Positive Bone Marrow Research Articles

Prognostic Importance of Bone Marrow Uptake on Baseline 18F-FDG Positron Emission Tomography in Diffuse Large B Cell Lymphoma.

To define the role of 18F-flourodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) in the detection of bone marrow (BM) involvement in patients with diffuse large B cell lymphoma (DLBCL). Fifty-four (mean age: 55.5 ± 18.3 years, 20 female and 34 male) DLBCL patients who underwent pretreatment 18F-FDG PET/CT were included to the study. Focal or diffuse BM 18F-FDG uptake that is higher than mediastinal blood pool uptake was accepted as positive. After staging of disease by CT and 18F-FDG PET/CT, all the patients received R-CHOP treatment after diagnostic blinded bone marrow biopsy (BMB). Presence of positive BM uptake in 18F-FDG PET/CT and histopathological examination results of BMBs were analyzed by Chi-square test. Sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of 18F-FDG PET/CT in the detection of BM involvement were calculated. Prognostic importance of the presence of BM 18F-FDG uptake was analyzed by Kaplan-Meier analysis. BM 18F-FDG uptake was detected in 8 patients. Histopathological examination of BMB revealed BM involvement in 6 out of 8 patients. BMB was negative in all patients who have no 18F-FDG uptake in the evaluation of PET/CT images. Sensitivity, specificity, accuracy, PPV, and NPV of 18F-FDG PET/CT in the detection of BM involvement were calculated as 100%, 96%, 96%, 75%, and 100%, respectively. In the Kaplan-Meier analysis, we found that presence of pretreatment 18F-FDG uptake in BM has a prognostic importance. Whereas mean time to progression (TTP) in patients with BM uptake was 32.25 ± 10.9 months and mean TTP in those without was 51.69 ± 3.6 months (p = 0.013). BM uptake in pretreatment 18F-FDG PET/CT is an important prognostic factor in DLBCL patients. Moreover, in consideration of high NPV, 18F-FDG PET/CT could eliminate unnecessary BMB in FDG-negative patients.

Detection of Leishmania (L.) infantum in stray dogs by molecular techniques with sensitive species-specific primers

ABSTRACTBackground: Canine visceral leishmaniasis (CVL) is a worldwide parasitic zoonosis caused by Leishmania (Leishmania) infantum around the world. Canids are the definitive hosts and sand flies the intermediate hosts.Objective: To test the hypothesis that a new species-specific primers (Lch14:Lch15, targeting a multiple alignment for L. infantum kDNA minicircle) is an efficient diagnostic tool for L. infantum.Methods: The presence of L. infantum DNA was assessed in blood samples of 69 stray dogs using the conventional PCR (cPCR) and quantitative PCR (qPCR). Additional 50 lymph nodes and 50 bone marrow samples (positive and negative samples for parasitological tests) from dogs from endemic and nonendemic areas for CVL were also used. Results: L. infantum strains, and all positive lymph node and bone marrow samples for parasitological test gave positive results for cPCR and qPCR, presenting analytical sensitivity of ∼100 parasite mL−1. For the blood samples, 40/69 (58%; CI 95%; 46%–69%) resulted positive for L. infantum in both tests. All positive samples were confirmed by sequencing.Conclusion: This study showed the importance of the specific detection of L. infantum based on species-specific primers by molecular techniques, highlighting the application as a confirmation method in epidemiological studies and to adopt the best control measures.

Reproducibility of SOX-11 detection in decalcified bone marrow tissue in mantle cell lymphoma patients

Mantle cell lymphoma (MCL) usually harbors the t(11;14)(q13;q32) with overexpression of CCND1 mRNA and transcription of the cyclin D1 nuclear protein. Regardless of CCND1 status, most MCLs also express the SOX11 nuclear protein, which is thus helpful in the diagnosis of the rare CCND1-negative MCLs. Recently, SOX11 has been reported to be often negative in MCLs clinically resembling marginal zone lymphoma and recently defined as "leukemic non-nodal" MCL in the incoming revision of the WHO classification of lymphoid tumors, for which the bone marrow biopsy is commonly the first diagnostic approach. Due to the less aggressive clinical behavior of the latter MCLs, the reliable determination of the SOX11 antigen in decalcified tissue is mandatory. To this end, since little data are available in the literature, four commercially available anti-SOX11 antibodies (two polyclonal and two monoclonal) were tested on 21 positive staging bone marrow (BM) biopsies from cyclin D1/SOX11-positive MCL patients (17 fixed in B5, 4 in 10% buffered formalin) and on 9 positive BM biopsies from leukemic non-nodal MCL patients. The results were compared for specificity, sensitivity, staining strength and degree of an additional staining on myeloid precursors, also evaluating possible impact of the different fixatives used. Non-mantle cell lymphomas were also tested to address specificity. All reagents showed high sensitivity but the monoclonal code CMC38221001 provided the highest specificity and the lowest degree of non-lymphoid staining on myeloid cells. Formalin fixation generally improved the performance of most antibodies when compared to B5 fixation.

