Minimal Bone Marrow Research Articles

Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies.Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs).Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance.Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity.The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response.Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment.We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials.Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma.ReferencesDrygin et al., Cancer Research 2011Bywater et al., Cancer Cell 2012Quin et al, Oncotarget, 2016Devlin et al., Cancer Discovery 2016Hein et al., Blood 2017 DisclosuresHarrison:Janssen-Cilag: Other: Scientific advisory board.

How We Manage Patients with Plasmacytomas.

To discuss the diagnostic approach, treatment options, and future considerations in the management of plasmacytomas, either solitary or in the context of overt multiple myeloma (MM). Advanced imaging techniques such as whole-body magnetic resonance imaging and positron emission tomography/computerized tomography are essential for the diagnostic workup of solitary plasmacytomas (SP) to rule out the presence of other disease foci. The role of flow cytometry and clonal plasma cell detection is currently under study together with other prognostic factors for the identification of patients with SP at high risk of progression to overt MM. Solitary plasmacytomas are treated effectively with local radiotherapy whereas systemic therapy is required at relapse. Clonal plasma cells that accumulate at extramedullary sites have distinct biological characteristics. Patients with MM and soft tissue involvement have poor outcomes and should be treated as ultra-high risk. A revised definition of SP that distinguishes between true solitary clonal PC accumulations and SP with minimal bone marrow involvement should be considered to guide an appropriate therapeutic and follow-up approach. Future studies should be conducted to determine optimum treatment approaches for patients with MM and paraskeletal or extramedullary disease.

Detection of Minimal Bone Marrow involvement of Blastic Plasmacytoid Dendritic Cell Neoplastic Cells - CD303 immunostaining as a diagnostic tool.

Blastic plasmacytoid dendritic cell (pDC) neoplasm (BPDCN) is a relatively rare hematological malignancy with significantly complex clinicopathological features that are still unclear. This study aimed to analyze the clinicopathological data of BPDCN and evaluate immunohistochemical detection of minimal bone marrow (BM) involvement. In this study, we examined skin and BM lesions from 6 patients with BPDCN. Neoplastic cells tested positive for CD303 (polyclonal, 100%; monoclonal, 40%) in the skin lesions and for CD303 (polyclonal, 100%; monoclonal, 67%) in the BM clots. Although immunostaining of CD4, CD56, CD123, CD303, and TCLl detected minimal BM involvement in 3 patients, morphological identification was challenging in the BM clots stained with hematoxylin-eosin. In conclusion, our results demonstrate the significance of observing BM smears to detect neoplastic cells and that immunohistochemical examination, including CD303 antibodies, is useful to detect minimal BM involvement. This study is the first to report the expression of thymic stromal lymphopoietin (TSLP) and its receptor in BPDCN cells. Therefore, the TSLP/TSLP receptor axis may be associated with the proliferation of BPDCN, and consequently, the survival of patients.

Calvarial Suture-Derived Stem Cells and Their Contribution to Cranial Bone Repair.

In addition to the natural turnover during life, the bones in the skeleton possess the ability to self-repair in response to injury or disease-related bone loss. Based on studies of bone defect models, both processes are largely supported by resident stem cells. In the long bones, the source of skeletal stem cells has been widely investigated over the years, where the major stem cell population is thought to reside in the perivascular niche of the bone marrow. In contrast, we have very limited knowledge about the stem cells contributing to the repair of calvarial bones. In fact, until recently, the presence of specific stem cells in adult craniofacial bones was uncertain. These flat bones are mainly formed via intramembranous rather than endochondral ossification and thus contain minimal bone marrow space. It has been previously proposed that the overlying periosteum and underlying dura mater provide osteoprogenitors for calvarial bone repair. Nonetheless, recent studies have identified a major stem cell population within the suture mesenchyme with multiple differentiation abilities and intrinsic reparative potential. Here we provide an updated review of calvarial stem cells and potential mechanisms of regulation in the context of skull injury repair.

Legumain correlates with neuroblastoma differentiation and can be used in prodrug design.

