Cells In Bone Marrow Aspirates Research Articles

The Presence of Multinuceated Plasma Cells in Bone Marrow Aspirates Has No Clinical Significance.

Abstract 4904There is no published literature on the clinical significance of multinucleated plasma cells in bone marrow aspirates. The few references in the literature that mention multinucleated plasma cells imply that their presence is pathologic. We decided to investigate the clinical significance of these cells. All bone marrow aspirates done at the Atlanta VAMC from 1/1/99 until 8/1/99 were examined by a skilled hematopathology technician for 2 minutes. The presence of multinucleated plasma cells were recorded if they were identified. We then determined clinical characteristics of the patients and compared the characteristics of those with multinucleated plasma cells and those without. RESULTS. There were a total of 61 aspirates for review. 34 of these had identified multinucleated plasma cells MNPCs. The average age of the group with MNPC was 58.7 years (SD 15.4) and the group without MNPC was 61.6 (SD 12.4). 11 of the 27 patients without MNPC were alive at 10 years (41%) while 14 of the 34 patients with MNPC were alive at 10 years (also 41%). Among the group with MNPC who were still alive at 10 years the average change in serum creatinine over the 10 year period was .23 mg/dl. Among the group without MNPCs the average change in serum creatinine over the 10 year period was .26 mg/dl. Finally, 12 of the 34 patients (35%)with MNPCs were found to have a clonal lymphoid disorder sometime over the 10 year period after their aspirate, while 10 of the 27 patients (37%, a nonsignificant difference)without MNPCs were found to have a clonal lymphoid disorder. CONCLUSIONMNPC have no impact on survival or changes in renal function over a 10 year period. Furthermore, their incidence is not impacted by age. Finally, they do not predict the occurrence of clonal lymphoid disorders. We conclude that MNPC are likely a normal variant of plasma cells DisclosuresNo relevant conflicts of interest to declare.

Correlation of Cytomorphology, Immunophenotyping, and Interphase Fluorescence in Situ Hybridization (FISH) in 381 Patients with MGUS and 310 Patients with Multiple Myeloma.