Abstract 3791: TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents

Abstract Background: Multiple myeloma and its associated bone disease are generally incurable. Hence, better therapies are needed, ideally targeting biomolecules implicated in the aberrant biology of the disease. Overexpression of transient receptor potential cation channel TRPV6, a highly selective calcium channel has been observed in breast, colon, thyroid, ovary and prostate cancer tissues, and in several tumour cell lines. TRPV6 expression has also been seen in osteoclasts, however its role in bone metabolism remains unclear. The reciprocal interaction between osteoclasts and myeloma cells is pivotal to the generation of bone lesions that characterize myeloma. While myeloma cells secrete factors promoting osteoclast activity, osteoclasts in turn are known to induce myeloma cell growth and survival. The TRPV6 peptide antagonist SOR-C13 is currently in phase I clinical trials as an anti-cancer therapy for advanced cancers. We investigated the expression and potential therapeutic significance of TRPV6 in human osteoclasts and myeloma cells using shrew venom derived peptide antagonists. Methods: High affinity TRPV6 peptide antagonists SOR-C13 and SOR-C27 derived from the C-terminus of venom from the short-tailed shrew, Blarina brevicauda were used. Human primary osteoclasts were generated in vitro from human bone marrow (BM) aspirates; characterized by Hoechst-phalloidin staining, Tartrate resistant acid phosphatase (TRAP) staining, TRAP enzyme activity and Cathepsin K expression. CD138 positive myeloma cells were isolated from patient bone marrow specimens by EasySep immunomagnetic separation, or examined in tissue microarrays of patient BM core biopsies. TRPV6 expression in primary human osteoclasts, myeloma cell lines and myeloma patient BM microarray was checked by qPCR, immunohistochemistry or immunoblotting. Anti-resorptive potential of SOR peptides using human osteoclasts was evaluated in Osteoassay plates that mimic bone, and myeloma cell growth inhibition was determined by prestoblue cell viability assays. Results: We found strong expression of TRPV6 protein in human myeloma cells and osteoclasts by immunohistochemical staining on myeloma patient bone marrow sections. Human osteoclasts generated in vitro and CD138 positive myeloma patient bone marrow plasma cells were found to express TRPV6. We saw dose-dependent inhibition of osteoclast activity in vitro by SOR-C13 and SOR-C27, including markedly reduced osteoclast formation, decreased TRAP activity and reduced osteoassay surface resorption. TRPV6 peptide antagonists were also found to inhibit the growth of human myeloma cell lines U266 and KMM-1. Conclusion: Anti-myeloma and anti-osteoclast activity of human TRPV6 antagonist peptides was seen in the current study. Taken together, our findings suggest a novel therapeutic approach for multiple myeloma involving TRPV6 inhibition to target both myeloma cells and osteoclasts. Citation Format: Alli Murugesan, Philippe Tremblay, Ming Han, Bithika Ray, Tyler Lutes, Tony Reiman. TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3791.

Expression of CDKN1C in the bone marrow of patients with myelodysplastic syndrome and secondary acute myeloid leukemia is associated with poor survival after conventional chemotherapy.

We tested the hypothesis that proliferative activity of hematopoietic stem cells has impact on survival in newly diagnosed patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (AML). RNA expression profiles of CD34(+) cells were analyzed in 125 MDS patients and compared to healthy controls. Prognostic impact on overall survival (OS) of mRNA proliferation signatures established for solid tumor cells was analyzed retrospectively. For validation on the protein level, immunofluorescence and immunohistochemistry analyses in bone marrow (BM) biopsies were performed, and an independent cohort of 223 MDS and secondary AML patients was investigated. Lower proliferative activity correlated with the expression of cyclin-dependent kinase inhibitor 1C (CDKN1C) and with shorter OS (p < 0.001). In multivariable analysis, higher CDKN1C expression was associated with worse OS (p = 0.02). On the BM level, a total of 84 (38%) patients showed CDKN1C protein expression before start of treatment. Patient, disease and treatment characteristics did not differ between CDKN1C-positive and -negative patients. Positive CDKN1C BM status was associated with shorter OS in multivariable analysis (HR 1.54, p = 0.04). There was an interaction between CDKN1C BM status and subsequent treatment with negative impact on OS being most pronounced in patients receiving conventional cytotoxic chemotherapy (n = 83, 2-year OS 30% versus 58%, p = 0.002). In conclusion, low-proliferative phenotype and CDKN1C expression were associated with shorter OS. CDKN1C protein expression in the BM of newly diagnosed, treatment-naïve MDS and secondary AML patients was identified as a prognostic factor for poor survival in patients treated with antiproliferative chemotherapy.

Evidence that CD146 positive bone marrow stromal cells in bone metastases have an inhibitory role in regulating tumor growth

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Muscle Tissue Engineering Using Gingival Mesenchymal Stem Cells Encapsulated in Alginate Hydrogels Containing Multiple Growth Factors.

Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p<0.05). Altogether, our findings confirmed that GMSCs encapsulated in RGD-modified alginate hydrogel with multiple growth factor delivery capacity is a promising candidate for muscle tissue engineering.

Novel Acylguanidine Derivatives Targeting Smoothened Induce Antiproliferative and Pro-Apoptotic Effects in Chronic Myeloid Leukemia Cells.

The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs) like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient’s life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC) which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it’s of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO), one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.