Neuroblastoma (NB) is a highly malignant solid tumor in children. The cysteine endopeptidase legumain is expressed in adult solid tumors, but its expression in NB has not been examined. In this study, we assayed legumain expression in two NB cell lines and in microarrays of tumor tissues collected from 46 children with undifferentiated NB, differentiated NB, and ganglioneuroblastoma. Correlation analyses showed that legumain was expressed in all NB cell lines tested and that expression correlated with the degree of tumor differentiation. The efficacy, specificity, and toxicity of EMC-AANL-DOX, a novel doxorubicin-based legumain-activated prodrug, were then evaluated in mouse model of NB. Compared with DOX, EMC-AANL-DOX showed greater inhibition of tumor growth and a lower toxicity at high doses. Neither leukocyte or platelet counts nor renal function or cardiac anatomy differed significantly between the EMC-AANL-DOX and control groups (p>.05), suggesting that the prodrug caused minimal bone marrow depression and did not induce renal or cardiac damage. The good specificity and efficacy of EMC-AANL-DOX and low toxicity recommend its use in the treatment of NB. Correlation analyses of NB differentiation and legumain expression may reveal a novel anti-tumor-related functions of the drug and a new strategy for the treatment of pediatric solid tumors.

The Clinical Impact of Minimal Bone Marrow Involvement on the Outcome of Patients with Follicular Lymphoma

IntroductionFollicular lymphoma (FL) is the second most common type of non-Hodgkin cell lymphoma, and usually manifests as a disseminated disease. Bone marrow (BM) involvement, which occurs in 40-70% of cases, is often seen in follicular lymphoma and thought to be associated with less favorable prognosis. Diagnosis of BM involvement has traditionally been based on morphological findings, and BM involvement has been determined using histology alone in most clinical trials. Immunocytologic or molecular studies, such as flow cytometry (FCM) and polymerase chain reaction (PCR), have become more readily available, and their usage has clearly documented minimal BM involvement reproducibly. In this study, we evaluated the impact of BM involvement detected by FCM and PCR on the outcome of patients treated for FL. MethodsPatients who were diagnosed with biopsy-proven FL between 2004 and 2015 at our institution were included in the study. All patients had received a staging bone marrow examination before treatment with immunotherapy-based regimen. Immunocytologic [FCM] and/or molecular [PCR] studies were always performed if the patients did not have morphological BM involvement. We used 4- or 6- color FCM, and performed PCR analysis of Bcl-2/IgH rearrangement and/or IgH rearrangement detected by modified BioMed-2 protocol. A total of 90 patients were included, and the median follow-up duration was 36 months (range, 6|122 months). The BM status was classified using into 3 categories: morphological, minimal, and negative BM involvement. Minimal BM involvement was defined as BM involvement detected by FCM or PCR without morphological evidence. Morphological and minimal BM involvements were detected in 37 (41%) and 38 (42%) patients, respectively. The primary outcome measure was progression-free survival (PFS). PFS curves were plotted using the Kaplan-Meier method and compared by the log-rank test. Multivariate analyses were performed using a Cox linear regression model. There were significant differences in gender, LDH levels, stage, nodal sites, and FL International Prognostic Index (FLIPI) between patients with and without morphological BM involvement (Table1). ResultsThe 3-year PFS rate for patients with negative BM involvement was significantly better than that for patients with minimal or morphological BM involvement (84.8% vs. 40.3% vs. 60.5%; p= 0.043) (Figure 1). There was no statistical difference in 3-year PFS between patients with morphological BM involvement and those with minimal BM involvement. The difference of 3-year PFS rate between patients with minimal BM involvement and those with negative BM involvement was significant for patients with FLIPI low-intermediate risk (88.9% vs. 51.5%; p= 0.032) and those with advanced stage disease (90.0% vs. 33.6%; p= 0.027), but there were no significant differences in patients deemed FLIPI high risk and those with limited stage disease. Multivariate analysis revealed that BM involvement, including morphological and minimal involvement, was a significant poor prognostic factor (hazard ratio 4.885 [95% confidence interval 1.16-20.56], p = 0.0305). ConclusionAt the start of treatment, bone marrow involvement was seen in most FL patients. Patients without any BM involvement had an excellent prognosis. Patients with minimal BM involvement had an equally poor prognosis as those with morphologic BM involvement. [Display omitted] [Display omitted] [Display omitted] DisclosuresIshikawa:Mundipharma KK: Research Funding.

Combined Hydration and Antibiotics with Lisinopril to Mitigate Acute and Delayed High-dose Radiation Injuries to Multiple Organs.