Abstract 2812Poster Board II-788From cytomorphological aspects, monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) are overlapping disorders. According to the WHO classification, a threshold of 10% of plasma cells (PCs) in bone marrow (BM) aspirates separates both categories. To clarify whether this separation is justified from cytogenetic aspects, we performed comparison of interphase fluorescence in situ hybridization (FISH) patterns in 691 patients with MGUS (n=381) or MM (n=310). Also, the results of cytomorphology and immunophenotyping using multiparameter flow cytometry (MFC) were correlated. 278 females and 413 males (22.8-92.8 yrs) at first presentation of MM/MGUS were analyzed between 2005-2009 in our laboratory with a combination of cytomorphology, MFC, and FISH following magnetic activated cell sorting (MACS) of CD138+ PCs (Robosep, STEMCELL Technologies, Vancouver). According to cytomorphological WHO criteria, 381 patients were categorized as MGUS (median BM PCs, 5%), 310 as MM (median PCs, 18.5%). The number of FISH probes being applicable to samples was depending on the amount of plasma cells yielded from MACS procedure. In the MM pts, a median of 12 probes was applied (range, 0-22) which was slightly higher than in the MGUS patients (median: 11; 0-18) (p=0.00002). FISH procedure was hampered by insufficient PC numbers more frequently in MGUS (79/381, 20.7%) than in MM pts (29/310; 9.3%) (p=0.0004). In MM pts, FISH revealed a median of 2 and a maximum of 7 cytogenetic alterations per patient in contrast to a median of 0 and a maximum of 5 in MGUS (p<0.0001). In more detail, in MM the maximum number of gains of genetic material per patient was 7 (MGUS: 5), of losses 7 (MGUS: 3), and of reciprocal rearrangements 2 (MGUS: 1). Subsequently, cytogenetic alterations were compared in those 527 pts (260 MM and 267 MGUS pts; 76.3% of all pts), in whom PC numbers allowed performance of at least 5 FISH probes (t(11;14), t(4;14), t(14;16), 13q14, TP53). Abnormal FISH results were detected in 145/260 MM (55.8%) and 106/267 MGUS pts (39.7%) (p=0.0002). In MM, 31/260 (11.8%) had a t(4;14)/IGH-FGFR3 in contrast to 5/268 (1.9%) in MGUS (p<0.0001). The t(11;14)/IGH-CCND1 (MM: n=41; 15.6%; MGUS: n=50; 18.7%; n.s.) and t(14;16)/IGH-MAF were similarly frequent in both cohorts (MM: n=8; 3.1%; MGUS: n=3; 1.1%; n.s.). Monosomy13/del(13)(q14) was more frequent in MM (n=103; 39.3%) when compared to MGUS (n=59; 22.1%, p=0.0001). Deletions of TP53/17p13 were seen in 16 MM (6.1%) and in 6 MGUS pts (2.2%) (p=0.029). Notably, in 7 MGUS cases with <1% PCs in cytomorphology, FISH revealed genetic alterations in 3 cases. PCs as quantified by cytomorphology vs. MFC ranged from 0-96% vs. 0-84% (median 8.5 vs. 2.0), respectively, with a highly significant correlation between both methods (Pearson, r=0.712, p<0.0001). However, as previously reported, MFC detected lower numbers in general: the median ratio of PCs by cytomorphology:MFC amounted to 4.25 (range 0.00-178.00). In 12 MGUS cases (1.7%) as defined with cytomorphology, MFC did not detect any PCs. Conversely, in 5 MGUS cases, MFC detected PCs while cytomorphology did not. In conclusion, cytogenetic patterns showed higher genetic complexity in MM cases when compared to MGUS, and both the t(4;14) as -13/del(13)(q14) were significantly more frequent in MM when compared to MGUS. However, the cytogenetic alterations showed no specific pattern for MM or MGUS categories. Therefore, the overlaps being seen from morphological aspects do also exist on the genetic level. This suggests that a cytogenetically based categorization of these cases might correlate better with the clinical profiles than the cytomorphological separation of MM/MGUS. Finally, this study supports the performance of FISH in MGUS cases. The demonstrated detection of malignant/monoclonal PCs by MFC also in cases in which cytomorphology fails to diagnose MM/MGUS emphasizes the inclusion of MFC in a standard diagnostic procedure. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Weiss:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Identification of Risk Factors Related to Poor Angiogenic Potency of Bone Marrow Cells From Different Patients

Therapeutic angiogenesis induced by the implantation of autologous bone marrow-derived cells has been used for the treatment of ischemic diseases. However, as the outcomes of cell implantation obviously vary among patients, it is essential to identify patients that would benefit the most from this treatment. We collected clinical and laboratory data from 25 patients scheduled to undergo sternotomy for various surgical procedures. Then, we aspirated bone marrow cells from the sternum during the operation and investigated the cell quality in vitro by cultivation, and their angiogenic potency in vivo using an ischemic limb model of mice. The angiogenic potency of bone marrow cells differed among patients. Aging, renal failure, anemia, and high serum levels of triglyceride, C-reactive protein, interleukin-6, and type I collagen cross-linked N-telopeptide (NTX) significantly correlated with poor angiogenic potency of bone marrow cells. We assigned scores to these risk factors, and found a strong correlation between the risk scores of patients and the angiogenic potency of their bone marrow cells (r=-0.883, P<0.001). These risk scores can predict the angiogenic potency of bone marrow cells for inducing therapeutic angiogenesis with an accuracy of 80%. We have identified the risk factors related to poor angiogenic potency of bone marrow cells and developed a new scoring system to predict their angiogenic potency for the treatment of ischemic diseases. Our results may help select patients for this treatment in future clinical trials.

