrajoli Alessandra , Cohen Evan N , Lee Bang-Ning , Tin Sanda , O’Brien Susan , Wierda William G. , Estrov Zeev , Faderl Stefan , Keating Michael J , Reuben James M.
Published: April 2020

Lenalidomide (Revlimid) is known to downregulate TNF-a, IL-6, IL-8, and VEGF, impair bone marrow microenviroment-neoplastic cell interaction, activate NK cells, and stimulate T-cell activity. Based on these properties, we sought to further evaluate the immumodulatory activity of treatment with lenalidomide in patients with previously untreated chronic lymphocytic leukemia age 65 and older. Nineteen patients enrolled in a phase II clinical trial were treated with lenalidomide, given orally at the dose of 5mg daily for 28 days followed by individual titration up of 5mg increments every 28 days to reach a maximum dose of 25mg daily. Patients were evaluated for response after 3 months and if an objective response or disease stability was obtained, the treatment was continued until obvious disease progression. Peripheral blood (15ml) was collected at baseline, after three months and every six months thereafter while on treatment in patients that agreed to optional correlative studies. Data at baseline and after three months of treatment are presented. Plasma cytokine levels were determined by Luminex Multiplex assay system. Percentages of CD3, CD4, and CD8 T cells and natural regulatory T cells (TR, CD4+CD25hiFoxP3+) cells, and the ability of anti-CD3 activated T cells to synthesize of IL-2, IFN-g, TNF-a, and IL-10 were measured by multi-color flow cytometry. Eleven patients had a partial response (responder, R) and 8 patients failed to respond (non-responder, NR) after 3 months of therapy. All patients had significantly higher percentages of CD3+ and CD4+ T-cells (P = 0.012) after three months of treatment respect to baseline, but only R patients had a significantly higher percentage of TR cells (P = 0.041). Both R (P = 0.016) and NR (P = 0.025) patients had higher percentages of IFN-g-producing CD8+ T cells after three months of therapy. Moreover in NR patients, the percentage of TR cells negatively correlated with the percentages of CD4+ T cells that produced IL-2 and TNF-a, and of CD8+ T cells that produced IFN-g and TNF-a. Following 3 months of therapy, plasma levels of IL-10 were significantly increased in both R (P =0.012) and NR (P = 0.05); IL-1b plasma level was elevated in NR (P = 0.012) but not in R; IL-17 plasma level was decreased in R (P = 0.006); neither R nor NR had changes in plasma levels of IL-1RA, IL-2, IL-6, TNF-a, TNF-R1, VEGF, and VEGF-R (R1, R2, R3). In conclusion treatment with lenalidomide increased the percentages of T-helper cells in both R and NR patients and of TR cells in R patients. Treatment with lenalidomide also increased the function of CD8+ T cells as exhibited by the synthesis of IFN-g in both R and NR patients. These preliminary data suggest that lenalidomide has a favorable immunomodulatory effect on patients with B-CLL. Sample collection is ongoing for further analyses.
Loss of the Y Chromosome in Philadelphia-Positive Cells Predicts a Poor Response of CML Patients to Imatinib Mesylate Therapy.
Authors: Lippert Eric , Laibe Sophy , Mozziconacci Marie-Joelle , Gervais Carine , Girault Stéphane , Gachard Nathalie , Tigaud Isabelle , Dastugue Nicole , Huguet Francoise , Fort Marie-Pierre , Eclache Virginie , Mahon Francois-Xavier
Published: April 2020