The NIAID Radiation and Nuclear Countermeasures Program is developing medical agents to mitigate the acute and delayed effects of radiation that may occur from a radionuclear attack or accident. To date, most such medical countermeasures have been developed for single organ injuries. Angiotensin converting enzyme (ACE) inhibitors have been used to mitigate radiation-induced lung, skin, brain, and renal injuries in rats. ACE inhibitors have also been reported to decrease normal tissue complication in radiation oncology patients. In the current study, the authors have developed a rat partial-body irradiation (leg-out PBI) model with minimal bone marrow sparing (one leg shielded) that results in acute and late injuries to multiple organs. In this model, the ACE inhibitor lisinopril (at ~24 mg m d started orally in the drinking water at 7 d after irradiation and continued to ≥150 d) mitigated late effects in the lungs and kidneys after 12.5-Gy leg-out PBI. Also in this model, a short course of saline hydration and antibiotics mitigated acute radiation syndrome following doses as high as 13 Gy. Combining this supportive care with the lisinopril regimen mitigated overall morbidity for up to 150 d after 13-Gy leg-out PBI. Furthermore, lisinopril was an effective mitigator in the presence of the growth factor G-CSF (100 μg kg d from days 1-14), which is FDA-approved for use in a radionuclear event. In summary, by combining lisinopril (FDA-approved for other indications) with hydration and antibiotics, acute and delayed radiation injuries in multiple organs were mitigated.

Significance of Minimal Residual Disease in Chronic Lymphocytic Leukemia

Newly introduced highly effective treatment options increase the importance of minimal residual disease measurement in chronic lymphocytic leukemia. Minimal residual disease is gaining interest mainly as a predictive marker; however, clinical significance of minimal residual dis-ease in chronic lymphocytic leukemia in many different situations remains unresolved. Factors with a possible impact on the clinical significance of minimal residual disease are as follows: technique for minimal residual disease quantification, treatment regimen, peripheral blood vs. bone marrow analysis or time -point for sampling. Highly sensitive methods now available to evaluate minimal residual disease can detect a single chronic lymphocytic leukemia cell in 10(-4)- 10(-5) leukocytes using either allele -specific oligonucleotide polymerase chain reaction or multicolor flow cytometry. Minimal residual disease quantification as a surrogate marker to assess treatment efficacy in routine hematological practice has to be further evaluated.

Potential Treatment of Marginal B-Cell Lymphoma With Antiviral Therapy

Background: The association between hepatitis C (HCV) and subtypes of B-cell lymphomas was initially described in epidemiological studies. These subtypes mainly are marginal zone lymphomas (MZLs) and lymphoplasmacytic lymphomas, both of which are indolent lymphomas. Hence there is a reported association - there is a potential role for antiviral strategies to influence the clinical course of these lymphomas. Case: We present a 39-year-old female patient with past medical history of hepatitis C with cirrhosis who underwent orthotropic liver transplantation. She was found to have periportal lymphadenopathy intraoperatively which were sampled. Pathology revealed marginal zone B-cell lymphoma. An MRI obtained one month postoperatively which showed persistent periportal, portocaval and gastrohepatic lymphadenopathy. She was asymptomatic without symptoms of night sweats, fever or weight loss. A subsequent bone marrow biopsy revealed small atypical lymphoid infiltrates suggestive of minimal bone marrow involvement. Given it was a low-grade B-cell lymphoma; patient was followed up and no treatment was initiated. One month later - she was noticed to have jaundice and worsening transaminases. Subsequently, she underwent liver biopsy, which showed changes consistent with severe recurrent hepatitis C. After thorough evaluation, she was started on Sofosbuvir and Ribavirin. The patient completed 24 weeks of therapy and achieved sustained viral response. Subsequently she had a PET scan, which showed no abnormal FDG uptake in the neck, chest, abdomen and pelvis or osseous structures. She remained in remission through her last follow up. Discussion: We report here the response to antiviral treatment of a liver transplant patient with low grade B-cell lymphoma associated with HCV infection. This further supports the potential link between some sub-types of B-cell lymphoma and HCV infection, and supports the use of antiviral treatment in this setting.