Neuroblastoma messenger RNA is frequently detected in bone marrow at diagnosis of localised neuroblastoma patients

IntroductionThe clinical importance of the detection of neuroblastoma messenger RNA (mRNA) in bone marrow (BM) of localised neuroblastoma patients at diagnosis remains unclear. In this prospective multicentre study, BM samples of a large cohort, were studied using real-time quantitative polymerase chain reaction (qPCR). MethodsBM samples at diagnosis from 160 patients with localised neuroblastoma were prospectively collected at Dutch and German centres between 2009 and 2013. qPCR was performed using five neuroblastoma specific markers. The association with other biological factors and the prognostic impact of BM positivity and clinical response was assessed. ResultsIn 58 out of 160 patients neuroblastoma mRNA was detected in BM. In 47 of the 58 positive samples only one marker was found positive. BM positivity was significantly associated with MYCN amplification (p = 0.02) and deletion of chromosome 1p (p = 0.04). In total 31 patients had an event, of which only five patients had progression to stage IV. BM positivity was not associated with an unfavourable outcome. However, the detection of more than one marker was associated with an unfavourable outcome (systemic or local relapse) (event free survival 48% versus 85%; p = 0.03) in the whole cohort and in the observation group. ConclusionsBM positivity was associated with unfavourable biological factors and might represent more aggressive tumours. Patients with qPCR positive BM should not be upstaged, because of very few systemic events in the cohort. However, for patients with more than one marker positive a more careful follow-up is advisable. These results need to be verified in a very large cohort of localised patients.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.

Metastatic Neuroendocrine Tumor with Extensive Bone Marrow Involvement at Diagnosis: Evaluation of Response and Hematological Toxicity Profile of PRRT with (177)Lu-DOTATATE.

The aim of this study was to evaluate the response and hematological toxicity in peptide receptor radionuclide therapy (PRRT) with lutetium (177Lu)-DOTA-octreotate (DOTATATE) in metastatic neuroendocrine tumor (NET) with extensive bone marrow metastasis at the initial diagnosis. A retrospective evaluation was undertaken for this purpose: Patients with NET with extensive diffuse bone marrow involvement at diagnosis who had received at least three cycles of PRRT with 177Lu-DOTATATE were considered for the analysis. The selected patients were analyzed for the following: (i) Patient and lesional characteristics, (ii) associated metastatic burden, (iii) hematological parameters at diagnosis and during the course of therapy, (iv) response to PRRT (using a 3-parameter assessment: Symptomatic including Karnofsky/Lansky performance score, biochemical finding, and scan finding), (v) dual tracer imaging features [with somatostatin receptor imaging (SRI) and fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT)]. Based on the visual grading, tracer uptake in somatostatin receptor (SSTR)-positive bone marrow lesions were graded by a 4-point scale into four categories (0-III) in comparison with the hepatic uptake on the scan: 0 - no uptake; I - clear focus but less than liver uptake; II - equal to liver uptake; and III - higher than liver uptake]. Hematological toxicity was evaluated using National Cancer Institute (NCI)-Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 score. A total of five patients (age range: 26-62 years; three males and two females) with diffuse bone marrow involvement at the diagnosis was encountered following analysis of the entire patient population of 250 patients. Based on the site of the primary, three had thoracic NET (two patients bronchial carcinoid and one pulmonary NET) and two gastroenteropancreatic NET (one in the duodenum and one patient of unknown primary with liver metastasis). Associated sites of metastases included the liver (n = 5), breast (n = 1), and aortocaval nodes (n = 1). On baseline diagnostic study [68 Ga-DOTANOC/TATE or the technetium (99mTc)-hydrazinonicotinamide (HYNIC)-tektrotyd (TOC)], tracer uptake in the bone marrow in all patients was Grade III. At the time of analysis, the patients received three to four cycles of PRRT and a cumulative dose of 16.1-25.6 GBq with a follow-up duration ranging 10-27 months. The response as assessed by three parameters: (i) Symptomatic: All patients (except for one) reported excellent symptomatic palliation and better quality of life with improvement of Karnofsky/Lansky scores; the single case with nonresponse had shown symptomatic response in the initial 6 months following which he had a progressive disease and death at 18 months (ii) biochemical: Three patients had shown more than 50% reduction in the serum chromogranin level, one had shown increase but had demonstrated clinical evidence of response with radiologically stable disease while the other who had shown slight increase of chromogranin A (CgA) level had shown progressive disease thereafter (iii) radiological: Three patients demonstrated partial response (on FDG-PET/CT), one patient had stable disease and one patient had progressive disease following initial clinical response. As per the NCI-CTCAE score, only one patient had persistent Grade I anemia without any deterioration with the administered dose at the time of analysis. FDG uptake in the bone marrow metastatic lesions showed no obvious FDG avidity on visual assessment except for two patients (low-grade FDG uptake). Interestingly, the associated metastatic lesions [except for patient I with Mib1 labeling index (LI): 1-2%], demonstrated high FDG avidity. Thus, we observed that the majority (in our series four out of five patients, i.e. 80%) of the patients had excellent symptomatic response with at least stabilization of the disease at a follow-up period of 10-27 months. The single patient who had a progressive disease also had a good symptomatic response in the initial 6 months from the first dose of PRRT. Despite the extensive bone marrow involvement, no hematological toxicity was observed (only one patient showed Grade I anemia), suggesting that PRRT is well-tolerated by this particular subgroup.

Tissue Is the Issue: Accuracy of PET Imaging to Detect Bone Marrow Clearance in Patients with Peripheral T-Cell Lymphoma