Loss of the Y chromosome (-Y) in bone marrow cells frequently occurs in elderly patients. It is thus unclear whether loss of the Y chromosome, when found in Philadelphiapositive cells, should be overlooked, or whether it represents a clonal evolution (a criterion of cytogenetic acceleration of the disease) or an additional cytogenetic abnormality (ACA), considered of little importance in patients treated with imatinib mesylate (IM). We have collected diagnostic characteristics, clinical, cytogenetic and molecular evolution data from 29 patients diagnosed with CML from 1991 to April 2007 in 6 participating centres, who presented a loss of the Y chromosome at diagnosis (n=20) or during evolution (n=9) and who were treated with imatinib in first (n=19) or second (n=10) line. They were compared to patients with a Philadelphia chromosome as sole anomaly, diagnosed at similar dates in the same centres, thus benefitting from the same management. Age and Sokal score at diagnosis were similar. In both -Y and control groups, two thirds of patients had received prior treatment (mostly interferon, hydroxyurea and aracytine). Median follow-ups were respectively 54 months (range: 7–146) and 58 months (range: 13–149), censored if therapy was changed for a second generation tyrosine-kinase inhibitor or bone marrow transplantation. The time to achieve complete cytogenetic remission (CCR) (22 months vs 7, p=0.02) and major molecular response (MMR) (not reached vs 30 months, p=0.0015) were significantly higher in -Y patients. Acceleration (8/29 vs 1/29, p=0.003), acutisation (4/29 vs 1/29, p=0.1) and death (3/29 vs 1/29) were more frequent in the -Y group, but owing to the rare occurrence of these events, statistical significance was clear for acceleration only. Interestingly, prognosis (time to CCR, MMR and occurrence of acceleration) was worse if loss of the Y chromosome was present in a subclone (suggesting a clonal evolution) rather than all mitoses (ACA) and also when it was observed at diagnosis rather than during the evolution of the disease. Consequently, survival was significantly reduced (55 months vs not reached, p=0.009) in those patients for whom -Y was present in a subclone at diagnosis. In conclusion, loss of the Y chromosome in Philadelphia-positive cells is an important finding since it affects the prognosis of CML patients, especially, but not exclusively, when present at diagnosis in a subclone. In this case, loss of the Y chromosome even predicts a decreased survival after imatinib treatment.
Favorable Outcome of Chronic Myeloid Leukemia Patients Treated with Imatinib Vs Early Allogeneic Stem Cell Transplantation.
Authors: Hasford Joerg , Lauseker Michael , Gathmann Insa , Pfirrmann Markus , Larson Richard A. , Guilhot François , O’Brien Stephen G , Druker Brian J. , Hehlmann Ruediger , Hochhaus Andreas ,   For the IRIS Study Group,   For the German CML Study Group
Published: April 2020

Background: Early allogeneic stem cell transplantation has been considered the only curative treatment for CML. The advent of imatinib provided a new chance to suppress long-lastingly the disease without risk of early deaths. As there is no randomized trial comparing transplantation with imatinib therapy, we compared the outcome between transplanted and imatinib-treated patients of two randomized trials.

Data base: We used the survival data of patients randomly allocated to imatinib in the IRIS trial (Druker et al. N Engl J Med2006;355:2408) and compared them with the genetically randomized transplanted (matched related donors) patients of the German CML III and IIIA studies (Hehlmann et al. Blood;109:4686).

Methods: Applying uniform inclusion criteria for age (18–55 yrs at diagnosis) to generate comparable samples, survival time was determined according to the intention-to-treat principle and after stratification for the Euro and EBMT scores using Kaplan-Meier curves. Information about Sokal and Euro score was missing for 123 and 128 patients from the IRIS trial and for 4 resp. 6 patients from the German CML III/IIIA studies.