Bone matrix vesicle-bound alkaline phosphatase for the assessment of peripheral blood admixture to human bone marrow aspirates

PurposePeripheral blood (PB) admixture should be minimized during numerical and functional, as well as cytokinetic analysis of bone marrow (BM) aspirates for research purposes. Therefore, purity assessment of the BM aspirate should be performed in advance. We investigated whether bone matrix vesicle (BMV)-bound bone alkaline phosphatase (ALP) could serve as a marker for the purity of BM aspirates. ResultsTotal ALP activity was significantly higher in BM serum (97 (176–124)U/L, median (range)) compared to PB serum (63 (52–73)U/L, p<0.001). Agarose gel electrophoresis showed a unique bone ALP fraction in BM, which was absent in PB. Native polyacrylamide gel electrophoresis revealed the high molecular weight of this fraction, corresponding with membrane-bound ALP from bone matrix vesicles (BMV), as evidenced by electron microscopy. A serial PB admixture experiment of bone cylinder supernatant samples, rich in BMV-bound ALP, confirmed the sensitivity of this proposed quality assessment method. Furthermore, a BMV ALP fraction of ≥15% is suggested as cut-off value for minimal BM quality. Moreover, the BM purity declines rapidly with larger aspirated BM volumes. ConclusionThe exclusive presence of BMV-bound ALP in BM could serve as a novel marker to assess purity of BM aspirates.

Bone marrow flow cytometry in staging of patients with B-cell non-Hodgkin lymphoma.

BackgroundBone marrow biopsies are routinely performed for staging patients with B-cell non-Hodgkin lymphoma (NHL). In addition to histomorphological studies, ancillary tools may be needed for accurate diagnosis. We investigated the clinical utility of multiparameter flow cytometric examination of bone marrow aspirates.MethodsA total of 248 bone marrow specimens from 232 patients diagnosed with B-cell NHL were examined. Monoclonal antibodies directed against CD19, CD20, CD10 (or CD5), and κ and λ immunoglobulins were used. Multi-stage sequential gating was performed to select specific cells of interest, and the results were compared with bone marrow histology.ResultsThe concordance rate between histomorphology and flow cytometry was 91.5% (n=227). Eight cases (3.2%) were detected by flow cytometry alone and were missed by histomorphology analysis, and 6 of these 8 cases showed minimal bone marrow involvement (0.09-2.2%). The diagnosis in these cases included large cell lymphoma (n=3), mantle cell lymphoma (n=3), and mucosa-associated lymphoid tissue (MALT) lymphoma (n=2). Thirteen cases were histopathologically positive and immunophenotypically negative, and the diagnoses in these cases included diffuse large cell lymphoma (n=7), T-cell/histiocyte-rich large B-cell lymphoma (n=2), anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (n=1), follicular lymphoma (n=1), MALT lymphoma (n=1), and unclassifiable lymphoma (n=1).ConclusionsMulti-color flow cytometry can be a useful method for assessing bone marrow in staging NHL and also plays a complementary role, especially in detecting small numbers of lymphoma cells.

Somatic Mutations in Splicing Factor 3b, Subunit 1 (SF3B1) Are a Useful Biomarker to Differentiate Between Clonal and Non-Clonal Causes of Sideroblastic Anemia