INTRODUCTION:Adult mature T- and NK-cell neoplasms are a heterogeneous group of aggressive lymphomas comprising approximately 10% to 20% of all non-Hodgkin lymphomas (NHL) with an estimated 5-year overall survival of 40%. Staging and treatment strategies are guided by malignant involvement on PET/CT scan and bone marrow (BM) biopsy. The Lugano Criteria, established in 2014, suggested that focal FDG uptake within the bone marrow was highly sensitive for both Hodgkin (HL) and diffuse large B-cell lymphoma (DLBCL) potentially obviating the need for a bone marrow biopsy if no lesions were seen. Limited data exist regarding the sensitivity of PET for BM involvement in T-cell lymphoma. This study compares PET/CT scans and repeat BM biopsy in patients who have undergone treatment for peripheral T-cell lymphomas (PTCL) by evaluating BM avidity on PET scans.METHODS:This is a single institution study using the Mayo Clinic Lymphoma Database between January 1, 2001 and January 1, 2015. We retrospectively identified all patients with a diagnosis of PTCL, biopsy proven BM involvement at diagnosis, and a concomitant PET for staging. Patients were then reviewed to assess completion of induction therapy and availability of both PET/CT and bone marrow results post-therapy. Evaluation for concordance and discordant BM involvement were then determined using BM biopsy as the gold standard.RESULTS:Sixteen patients had both PET/CT and BM biopsy after completing induction therapy. Median age at diagnosis was 63 years (range 34-72) and 69% were male. PTCL subtype was peripheral T-cell lymphoma, not otherwise specified in seven patients; ALK negative anaplastic large cell lymphoma in one patient; and angioimmunoblastic in 8 patients. Pre-treatment PET/CT scans demonstrated eight patients (50%) with false negative scans. Post-treatment biopsy results demonstrated that ten (62.5%) had biopsy proven residual bone marrow involvement after induction. Eight patients (50%) were found to have BM biopsy proven disease with a negative PET scan. Two patients (12.5%) had both positive BM biopsy and PET scans; 5 patients (31.3%) had negative BM biopsies and PET scans. One patient (6.25%) had a negative BM biopsy, but had a PET scan that revealed positive disease. This patient was considered to have had a false positive PET scan and indeed has remained in remission since April 2009 without any further relapse or treatment. Sensitivity of PET for BM involvement was very poor at 20% (2/10) with a specificity of 50% (2/4) (Table 1 and 2).CONCLUSIONS:This study in PTCL indicates that PET scans at the completion of therapy have a 50% false negative rate. These patients should not be assumed to have negative bone marrow involvement based solely on PET/CT scans. A bone marrow biopsy at the end of therapy is necessary in PTCL patients to confirm complete response.Table 1Bone Marrow InvolvementPre-Therapy PET Scan ImagingPET NegativePET PositiveTotal PatientsBone Marrow NegativeNANANABone Marrow Positive8816Total Patients8 (50%)8 (50%)16Abbreviations: NA, not applicable. The study defined that all patients had to have a positive bone marrow at baseline to be eligible for the study.Table 2Bone Marrow InvolvementPET NegativePET PositiveTotal PatientsBone Marrow Negative426 (37.5%)Bone Marrow Positive8210 (62.5%)Total Patients12 (75%)4 (25%)16Table 1 and 2. Involvement of bone marrow with peripheral T-cell lymphoma based on evaluation by PET scans versus bone marrow biopsy. DisclosuresMaurer:Kite Pharma: Research Funding.

Bone Marrow Involvement Detected By Ig Heavy Chain Rearrangement Has an Negative Impact of Progression Free Survival Independently of IPI and FDG-PET in Diffuse Large B-Cell Lymphoma

Introduction Bone marrow (BM) involvement is an important prognostic factor in DLBCL, which is incorporated into the new prognostic index of NCCN-IPI in rituximab era. Although BM involvement has been evaluated with bone marrow biopsy (BMB), the recent international criteria for staging of non-Hodgkin lymphoma and Hodgkin lymphoma (Lugano criteria) proposed omission of BMB as pre-treatment evaluation in selected cases. However, validity of omission of BMB remains uncertain. Our institute previously reported that the detection of BM clonality using flow cytometry (FCM) or immunoglobulin heavy chain rearrangement (IgR) has prognostic value in DLBCL. Aim of this study is to inspect prognostic value of PET-defined BM involvement combined with clonality of BM.Patients and Methods We studied consecutive DLBCL patients who were diagnosed in our institute in 2007-2014, received rituximab combined with CHOP-like regimen and undertook pre-treatment staging by FDG-PET/CT, FCM and PCR using BM aspirates. We reviewed the medical records, the staging FDG-PET/CT reports, DICOM images (if available), FCM and IgR reports. BM uptake of FDG was classified as focal or diffuse pattern. Diffuse uptake pattern was not regarded as lymphoma involvement on staging unless lymphoma was proven histologically or cytogenetically. B-cell clonality of BM was defined as positive, either when PCR amplification detected clonal immunoglobulin heavy chain (FR1, FR2, FR3) gene rearrangements with multiplex BIOMED-2 primer sets or when FCM detected κ/λ deviation among B-cell population.Results One-hundred thirty four patients were identified: median age 69.5 years old (range, 19 - 89), 76 male (57%), LDH above the upper limit in 60(45%), stage ≥ 3 in 69(51%), bulky mass in 10(7.5%), abnormal lymphocytes in BM detected microscopically in 13(9.7%), IPI score were distributed as 0-1 in 46(34%), 2 in 31(23%), 3 in 30(22%) and 4-5 in 27(20%). Thirty patients (22% of the whole cohort) were positive for BM FDG-PET uptakes; the uptake patterns were focal in 23(77%) and diffuse in 7(23%). Follow-up time was median 741 days (range, 31 - 1766). B-cell clonality of BM was found in 20 patients (15%) with FCM and in 27 patients (20%) with IgR. B-cell clonality was more frequently observed in the patients with BM FDG-PET uptake (FCM-based clonality: 37% for positive vs 10% for negative, p-value, 0.002; IgR-based clonality: 38% for positive vs 18% for negative, p-value, 0.034). In addition, IPI score was significantly worse in the BM FDG-PET uptake patients (IPI ≥2: 93% in positive BM FDG-PET uptake vs 58% in negative BM FDG-PET uptake, p-value, < 0.001). Abnormal lymphocytes by BM aspiration did not affect PFS in our cohort significantly (0.14). Patients with BM FDG-PET uptake had more extranodal lesions than patients without BM FDG-PET uptake (mean 0.66 vs 1.6, p-value, < 0.001). In entire cohort, 2-year PFS and 2-year OS were 70% and 83%, respectively. Pretreatment BM FDG-PET uptake had negative impacts on PFS (2-year PFS, 74% for negative vs 53% for positive, p-value, 0.017). Clonality in BM detected with IgR had negative effect on PFS (2-year PFS, 74% for negative vs 55% for positive, p-value, 0.017)(Figure). IPI were also a significant prognostic factor for PFS (2-year PFS, 92% for IPI<2 vs 59% for IPI ≥2, p-value, <0.001). In contrast, clonality using FCM was not a prognostic factor for PFS (2-year PFS, 72% for negative vs 69% for positive, p-value, 0.426). In multivariate analysis for PFS, in which BM FDG-PET focal uptake, clonality using PCR and IPI ≥ 2 (vs <2) were incorporated as covariates, Cox proportional hazard model revealed that clonality using IgR and IPI≥2 (vs <2) into Cox proportional hazard model. As a result, clonality using IgR and IPI≥2 had significant impact on PFS (IgR: HR, 2.27(range, 1.14-4.55, p-value, 0.02); IPI≥2: HR, 7.27 (range, 2.20-24.11, p-value, 0.001). BM FDG-PET focal uptake in BM was not significant possibly because of confounding with IgR and IPI (HR, 1.73(range, 0.81-3.68, p-value, 0.156).Conclusion Some previous studies showed that BM examination, as well as FDG-PET, had merits for more precise survival prediction. In our study, B-cell clonality of BM is an adverse prognostic factor for PFS that is independent of IPI and the impact of BM FDG-PET status was limited. Although BM clonality was more frequent in the patients whose FDG-PET uptake of BM were focal, BM clonality should be examined combined with FDG-PET. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Study of IgG Subclass of the Patients with Immuno-Related Hemocytopenia