Results: 377 CML patients in chronic phase treated with imatinib and 285 patients with early allogeneic stem cell transplantation were analyzed. In the imatinib arm of the IRIS trial, 42 patients had been transplanted and 12 patients have died subsequently. Five-year-survival in this subgroup of 377 younger patients of the IRIS trial was 94.7%. The patients’ characteristics of the two groups were comparable (CML III/IIIA vs IRIS), median age: 38 vs 44 yrs., female sex: 40% vs 39%, Sokal Score low 49.8% vs 58.3%, intermediate 30.2% vs 25.2%, high risk 19.9% vs 16.5%; Euro Score low 62.7% vs 58.6%, intermediate 31.5% vs 34.9%, high risk 5.7% vs 6.4%. Median observation time was 75 months for transplanted patients and 61 months for imatinib-treated patients. Patients of both groups have not yet reached median survival times. Five-year survival rates were 94.7 % (all imatinib treated patients) and 71.1 % (all transplanted patients). In all prognostic strata, irrespective of the prognostic score used, five-year-survival with imatinib was evidently superior compared to transplantation: Euro score: low risk 97.8% vs 78.3%, intermediate risk 93.6% vs 58.9%, EBMT score 0–2: 94.7% vs 78.8%, EBMT score 3–4: 94.7% vs 52.2%. There were just 5 transplanted patients with an EBMT score >4. Neither the censoring of the 42 transplanted patients of the IRIS trial affected the results nor the in- or exclusion of the 12 subsequent deaths.

Conclusions: We could not identify any subgroup of patients with CML who clearly showed a benefit from early transplantation compared to treatment with imatinib, given an observation time of 5–6 years.
Long-Term Immune Deficiency (ID) after Allogeneic Stem Cell Transplantation (allo-SCT) Following a Myeloablative (MA) Conditioning Regimen.
Authors: Corre Elise , Carmagnat Maryvonnick , Busson Marc , de Latour Regis Peffault , Robin Marie , Ribaud Patricia , Toubert Antoine , Rabian Claire , Socie Gerard
Published: April 2020

Background; although numerous studies have described ID in the short-term post transplant, few include data on long-term ID following allo-SCT.

Aim; describe longitudinally the evolution of T-, B-, and NK-cells in a cohort of 140 consecutive pts following allo-SCT and to correlate this evolution with the advent of late clinical infections.

Patients & Methods; 140 pts with a median follow-up of 38 months (mo) were studied [median age 27 (39 less than 16) & 77% with malignancies]. Bone marrow (81%) from sibling (66%) was used before MA conditioning regimen (TBI 42%). 51% had grade II–IV acute- and 30% extensive-GvHD. All pts were monitored before and at 3, 6, 12, & 24 mo post transplant for both CD4 & CD8 subsets [CD45RA (naïve), 45RA+62L- (terminally differentiated), 45RO+ (memory), 28+ (competent), and Dr+ (activated)],NK cells (CD3-, 16+56+), and B cells CD19+/5+, 19+/27- (naives) and 19+27+ (memory).

Results; we observed; for both CD4 CD8 a 1- year ID of the naive, memory and competent and above limits value for competent subsets; NK normalization at 6 mo; expansion of CD19+/5+ after 3 mo and persisting (till 24 mo) ID of memory B cells. Chronic GvHD did not influence significantly those parameters for CD8 subsets while strongly affecting the naïve and competent CD4 subsets, but the most profound defect was on B cell subsets especially on memory B. Contrasting this profound ID the cumulative incidence of late severe infections (LSE) was rather low (14% at 4 years). The only clinical factor that was marginally statistically linked to these was the use of PBSC instead of marrow. We then finally constructed Cox models to search for putative clinical or immunological risk factors for these LSE. Only low B cell counts at 6 (p=.05) & 24 mo (p=.016) were associated with the hazard of developing SLE, in particular if patients did not developed chronic GvHD (see Figure)

Figure Figure

Conclusion; despite a long lasting ID post transplant following MA conditioning the rate of LSI remains low with modern antimicrobial prophylaxis; only B cell defect correlated with LSI despite immunoglobulin supplementation in pts with low Ig level.
Genomic Instability Is Frequent in Oral and Rare in Nasal Mucosal Cells of Allogeneic HCT Recipients.
Authors: Khan Faisal , Sy Sarah , Louie Polly , Lee Robyn , Stewart Douglas A. , Russell James A. , Storek Jan
Published: April 2020