Background: Sideroblastic anemia (SA) can present as congenital SA (CSA), acquired clonal SA, and acquired reversible SA. Patients (pts) with SA have anemia and ring sideroblasts (RS). Acquired clonal SA is often linked to myelodysplastic syndromes (MDS) or myelodysplastic/ myeloproliferative neoplasms (MDS/MPN). Clinico-pathologic overlap features, unmet morphologic and/or cytogenetic criteria complicate the diagnosis of SA leading to delayed therapies. Currently the diagnosis of SA is based on bone marrow (BM) examination and routine blood tests. There is a need to find easily testable biomarkers that can lead to faster diagnosis of clonal and non-clonal SA. Somatic mutation in splicing factor 3b, subunit 1 (SF3B1) are frequent in MDS-RS and MDS/MPN-RS and have been closely associated with RS.Objective:SF3B1 mutations can be a useful diagnostic biomarker for pts with acquired clonal SA who present with cytopenias and/or minimal morphologic changes suspicious of MDS and MDS/MPN but not sufficient to make a definitive diagnosis.Patients and Methods: Six pts with SA at presentation and seen at Cleveland Clinic were included in this study. The median age was 38 years (range, 6-75). Blood tests and BM biopsy showed persistent anemia [Hgb, 10.5 g/dL (range, 8.8-13)], RS [numerous (3 pts), 15% (1 pt), rare (1 pt) and >15% (2 pts)], 3/6 pts had minimal erythrodysplasia with 1 pt having a mild megakaryocytic dysplasia, 3 pts had hypercellular (60-90%), 2 pts had normocellular (50%, 80%) and 1 pt had hypocellular BM (30%) for age, and < 5% BM (2%=2 pts; 1%=1 pt; 3%=1 pt). Two pts had PLT count > 400 k/uL, 3 pts had > 100 k/uL, and 1pt <100 k/uL. Leukocytosis was observed in 2, leukopenia in 1, and neutropenia in 2 pts. Iron studies were unremarkable Molecular analysis was performed on SF3B1 (all exons), PRPF8 (exons 15, 25, 33), U2AF1 (exons 1, 6, 7), SRSF2 (exons 1, 2), DNMT3A (18-23), EZH2 (exons 18, 19) and IDH1/2 (exon 4). BCR/ABL p210 transcripts were assessed by RT-PCR. JAK2 (exon 14) was assessed by melting curve analysis.Results: All pts presented with sustained anemia, RS w/o or with minimal BM dysplasia, and normal karyotype. SF3B1 mutations (K666N and K700E) were detected in 2 pts (pts#2 and 6) before the clonal disease manifested. The first pt was a 75-year-old man with leukocytosis, transfusion-independent anemia, and fatigue. At the time of SF3B1 molecular testing the BM was hypercellular (60%) with <10%, megakaryocytic dysplasia, 2% blasts and 15% RS. JAK2 and BCR-ABL were wild type (WT). Later on, the patient developed hepatomegaly, his cytopenias worsened requiring RBC transfusions, and a repeat BM biopsy showed more prominent dysplasia of erythroid cells and megakaryocytes. The second pt was a 51-year-old man with fatigue, low WBC= 3.0 k/uL, ANC=0.7 k/uL, and PLTs=113 k/uL and macrocytic anemia (MCV=106 fL and Hgb=12.2 g/dL). His spleen was borderline enlarged (14 cm). BM biopsy showed 90% cellularity, erythroid hyperplasia, rare dyserythropoiesis and 60% RS. Suspicion for a possible MDS was raised although morphology was inconclusive. SF3B1 mutation (K700E) confirmed the clonality of the disease. A few months later, the pt developed worsening counts and overt MDS was confirmed by BM biopsy. All SF3B1WT pts were WT also for other genes known to be mutated in MDS and MDS/MPN like PRPF8, U2AF1, SRSF2, DNMT3A, IDH1/2, and EZH2. Only genetic tests showed an ALAS2 mutation in pt#1 and a paternally inherited duplication of 10q23.31 in pt#3. Both pts were diagnosed with CSA. Clinical history and work up assessed the same diagnosis for pt#4. Pt#5 presented with neutropenia and anemia. BM biopsy showed no dysplasia, 50% BM cellularity, trilineage maturation, and 25% RS. The presence of RS appeared to be induced by the use of an antibiotic prior the BM biopsy. Patient’s cytopenias and RS resolved after drug cessation.Conclusion: This case series although conducted in few pts provides important insights in the diagnostic use of molecular genetics in clinical practice. Often SA pts come to the clinic with inconclusive morphologic features of MDS or MDS/MPN. The presence of SF3B1 mutations may serve as an additional tool that can help differentiating between clonal and non-clonal cases of SA. In both pts, SF3B1 mutations anticipated the subsequent overt manifestation of clinical MDS or MDS/MPN phenotype. The collection of a larger cohort of pts is ongoing in order to further confirm this finding. DisclosuresNo relevant conflicts of interest to declare.

Bcl-2/IgH Expression of Diffuse Large B-cell Lymphoma Cells in Minimal Bone Marrow Infiltration