Objective: To investigate the immunoglobulin G (IgG)sublass on the membrane of bone marrow(BM) hematopoietic cells and IgG levels in BM supernatant of patients with immuno-related hemtocytopenia(IRH).Method: Newly diagnosed IRH patients and ICUS patients were enrolled in this study, as well as healthy donors as controls. The categories of auto-antibodies(IgM and IgG) on the membrane of BM hemmatopoietic cells(CD15+,CD34+ and GlyCoA+ cells)were assayed by Flow cytometry(FCM). The categories of auto-antibodies(IgG1 and IgG3) on the membrane of BM hemmatopoietic cells were assayed by FCM.BM nucleated cells antibodies (IgG1AIgG2AIgG3 and IgG4)were detected by immunohistochemical staining. The IgG1AIgG2AIgG3 and IgG4 levels in BM supernatant were detected by Cytometric Bead Array (CBA)GThe ratio of CD5+CD19+/CD19+ in blood were tested by FCMGThe mRNA expression of IgG1 in peripheral blood were detected by RT-PCR.Result: The positive rate of antibodies (IgG) on BM hemmatopoietic cells of IRH patients was 58.69(27/46), which was significantly higher than that in ICUS group(0%) and healthy control group(0%). The positive rate of IgG1 on CD15+ cells was (3.13±1.80)% in IRH group, which was significantly higher than that in healthy control group. The positive rate of IgG3 on CD15+ cells was(2.73±2.09)% in IRH group, which was significantly higher than that in healthy control group. The positive rate of IgG1 on GlyCoA+ cells was( 3.97±2.21)% in IRH group,which was significantly higher than that in healthy control group. The positive rate of IgG3 on GlyCoA+ cells was (2.24±1.82)% in IRH group,which was significantly higher than that in healthy control group. IgG subclass(IgG1AIgG2AIgG3 and IgG4)on BM nucleated cells were detected by Immunohistochemistry. 11/15 IRH patients' IgG1 positive cells≥3(count 100 bone marrow nucleated cells). There were rarely IgG1+ cells in ICUS patients and healthy donors' bone marrow smears. IgG2,IgG3 and IgG4 positive BM nucleated cells were rarely detected in three groups by immunohistochemistry. The IgG1 level in IRH group was (6998±2498)mg/L which was higher than remission group (4702±1235)mg/L, healthy control group (5613±1640) mg/L and ICUS group (5166±1761) mg/L, P<0.05. The IgG3 level in IRH group was (637±252)mg/Lwhich was higher than remission group(415±196)mg/L,healthy control group (422±212) mg/L andICUS group (485±197) mg/L, P<0.05. IgG2 levels in four groups were no statistical difference. IgG4 levels in these four groups were no statistical difference. The total IgG level in IRH group was (10903±2576)mg/L which was higher than remission group(8468±1957)mg/L, P<0.05. The ratio of CD5+CD19+/CD19+ in IRH group was(23.34±9.36)% which were significance higher than healthy control group(13.67±5.10)% and ICUS group(12.40±3.63)% (P<0.01). IgG1 level had a positive correlation with the proportion of CD5+ B lymphocytes(r=0.392, P =0.043). There was an inverse correlation between the IgG1 level and the count of neutrophil (r = -0.459, P = 0.016) in IRH patients. There was an inverse correlation between the level of IgG3 and hemoglobin(Hb) (r = -0.389, P = 0.045) in IRH patients. In IRH group, the mRNA expression of IgG1 in peripheral blood is 5.27±4.83 which is higher than healthy control 1.58±1.50, P<0.05.Conclusion: There are auto-antibodies IgG on the membrane of BM hematopoietic cells of IRH patients. IgG1 and IgG3 on BM nucleated cells increased significantly in BMMNC-Ab IgG positive IRH patients. DisclosuresNo relevant conflicts of interest to declare.