Genomic Instability (GI), appraised by microsatellite length alteration, is a precancerous or cancerous condition. We hypothesized that genomic instability is frequent in oral but not nasal mucosal cells of HCT recipients as oral but not nasal carcinoma is frequent in long-term HCT survivors.We examined epithelial cells from buccal and nasal mucosa of 35 subjects for the presence of GI at 15 microsatellite loci spanning 14 human autosomes. The study population included long-term allo-HCT survivors (4–22 yrs, n=18) and short-term allo-HCT survivors (2–3 months, n=5), and long-term auto-HCT survivors (4–12 yrs, n=5). Controls also included 5 patients treated with intensive chemotherapy and 2 healthy volunteers. DNA extracted from peripheral blood leukocytes and cells of nasal and buccal mucosa was PCR amplified for a panel of 15 Short tandem repeat (STR) markers (ABI-Identifiler TM). The amplicons were subjected to capillary electrophoresis and fragment size analysis was performed to identify novel allele peaks (one that was absent in donor and recipient blood pre-transplant but present in recipient mucosa post-transplant) indicative of microsatellite instability (MSI). MSI was observed in buccal mucosal cells of 56% long-term survivors of allo-HCT; the number of STR loci showing novel allele peaks ranged from 1–6 (median=3). None of the short-term survivors of allo-HCT, long-term survivors of auto-HCT, or control individuals showed MSI in the buccal mucosal cells. Only one allogeneic HCT survivor showed MSI (at one STR locus) in nasal mucosal cells. No genomic alterations were observed in blood leukocytes of any of the above patients or controls. In conclusion, genomic instability is frequently observed in long-term allo-HCT suvivors in oral mucosal cells but rarely in nasal mucosal cells. The facts that oral but not nasal mucosa is frequently involved in chronic GVHD and that MSI was not detected in recipients of auto-HCT suggest that graft-vs-host reaction induces genomic instability.
Reduced Intensity Conditioning Allogeneic Stem Cell Transplantation (RIC allo-SCT) for Patients with High-Risk Multiple Myeloma (MM): A Survey from the Societe Francaise De Greffe De Moelle Et De Therapie Cellulaire.
Authors: Mohty Mohamad , Quoc-Hung Le , Nicolini Franck E , Garban Frederic , Socie Gerard , Attal Michel , Milpied Noel- Jean , Lioure Bruno , Bordigoni Pierre , Yakoub-Agha Ibrahim , Bourhis Jean-Henri , Rio Bernard , Deconinck Eric , Renaud Marc , Harousseau Jean-Luc , Blaise Didier , Michallet Mauricette
Published: April 2020

The results of RIC allo-SCT for MM are still under considerable debate. While EBMT data did not support the universal use of RIC for MM allografts, the Italian and spanish randomized multicenter trials suggested that in newly diagnosed myeloma, outcome in recipients of a hematopoietic stem-cell autograft followed by RIC allo-SCT from an HLA-identical sibling is superior to that in recipients of tandem stem-cell autografts. The aim of this multicenter retrospective national study was to identify prognostic factors for outcome of high-risk patients with MM after allo-SCT prepared by RIC. Data from 219 patients (median age 52 years, range 27–66), who received grafts from a sibling (n=197) or unrelated donor (n=22) were analyzed. At time of transplant, only 37 patients (17%) received RIC allo-SCT in CR or VGPR, while 134 patients (61%) were transplanted in PR. 48 patients were transplanted either in stable disease (n=15) or were in refractory/ progressive disease (n=33). All patients have received at least one autologous transplant prior to RIC allo-SCT. The graft source was PBSCs in the majority of patients (n=183). 21% of the patients received the Seattle Fludarabine and low dose TBI RIC regimen, while 53% of patients received Fludarabine, Busulfan and ATG. 32 patients (15%) died of transplant-related complications. The incidences of grade 2–4 acute GVHD and extensive chronic GVHD were 37% and 20% respectively. At 3 years, overall and progression free survivals (OS, PFS) were 41% (95%CI, 34–49) and 19% (95%CI, 14–27) respectively. Disease status (CR, PR, SD vs. progressive) was significantly associated with overall survival (P=0.0002). In multivariate analysis, disease status at time of RIC allo-SCT, was the strongest parameter associated with an improved OS and PFS (P=0.005 and P=0.004 respectively). Despite its obvious caveats, the relatively low TRM observed in this series, suggest that there is still space to investigate RIC allo-SCT for MM. However, RIC allo- SCT appears to result in a durable response only if it is applied early in the disease history, especially when patients are still chemosensitive. Since the latter results are also expected to be further improved with the systematic and early use of maintenance therapies (Bortezomib and/or Lenalidomide) after RIC allo-SCT, randomized or quasirandomized prospective studies are still warranted.
Prognosis Value of [18f] FDG-PET Prior Versus after Autologous Stem Cells Transplantation in High Grade Non-Hodgkin’s Lymphoma.
Authors: Roland Virginie , Milin Caroline Bodet , Moreau Anne , Gastinne Thomas , Mahe Beatrice , Dubruille Viviane , Maisonneuve Hervé , Morineau Nadine , Moreau Philippe , Jardel Henri , Planche Lucie , Moreau Philippe , Harousseau Jean- Luc , bodere Françoise Kraeber , Le Gouill Steven
Published: April 2020