We sought to investigate the role of the Bcl-2 translocation at the chromosomal and protein levels in minimal bone marrow (BM) infiltration by diffuse large B-cell lymphomas (DLBCL). The presence of the Bcl-2/IgH fusion gene was detected in BM samples and paraffin-embedded lymph node (LN) samples from 103 patients with DLBCLs using FISH. Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using immunocytochemistry (ICC) and immunohistochemistry (IHC), respectively. Bcl-2/IgH translocation in paraffin-embedded LN tissue sections was observed in 43 (41.7%) patients by FISH. Of the 43 patients, the Bcl-2/IgH rearrangement in the bone marrow specimens occurred in 34 patients. The Bcl-2/IgH recombination rate in stage III cancers was not significantly different compared to the rate observed in stage I/II cancers (p = 0.101), respectively showing no statistical differences between stage IV and I/II (p = 0.179). In 64.7% (22/34) of the cases with t(14;18), Bcl-2 was detected based on ICC analysis. Positive Bcl-2 ICC staining and the t(14;18) translocation were positively correlated (p < 0.001). We then applied our FISH method to slides with at least one abnormal cell and were subjected to FISH analysis after staining. During the follow-up, no infiltration by cytomorphology for 16 DLBCL patients whose bone marrow presented Bcl-2/IgH gene rearrangement at diagnosis, and two cases were positive by morphology compared to FISH-positive results 6 months later; 9 out of 16 patients (56.3%) presented with positive Bcl-2/IgH results earlier than the morphology evaluation after 12 months. Utilizing both FISH and cytologic morphology, the assessment of Bcl-2/IgH translocation status could contribute to the better detection of minimal bone marrow infiltration and relapse receiving treatment by DLBCL cells.

Immunohistochemical analysis of CD123, CD56 and CD4 for the diagnosis of minimal bone marrow involvement by blastic plasmacytoid dendritic cell neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive haematological malignancy derived from plasmacytoid dendritic cell precursors. Almost all patients show cutaneous manifestations, and >60% show bone marrow (BM) involvement at initial presentation. In cases where there is BM involvement by only a small number of tumour cells, such involvement is difficult to ascertain solely on morphological examination. In such situations, immunohistochemistry (IHC) may be useful in revealing minimal BM involvement by BPDCN. We investigated six patients with BPDCN. Initial morphological diagnosis disclosed BM involvement in only one of the six patients. To confirm BM involvement, IHC for CD4, CD56 and CD123 was performed on BM biopsies or clot sections. IHC revealed minimal BM involvement (CD123, 3/3; CD56, 2/3; CD4, 2/3) in three patients with BM that appeared morphologically normal. Our data clearly support the utility of IHC in diagnosing minimal BM involvement by BPDCN. Accordingly, we highly recommend immunohistochemical analyses for CD123, CD56 and CD4 in BPDCN patients, particularly in cases where the initial BM study indicates normal morphology.

Acute Gastrointestinal Syndrome in High-Dose Irradiated Mice

The most detailed reports of the response of the gastrointestinal system to high dose acute radiation have focused mainly on understanding the histopathology. However, to enable medical countermeasure assessment under the animal rule criteria, it is necessary to have a robust model in which the relationship between radiation dose and intestinal radiation syndrome incidence, timing, and severity are established and correlated with histopathology. Although many mortality studies have been published, they have used a variety of mouse strains, ages, radiation sources, and husbandry conditions, all of which influence the dose response. Further, it is clear that the level of bone marrow irradiation and supportive care can influence endpoints. In order to create robust baseline data, the authors have generated dose response data in adult male mice maintained under identical conditions and exposed to either total or partial-body irradiation. Partial-body irradiation includes both extensive (40%) and minimal (5%) bone marrow sparing models, the latter designed to correlate with an established primate model and allow assessment of effects of any medical countermeasure on all three major radiation syndromes (intestinal, bone marrow, and lung) in the surviving mice. Lethal dose (LD(30), LD(50), and LD(70)) data are described in the various models, along with the impact of enteric flora and response to supportive care. Correlation with diarrhea severity and histopathology are also described. These data can be used to aid the design of good laboratory practice (GLP)-compliant Animal Rule studies that are reflective of the conditions following accidental radiation exposure.

Gene expression profiling with principal component analysis depicts the biological continuum from essential thrombocythemia over polycythemia vera to myelofibrosis

The recent discovery of the Janus activating kinase 2 V617F mutation in most patients with polycythemia vera (PV) and half of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF) has favored the hypothesis of a biological continuum from ET over PV to PMF. We performed gene expression profiling of whole blood from control subjects (n = 21) and patients with ET (n = 19), PV (n = 41), and PMF (n = 9) using DNA microarrays. Applying an unsupervised method, principal component analysis, to search for patterns in the data, we demonstrated a separation of the four groups with biological relevant overlaps between the different entities. Moreover, the analysis separates Janus activating kinase 2-negative ET patients from Janus activating kinase 2-positive ET patients. Functional annotation analysis demonstrates that clusters of gene ontology terms related to inflammation, immune system, apoptosis, RNA metabolism, and secretory system were the most significantly deregulated terms in the three different disease groups. Our results yield further support for the hypothesis of a biological continuum originating from ET over PV to PMF. Functional analysis suggests an important implication of these gene ontology clusters in the pathogenesis of these neoplasms and in disease evolution from ET over PV to PMF.