Validation of MRD Quantification By Flow Cytometry for Pediatric BCP ALL Relapsed Patients Treated on the Intreall Protocol

At acute lymphoblastic leukemia (ALL) relapse, about 40% of children can be salvaged with intensified multi-agent, high dose chemotherapy and in very high risk patients, with additional stem cell transplantation (SCT). To improve on this, the International BFM Study Group has led a consortium of 19 countries to develop the world's largest trial for relapsed ALL (IntReALL). Standard risk patients will be randomized to receive the ALL-REZ BFM 2002 or UK ALL R3 therapy and post induction will be randomised to receive the additional targeted anti-CD22 drug, Epratuzumab, during consolidation, to clear residual disease. Children with end of re-induction MRD positive bone marrow will undergo SCT following consolidation. Both ALL-REZ BFM 2002 and UK ALL R3 used MRD PCR-based quantification of clonal Ig/TCR rearrangements, with different cut offs (10-3 for ALL-REZ BFM 2002 and 10-4 for UK ALL R3) and this is the reference assay for IntReALL. However, flow MRD may also play a role for patients without PCR targets. Flow MRD relies on the discrimination of leukaemic blasts from hematogones and this can be hampered depending on the degree of haematopoietic regeneration which varies depending on the treatment protocol and is especially important after intensive induction treatment in relapsed protocols.Thus, prospective MRD quantification of patients entered onto the UKALLR3 and ALL-REZ BFM 2002 clinical trials was performed by a standardised, quality assured, 4-8colour Flow MRD assay in end of re-induction bone marrow aspirates, by laboratories in the IBFM FLOW consortium (n=221). Flow MRD in both treatment protocols was classed as a prospective biological add on study and not used for clinical decision making. Median MRD levels were 0.026 +/-9.9% SD for BFM versus 0.027+/-18% SD for UK protocols, with comparative MRD positivity rates of 45% versus 54%, respectively. Comparison with MRD levels as assessed by molecular analysis of antigen receptor gene rearrangements was performed in 170 samples (BFM,128; UK R3, 42). The Spearman rank correlation of all samples was 0.90 (p<0.0001) for patients treated on the BFM protocol, compared to 0.82 (p<0.0001) for those on UK ALL R3. Risk category concordance was 88% (ALL-REZ BFM) and 88% (UKALLR3). For the 21 discordant samples, 5 were MRD positive by flow but negative by PCR and 17 were negative by flow and positive by PCR. When analysing the accuracy, with which flow MRD classified specimens identically as PCR, the sensitivity of flow MRD in the ALL-REZ BFM protocol was 81% (cut off 0.1%) and in UK ALL R3 was 79% (cut off 0.01%). Specificity values were 93% versus 100%, respectively.Although sample processing and quantification of MRD differ between PCR and FC MRD, in both re-induction protocols, there was good correlation of MRD levels assessed by flow cytometry and PCR, validating the use of Flow MRD as a method of choice in patients without PCR targets in the IntReALL trial. Flow MRD also has the advantage of enabling levels of CD22 to be assayed on MRD cells, prior to treatment with Epratuzumab.This research has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278514 - IntReALL", Deutsche Kinderkrebsstiftung for its funding support of the ALL-REZ BFM 2002 clinical trial and the minimal residual disease studies by PCR and the Deutsche Jose Carreras Leukämiestiftung for support of the international principal investigator, Leukaemia and Lymphoma Research and North East Children's Cancer Research Fund, NT 13462-4, NV15-28525A, NV15-26588A, UNCE 204012. DisclosuresNo relevant conflicts of interest to declare.

KIT D816V Mutation Positive Bone Marrow Mesenchymal Stem Cells in Indolent Systemic Mastocytosis Are Associated with Disease Progression

PURPOSEMultilineageinvolvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here we investigated the potential involvement of BM mesenchymal stem cells (MSC) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. METHODSThe KIT D816V mutation was investigated in highly-purified BM MSC and other BM cell populations from 83 ISM patients followed for a median of 116 months. MC clonality was further evaluated in female patients by the pattern of inactivation of the X chromosome (XCIP). RESULTSKIT D816V-mutated MSC were detected in 22/83 (27%) ISM patients. All MSC-mutated patients had multilineage KIT mutation (100% vs. 30%, p=0.0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P =.03) and a polyclonal XCIP of the KIT- mutated BM MC (64% vs 0%; P =0.01) vs other multilineage ISM cases. Moreover, presence of KIT D816V-mutated MSC was associated with more advanced disease features of ISM, a greater rate of disease progression (50% vs 17%; P =.04) and a shorter progression-free survival at 10, 20 and 30 years (P ≤.003). CONCLUSIONOverall, these results support the notion that ISM patients with mutated MSC may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSC and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. DisclosuresNo relevant conflicts of interest to declare.