Positron emission tomography using fluorine 18-fluorodeoxyglucose (FDG-PET) is required in the assessment of therapeutic response in diffuse large B-cell lymphoma. Limited studies addressed the interest of FDG-PET prior autologous stem cells transplantation (auto-SCT) in High-grade non Hodgkin’s lymphoma (HG-NHL). Between April 2002 and December 2005, 42 HG-NHL (DLBCL in 35 cases and aggressive transformation in 7 cases) patients underwent auto-SCT in our institution and were systematicaly explored prior and after auto-SCT using computed tomography (CT) and FDG-PET. The therapeutic strategy included R-CHOP or R-DHAP as induction chemotherapy followed by auto-SCT. In note, auto-SCT was always included in the initial strategy and scheduled after completion of induction chemotherapy for patients at least in partial response (PR) according CT assessment. Pre-auto-SCT FDG-PET results did not influence the scheduled strategy. Auto-SCT was performed as part of the first-line therapy in 35 cases (83 %) or at the time of relapse in 10 cases. Prior auto-SCT, 14 patients were in PR and 24 in CR/Cru. Conditioning regimen consisted of BEAM in all cases (except one patient). The median follow-up (calculated from the time of auto-SCT) for living patients was 34,5 months [19–74 months]. The 2-years EFS and OS estimates were 79,6 % [63,2%; 89,3%] and 90,5 % [76,6%; 96,3%], respectively. According to pre and post auto- SCT FDG-PET results, patients were classified into 3 groups: NEG/NEG (n=25) POS/ POS (n=8) and POS/NEG (n=9). The 2-years EFS and OS estimates for NEG/NEG, POS/ POS and POS/NEG patients were 88 % versus 19 % versus 100 % (p= 0,0001) and 92 % versus 75 % versus 100 % (p = 0,19), respectively. According to FDG-PET response after auto-SCT, the 2-years EFS estimates for NEG versus POS patients were 90, 9 % versus 19 % (p<0,0001). In contrast, pre auto-SCT FDG-PET status has no prognosis value on both EFS (2-years EFS: 88% versus 65%) and OS (2-years EFS: 92% versus 88%). We conclude that only post and not pre-auto-SCT FDG-PET results has a prognostic value in HG-NHL. This result suggests that an pre-transplant intermediate positive FDGPET should not delay a scheduled ASCT which could clean off the negative value of an intermediate positive FDG-PET.
Early Pre/Post Fluoro-Deoxyglucose Positive Emission Tomography (PET) Does Not Predict Outcome of Patients Undergoing Hematopoietic Stem Cell Transplantation in Hodgkins Disease and Non-Hodgkins Lymphoma.
Authors: Palmer Jeanne , Goggins Timothy , Broadwater Gloria , Chao Nelson J. , Chute John , Coleman R. E. , Sullivan Keith M , Long Gwynn , Gasparetto Cristina , Beaven Anne , Rizzieri David
Published: April 2020