Abstract 3803: Anti-cancer activity of Capridine, a novel nitroacridine chemotherapeutic, in glioblastoma

Abstract Glioblastoma is characterized by highly proliferative tumor cells, increased cellularity, necrosis, and proliferation of capillary endothelial cells. The incidence rate of glioma in adults is 50% greater in men than in women suggesting that there may be a male hormonal component that modulates glioblastoma progression. Our preclinical developmental program identified Capridine as a chemotherapeutic agent that had specificity towards prostate cancer with minimal bone marrow toxicity. Since the DNA intercalating activity of Capridine does not completely correlate with its cytotoxic activity, we examined other alternative cellular targets. Our investigation identified the androgen receptor (AR) and its dependent signal transduction mechanism as a possible cellular mediator of its cytotoxic activity. In this study, our analysis revealed that glioblastoma cell lines T98G and MO59K expressed AR and their growth was responsive to the AR-mediated signal transduction. Capridine inhibited the growth of glioblastoma cells by down regulating the AR and inhibiting the phosphorylation of Focal adhesion kinase (FAK). Since FAK is associated with integrin and neuropeptide mediated signaling, critical for cell migration and invasion, we examined the activity of Capridine on cell invasion and migration using a Boyden Chamber assay. Capridine inhibited the migration and invasion of the glioblastoma cell lines T98G and MO59K. Our data not only validate AR as a target of Capridine as with prostate cancer but also implicate a novel target, FAK in glioblastoma. Combination approaches using Capridine and anti-hormonal therapy may prove to be viable chemotherapeutic regimen for primary glioblastoma as well as an inhibitor of metastatic propensity. These preclinical studies are being extended in an in vivo animal model with combination treatment approaches with a view to develop novel chemotherapy based treatment for glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3803. doi:1538-7445.AM2012-3803

Extramedullary Plasmacytoma of the Lung – Rather a Disseminated than a Localized Disease? – A Case Report of a Primary Pulmonary Plasmacytoma Showing Distinct Signs of Systemic Spread

Objective: We report an unusual case of a patient with a plasma cell tumor presenting first as a primary pulmonary plasmacytoma (PPP) but then showing distinct signs of systemic spread. Case Summary: A 58-year-old woman presented with an extramedullary plasma cell tumor of the lung with additional affection of lymph nodes above and underneath the diaphragm, without apparent infiltration of the bone marrow by malignant plasma cells, but with evidence of a small proportion of clonal plasma cells in FACS analysis. Due to the fact that the patient showed excretion of lambda light chains in the urine in addition to the solid manifestations on multiple sites and minimal bone marrow involvement, we verified the systemic spread. This is in contrast to primary extramedullary plasmacytoma presenting as solitaire plasma cell tumors mostly occurring in the upper aerodigestive tract or extramedullary myeloma, which describes relapses or extramedullary progression of multiple myeloma emerging in various organs. We herein present all features of a, to our opinion, systemic disease by means of affection pattern, laboratory values, and bone marrow infiltration. Conclusion: This unusual case demonstrates the presentation of an extramedullary plasma cell tumor that appeared as a PPP but showed distinct signs of dissemination as well as uncommon features such as a monoclonal component of IgM type. This example reveales that extramedullary manifestations of plasma cell dyscrasia should be surveyed carefully as they can conceal an underlying systemic disease.