Low Dose, Single Fraction, Whole Lung Irradiation for Extramedullary Hematopoiesis Associated with Myelofibrosis with Myeloid Metaplasia

IntroductionPolycythemia vera (PV) and Essential thrombocytosis (ET) progress to myelofibrosis (MF). Extramedullary hematopoeisis (EMH) is common in patients with primary or secondary MF, and can occur in the lungs. Pulmonary EMH can cause recurrent pleural effusions, pulmonary hypertension, and right heart failure with symptoms of dyspnea, cough, and fatigue. Low dose single fraction whole lung irradiation (WLI) has been utilized at our institution, and our preliminary report of 4 patients noted symptomatic improvement with no reported acute side effects. Here we report on a larger cohort of 57 patients as well as long term outcomes for 20 of those patients, including the original 4 patients.MethodsWe performed a retrospective review of 57 patients with myelofibrosis and pulmonary EMH who received single fraction WLI to a dose of 100 cGy at the Mayo Clinic from March 2001 to March 2014. Data related to the following parameters was collected: initial diagnosis, age at initial diagnosis, date of progression to myelofibrosis, initial treatment prior to radiation therapy, whole body bone marrow scan findings if available, and response to WLI. Overall survival was measured using the Kaplan Meier method. Chi-square analysis was used to evaluate predictors of response to WLI.ResultsThe median age at first WLI was 67 years (45-84 years), and 33 patients (58%) were male. Twenty-two patients (39%) had a diagnosis of primary MF, 27 patients (47%) had PV or ET, and 8 patients (14%) had another cause of secondary MF. At the time of WLI, 27 patients (47%) were on supplemental oxygen, and 3 patients (5%) were in the intensive care unit. Hydroxyurea (n=14, 25%), JAK2 inhibitors (n=9, 16%), Anagrelide (n=3, 5%), and Thalidomide and Prednisone (n=3, 5 %) were the most frequent treatments prior to WLI. EMH was confirmed on bone scan in 38 patients (67%). In the remaining 19 patients, a diagnosis of EMH was made based on clinical impression. This included symptoms of dyspnea, cough, and fatigue, echocardiographic findings of pulmonary hypertension, and in some patients recurrent pleural effusions (n=13), positive lymph node biopsy (n=2), or thoracentesis (n=1). Twenty-eight (49%) patients had other active cardiac or pulmonary conditions that likely contributed to their clinical symptoms. These patients were receiving concurrent treatment for their other conditions. In some patients there were multiple coexisting conditions.Clinical improvement occurred in 30 patients (53%). The median time from WLI to symptomatic improvement was 10 days (1-174 days). Twenty-four patients (42%) did not have clinical improvement. Nine patients (16%) had stable symptoms, 15 patients (26%) had progressive symptoms, and 3 patients (5%) had insufficient follow up. In the group of patients with concurrent active cardiac or pulmonary conditions, 15 patients (54%) had clinical improvement following WLI. In the 29 patients who had solitary EMH, 15 (52%) patients had clinical improvement. There was no difference in response rates related to oxygen use at the time of WLI. Six patients (11%) received WLI on multiple occasions. There was no difference in the percentage of patients with positive bone marrow scans (67%) in the 2 groups. The median overall survival was 259 days for all patients. Patients who improved after WLI had a median survival of 325.5 days compared to 122.5 days for patients who did not improve.No new hematologic abnormalities temporally related to WLI were reported. Long term follow up beyond 1 year was available for 20 patients (35%). No patients developed pneumonitis or pulmonary fibrosis that was considered related to WLI. One patient received a diagnosis of an upper esophageal squamous cell carcinoma 6 years after WLI and allogeneic stem cell transplant.ConclusionOur prior study showed WLI is safe and effective in a small number of patients with isolated pulmonary EMH from MF. The current study confirms the long term safety of this approach. Our results suggest WLI may contribute to symptomatic improvement in 1/2 of patients, even in the common clinical situation of multiple coexisting cardiac and pulmonary conditions. Repeat WLI is also well tolerated and can result in symptomatic improvement. We did not find any factors that predicted response to WLI. WLI should be considered in patients who have clinically proven pulmonary EMH and associated symptoms, even in the presence of other conditions, and can be repeated safely. DisclosuresNo relevant conflicts of interest to declare.

Prospective long-term minimal residual disease monitoring using RQ-PCR in RUNX1-RUNX1T1-positive acute myeloid leukemia: results of the French CBF-2006 trial

In t(8;21)(q22;q22) acute myeloid leukemia, the prognostic value of early minimal residual disease assessed with real-time quantitative polymerase chain reaction is the most important prognostic factor, but how long-term minimal residual disease monitoring may contribute to drive individual patient decisions remains poorly investigated. In the multicenter CBF-2006 study, a prospective monitoring of peripheral blood and bone marrow samples was performed every 3 months and every year, respectively, for 2 years following intensive chemotherapy in 94 patients in first complete remission. A complete molecular remission was defined as a (RUNX1-RUNX1T1/ABL1)×100 ≤ 0.001%. After the completion of consolidation therapy, a bone marrow complete molecular remission was observed in 30% of the patients, but was not predictive of subsequent relapse. Indeed, 8 patients (9%) presented a positive bone marrow minimal residual disease for up to 2 years of follow-up while still remaining in complete remission. Conversely, a peripheral blood complete molecular remission was statistically associated with a lower risk of relapse whatever the time-point considered after the completion of consolidation therapy. During the 2-year follow-up, the persistence of peripheral blood complete molecular remission was associated with a lower risk of relapse (4-year cumulative incidence, 8.2%), while molecular relapse confirmed on a subsequent peripheral blood sample predicted hematological relapse (4-year cumulative incidence, 86.9%) within a median time interval of 3.9 months. In t(8;21)(q22;q22) acute myeloid leukemia, minimal residual disease monitoring on peripheral blood every 3 months allows for the prediction of hematological relapse, and to identify patients who could potentially benefit from intervention therapy. (ClinicalTrials.gov ID #NCT00428558).