This study was designed to investigate the predictive value of early pre/post stem cell transplant (SCT) FDG-PET on outcomes in non hodgkins (NHL) or Hodgkins lymphoma (HL). We analyzed patients who received SCT for treatment of relapsed or refractory NHL or HL at Duke Medical Center between the years of 1996–2007. Patients who had either a FDG-PET scan following salvage chemo- or radio- therapy and within 14 weeks of transplantation (pre-PET)or a FDG PET scan within 6–14 weeks following transplantation (post-PET) were included in the analysis. PET was determined to be positive or negative based on chart review of radiology reports. Survival times are estimated using Kaplan- Meier method. Hazard ratios (HR) are from univariate Cox proportional hazards models. Overall survival (OS) was measured from the time of transplant until death, and for those patients still alive, it was censored at the last follow up date. Disease free survival (DFS) was measured from the time of transplant until first progression or death, whichever occurred first, and was censored at the date of last follow up for those alive without progression. A total of 102 patients were identified with PET in the appropriate time period. Median age was 48.5 (18–78); 71 patients had NHL, 31 patients had HL. Ninety-eight (96%) of the patients had chemosensitive disease, and had a documented PR (62) or CR (36) prior to transplant. The median DFS and OS of this population was 55 and 73 mo respectively. Median time to follow up was 38 mo (1.5 – 145 mo). Of the 75 pre-PET scans, 32 (43%) were positive; of the 78 post-PET scans, 22 (28%) were positive. At the time of this analysis, 28 (27%) of the patients had died from disease progression. There were 8 (10.6%) pre-PET, and 7 (9%) post- PET readings that were not clear. Our analysis revealed that neither pre-PET nor post-PET results were predictive of DFS (HR 1.73 p=0.16, HR 1.97 p = 0.052) or OS (HR 1.92 p=0.13; HR1.65 p=0.21). Further, for the patients who had measurable disease at the entry to SCT, post-PET was not predictive for DFS (HR 1.62, p=0.29) or OS (HR 1.59 p=0.35). Exclusion of unclear PET readings did not have any effect on these results. In subset analysis, post- PET did predict DFS in patients with NHL (HR 2.6 p=0.034). Our results suggest that early pre/post SCT PET analysis is not beneficial in this algorithm for predicting overall survival in these patients. Negative post-PET may predict DFS in patients with aggressive NHL, however, did not predict OS in this group. Our analysis indicates that other methods of determining risk of relapse and transplant outcome are needed.
HIV Status Does Not Affect the Outcome of Autologous Stem Cell Transplantation (ASCT) for B Cell Non Hodgkin Lymphoma (NHL).
Authors: Krishnan Amrita , Zaia John , Alvarnas Joseph , Kogut Neil , Spielberger Ricardo , Wardlow Michelle , Tsai Nicole , Paris Tanya , Forman Stephen J
Published: April 2020

Randomized trials have demonstrated superior progression free (PFS)and overall survival (OS) with autologous stem cell transplant (ASCT) for patients with relapsed chemosensitive non-Hodgkin lymphoma (NHL) compared with conventional dose salvage chemotherapy. More recent trials have proven that ASCT is also feasible in pts with AIDS associated NHL (ARL). However, the impact of the HIV infection on long term outcome is unknown. We therefore, undertook a retrospective case control analysis of ASCT for high risk B cell NHL in HIV positive (HIV Pos) and HIV negative (HIV Neg) pts. Twenty-nine pts with ARL who underwent ASCT between 1998 and 2007 were matched with HIV Neg controls. Pts were matched for gender, time from NHL dx to ASCT, age at ASCT, dz status at ASCT, number of prior regimens, conditioning regimen (chemo vs. FTBI). Histology was matched as closely as possible with the exception that there were more high-grade NHL pts in the HIV Pos group.