Bcl-2/IgH expression in minimal bone marrow infiltration by follicular lymphoma cells

The purpose of this study was to investigate the roles of bcl-2 chromosomal translocation and Bcl-2 protein expression in follicular lymphoma (FL) minimal bone marrow (BM) infiltration. We identified the same bcl-2/IgH fusion gene in paraffin-embedded lymph node (LN) samples and BM samples using immunohistochemistry (IHC), immunocytochemistry (ICC), cytologic morphology and fluorescence in situ hybridization (FISH). The presence of the Bcl-2/IgH fusion gene in the BM samples and paraffin-embedded LN samples from 56 patients with follicular lymphomas was detected using FISH. The Bcl-2 protein levels in BM and paraffin-embedded tissues were quantified using ICC and IHC, respectively. Approximately 78.6% (44/56) of the paraffin‑embedded LN tissue sections that underwent FISH analysis had a bcl-2/IgH translocation. The primary lesion was also positive for the bcl-2/IgH fusion gene, as were the BM minimal infiltrates. The bcl-2/IgH rearrangement occurred in 88.6% (39/44) of the BM specimens. The bcl-2/IgH recombination rate in stage III/IV cancers was significantly different to that observed in stage I/II cancers (p=0.041). In 59% (23/39) of the cases with t(14;18), Bcl-2 was found to be present as assessed by ICC. Positive Bcl-2 ICC staining and the t(14;18) translocation (as detected using FISH) were positively correlated (p=0.028). We then applied the FISH method to slides that had previously been morphologically evaluated using Wright-Giemsa staining; any slides with at least one abnormal cell were subjected to FISH analysis following staining. The assessment of bcl-2/IgH translocation status may contribute to the better detection of minimal BM infiltration by FL cells. Utilizing FISH and cytologic morphology techniques allows for earlier and more accessible BM examination.

Gene Expression Profiling with Principal Component Analysis Depicts the Biological Continuum From Essential Thrombocythemia Over Polycythemia Vera to Myelofibrosis

AbstractAbstract 4115 Introduction:The classical Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs) encompass polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). After several years, a proportion of patients with PV may develop increasing splenomegaly and bone marrow fibrosis to terminate in postpolycythemic myelofibrosis, which then has a very poor prognosis with median survival of only a few years. A high proportion of the ET-population has been shown to have prefibrotic PMF or a “PV-like” phenotype, which after a variable time may terminate in myelofibrosis with myeloid metaplasia (MMM). Although the concept of prefibrotic myelofibrosis as a separate disease entity to be distinguished from ET by distinct histopathological features has been questioned, it seems logical that a continuum exists from “early” myelofibrosis with no or minimal bone marrow fibrosis to the advanced stage of myelofibrosis with myeloid metaplasia. Accordingly, a new concept of these diseases as a biological continuum from ET over PV to PMF has emerged. Indeed, this biological continuum reflects the various clinical stages as also described for chronic myelogenous leukaemia (CML) – a chronic stable phase (ET-PV), an accelerated phase (transitional phase), and a terminal myelofibrosis or blast phase of the disease (PMF-BP).It is evident that the JAK2 V617F mutational load is an important determinant of phenotype, but other genetic and epigenetic events contribute to the phenotypic presentation. Several gene expression studies have identified genes which might be of pathogenetic relevance for the development of CMPNs. However, no study has applied an unsupervised method as Principal Component Analysis (PCA) to discover unknown trends in the data. We have carried out gene expression profiling with PCA to ascertain if this analysis might unravel distinct grouping of disease entities supporting the biological continuum from early ET over PV to the advanced myelofibrosis phase. Patients and Methods:Using HG-U133 Plus 2.0 microarray from Affymetrix, recognizing 54675 probe sets (38500 genes), gene expression microarray studies have been performed on control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). An unsupervised statistical method, PCA, which is an exploratory tool used to uncover unknown trends in the data based on gene expression profiles, was applied to the data. Results:The results from the PCA confirm the hypothesis of a biological continuum from control subjects over ET to PV and finally PMF (figure 1). The figure also reveals that patients with PV in a transitional stage (PVtrans) with pancytosis, huge splenomegaly, and bone marrow fibrosis have a gene profile between PV and PMF. Furthermore, single gene analysis revealed that several genes are highly dysregulated in patients with ET, PV, and PMF compared to control subjects even after correcting for multiple testing. Discussion and Conclusions:Using global gene expression profiling with PCA in patients with different CMPNs (ET, PV, PMF), we have yielded support to the concept of a biological continuum from early ET to the advanced myelofibrosis stage. Studies are in progress to identify single genes or clusters of genes accounting for the biological continuum and accordingly the progression from ET over PV to myelofibrosis. Disclosures:No relevant conflicts of interest to declare.