Nestin Positive Bone Marrow Derived Cells Responded to Injury Mobilize into Peripheral Circulation and Participate in Skin Defect Healing

Exogenously infused mesenchymal stem cells (MSCs) are thought to migrate to injury site through peripheral blood stream and participate in tissue repair. However, whether and how endogenous bone marrow MSCs mobilized to circulating and targeted to tissue injury has raised some controversy, and related studies were restricted by the difficulty of MSCs identifying in vivo. Nestin, a kind of intermediate filament protein initially identified in neuroepithelial stem cells, was recently reported as a credible criteria for MSCs in bone marrow. In this study, we used a green fluorescent protein (GFP) labeled bone marrow replacement model to trace the nestin positive bone marrow derived cells (BMDCs) of skin defected-mice. We found that after skin injured, numbers of nestin+ cells in peripheral blood and bone marrow both increased. A remarkable concentration of nestin+ BMDCs around skin wound was detected, while few of these cells could be observed in uninjured skin or other organs. This recruitment effect could not be promoted by granulocyte colony-stimulating factor (G-CSF), suggests a different mobilization mechanism from ones G-CSF takes effect on hematopoietic cells. Our results proposed nestin+ BMDCs as mobilized candidates in skin injury repair, which provide a new insight of endogenous MSCs therapy.

Prognostic Significance of Bone Marrow Infiltration Detected By PET-CT in Newly Diagnosed Diffuse Large B Cell Lymphoma

PurposeThe aim of this study was to examine the prognostic value of bone marrow involvement (BMI) assessed by PET-CT in treatment-naïve patients with diffuse large B-cell lymphoma (DLBCL).Patients and methodsAll patients from a single centre diagnosed as DLBCL between 2005 and 2014 had data extracted from staging PET-CT, bone marrow biopsy (BMB), and treatment records. The final diagnosis of BMI was defined as: (i) positive bone marrow biopsy; (ii) positive PET-BMI confirmed by guided biopsy or targeted MR imaging; (iii) concomitant disappearance of bone marrow uptake and uptake in other lymphoma lesions on PET-CT after R-chemotherapy.ResultsOf 169 patients, 20 patients (12%) had BMI on BMB, whereas 35 patients (21%) had positive BMI according to PET-CT findings (PET-BMI(+)). Thirty-three out of the 35 patients with PET-BMI(+) showed a focal pattern and 2 a diffuse pattern, respectively. In multivariate analyses, PET-BMI(+) remained significant for overall survival (OS) (HR 2.90, 95%CI 1.21−6.96, P =0.017) while progressive-free survival (PFS) was significant only in univariate analysis (P<0.001) (Table 1). Among patients with PET-BMI(+) at diagnosis (N=35), patients with SUVmax of bone marrow (SUVmax(BM)) more than 8.6 were significantly associated with high IPI score (3−5) (P =0.002) and worse PFS and OS (P =0.025 and P =0.002, respectively) (Figure 1). In the 68 stage IV cases, three-year OS was higher for patients with negative PET-BMI (PET-BMI (−)) than patients with PET-BMI(+) (84.2%¡À6.5% vs. 44.1%¡À8.6%, respectively; P =0.003) while PFS only shown a trend of statistic significance (P =0.077) between the 2 groups, with estimates of 3-year PFS at 49.3%¡À9.2% and 28.6%¡À7.6%, respectively (Figure 2). Among the 69 patients with inter-risk of IPI (2−3), patients with PET-BMI(+) (N=21) had significantly inferior PFS and OS than patients with PET-BMI(−) (N=48) (P =0.009 and P<0.001, respectively) (Figure 3).ConclusionsOur data raised several important issues about the predictive significance of BMI assessed by PET-CT in DLBCL: (i) The bone marrow status assessed by PET-CT is an independent predictor of OS independent of IPI; (ii) For baseline PET-BMI(+) patients, the optimal cutoff value of SUVmax(BM) to predict the survival outcomes was 8.6; (iii) In patients with stage IV disease, worse survival outcomes were observed in patients with BMI than that without BMI; (iV) Patients with PET-BMI(+) in the intermediate risk-group should be managed as high-risk group patients.Table 1Cox regression analysis for PFS and OS for all the patients with DLBCL (N=169)PFSOSUnivariate analysisMultivariate analysisUnivariate analysisMultivariate analysisHR (95%CI)PHR (95%CI)PHR (95%CI)PHR (95%CI)PPET-BMI+3.96 (2.38-6.59)<0.001--6.73 (3.40-13.34)<0.0012.90 (1.21-6.96)0.017BMB-BMI+4.49 (2.53-7.98)<0.001--6.24 (3.06-12.73)<0.001--IPI>27.27 (4.19-12.63)<0.0013.12 (1.31-7.47)0.0109.02 (3.94-20.61)<0.0013.62 (1.01-13.03)0.049Age >601.61 (0.98-2.64)0.0601.18 (0.61-2.27)0.627Stage III or IV6.08 (2.77-13.36)<0.001--6.78 (2.08-22.12)0.002--ECOG 2-42.79 (1.65-4.71)<0.0011.97 (1.12-3.47)0.0193.39 (1.75-6.55)<0.001--LDH>ULN4.68 (2.82-7.78)<0.001--4.31 (1.96-9.48)<0.001--Extranodal site >13.15 (1.91-5.18)<0.001--3.04 (1.58-5.86)0.001-- [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.