Patient HIV Pos HIV Neg P value* (* P value by Fishers exact test or Wilson signed rank sum test) Age at ASCT 42 (11–68) 48(21–65) .06 HISTOLOGY Large cell 19 25 Burkitts 10 0 Follicular gr 3 0 3 Marginal zone 0 1 Disease status 1CR/PR 16 16 IF 2 2 1.0 Relapse 11 11 Prior regimens 2(1–4) 2(1–4) .33 Conditioning - - FTBI 4 4 1.0 Non TBI 25 25

Median followup for HIV Pos pts is 46.7 months and 43.3 months for HIV neg controls (NS). Pts engrafted WBC at a median of 10 days (HIV Pos) and 11 days (HIV Neg) (NS) respectively. Non-relapse mortality (NRM) was 8% (95% CI 2–26) in HIV Pos pts and 4% (95%CI 0.7–25)in HIV Neg controls (NS). Two year PFS for the HIV Pos cohort was 76% (95% CI 62–85) and 56% (95% CI 45–66) for the controls (NS). OS was also similar at 75% (95% CI 61–85) versus 75% (95% CI 60–85)respectively{ see figure }. In conclusion, despite the inclusion of more poor risk pts by histology in the HIV Pos cohort, our series demonstrated matching OS for HIV Pos and HIV Neg pts undergoing ASCT for NHL The equivalent NRM also provides further evidence that HIV status does not affect the outcome of ASCT for NHL and therefore, should be considered a standard approach for select pts with ARL.

Figure Figure
Hemorrhagic Cystitis and BK Virus Infection in Children after Hematopoietic Stem Cell Transplantation.
Authors: Gruhn Bernd , Maier Loreen , Egerer Renate , Fuchs Dietlinde , Zintl Felix , Beck James
Published: April 2020

Hemorrhagic cystitis (HC) is a serious complication after hematopoietic stem cell transplantation (HSCT). The pathogenesis of HC in adults is not fully understood and may be influenced by BK virus infection, type of transplant, conditioning regimen, stem cell source, and graft-versus-host disease. Little is known about the development of HC in children after HCST, especially about the association with BK virus infection. Therefore, we retrospectively analyzed the incidence, risk factors and BK virus association of HC in 165 consecutive children (median age, 12 years) who underwent peripheral blood stem cell (n=97; T-cell depleted: n=48) or bone marrow transplantation (n=68) between 1/2000 and 12/2006 in a single center. Fifty nine patients received autologous HSCT and 106 patients underwent allogeneic HSCT. Nineteen of the 165 patients (11.5%) developed HC after a median of 33 days (range, 1–98 days). All 19 patients with HC underwent allogeneic HSCT and showed BK viruria after transplantation. An acute graft-versus-host disease was significantly more frequent in children with HC (P < .001). Significant risk factors in univariate binary logistic analyses were age > 12 years (OR, 3.275; P < .031), use of busulfan (OR, 3.514; P < .013), use of busulfan and cyclophosphamide in combination (OR, 4.935; P < .002), and an unrelated donor (OR, 3.309; P < .043). Independent risk factors in multivariate binary logistic analyses were age > 12 years and the combination of busulfan and cyclophosphamide. We suggest that cyclophosphamide is toxic to the urinary bladder and busulfan enhances this effect. Furthermore, we analyzed the BK virus load in urine by real-time polymerase chain reaction. We found in patients without HC a significantly increased number of BK virus copies in urine in children older than 12 years (P < .009) and in children who received antithymocyte globulin (P < .001). In addition, BK virus load in urine was significantly increased in children who suffered from HC. Thirteen of 14 children with HC had a BK virus load in urine >107 copies/mL (P < .001). We observed in individual BK virus profiles an increase of BK virus copies in urine before the onset of HC. We conclude that HC in children is a disease of multiple etiologies. Allogeneic HSCT, the combination of busulfan and cyclophosphamide, age > 12 years, and an unrelated donor are risk factors for the development of HC in childhood. Increased BK virus load in urine of more than 107 copies/mL may lead to HC. Therefore, it is useful to quantify BK virus in urine in those children with above mentioned risk factors to initiate early treatment or to prevent the development of HC.