Disseminated tumor cells in primary breast cancer: Evaluation of the percentage of breast cancer stem cells in bone marrow aspirates of patients receiving neoadjuvant chemotherapy

505 Background: Disseminated tumor cells (DTC) in the bone marrow (BM) are related to poor prognosis of primary breast cancer (PBC). Cancer-initiating stem cells (CSC) have been detected in BM of PBC patients. Downregulation of the Notch pathway leads to murine mammary stem cell expansion and its expression is an early event in breast cancer. We enumerated CSC in BM mononuclear cells (BMC) and assessed the expression of Notch-1, ER, and HER-2 to study the differences between untreated patients and those treated with neoadjuvant chemotherapy. Methods: BM was collected from 90 PBC patients at time of surgery to assess DTC (CD326+CD45-) and CSC (CD326+CD45-CD44+CD24-) by FACS analysis. BM samples were also interrogated for the presence of CSC with ALDH activity using the Aldefluor® detection kit. RNA extracted from 61 BMC was analyzed for Notch-1, ER, and HER-2 transcripts by RT-PCR; the level of each transcript was normalized to that of the housekeeping gene, GAPDH. Results: The median age of patients is 51 years old (range, 27–77 years). A total of 29 patients (32%) received neoadjuvant chemotherapy, including 8 patients with anti-HER-2 targeted therapy. BMC of neoadjuvant-treated patients had a significantly higher median percentage of CSC in BMC (P= 0.046) and a significantly higher proportion of CSC cells within the Aldefluor+ population (P= 0.013) than those of untreated patients. Median level of Notch-1 transcripts in BMC was lower in neoadjuvant-treated patients compared with that of untreated patients (P= 0.07). There was an inverse correlation between the level of Notch-1 transcripts and the percentage of CSC in BMC of untreated (Spearman's rho= -0.519, P &lt; 0.001), but not in neoadjuvant treated (Spearman's rho= -0.483, P= 0.058) patients. Conclusions: Neoadjuvant chemotherapy is associated with expansion of CSC in the BMC of patients with PBC. Untreated patients with higher level of Notch-1 transcripts in the BMC tend to have lower percentages of CSC and suggest that Notch signaling may be crucial for differentiation of CSC. The prognostic value of this finding is under investigation and gene expression profile of CSC may provide indications for novel neoadjuvant therapies. No significant financial relationships to disclose.

Caspase-cleaved cytokeratin 18 fragment (M30) as marker of postoperative residual tumor load in colon cancer patients

Background Soluble cytokeratin 18 (CK18; M65) and a caspase-cleaved fragment of CK18 (M30) have been used as biomarkers, corresponding to tumor cell death and apoptosis, respectively. Methods In the present study, M30 was quantified for the first time in serum samples of colon cancer patients pre- and postoperatively as well as during chemotherapy. Minimal residual disease (MRD) was assessed preoperatively by detection of pan-cytokeratin antibody A45-B/B3-positive cells in bone marrow aspirates. Results Out of 46 patients, those with colon tumors of stages I and IV had significantly elevated M30 serum concentrations compared to controls (n = 23). In 31 colon cancer patients, M30 determinations were performed prior to and seven days after tumor surgery. A group of 24 patients exhibited a significant decrease of M30 in response to tumor removal, in contrast to seven patients who revealed either persistent or higher M30 levels postoperatively. The frequency of MRD was not significantly different for patients with decreasing (4/24) and persisting (3/7) M30. However, M30 correlated significantly with the increased number of recurrences within 36 months in the group with persisting M30 (4/7 versus 2/24, p = 0.032; hazard ratio 8.3, p = 0.016). In a group of patients (n = 10) receiving capecitabine/oxaliplatin chemotherapy (CapOx), transient increases in M30 did not correlate with responses. Conclusion The data obtained within the present limited pilot study in colon cancer patients demonstrate that perioperative changes of M30 may indicate systemic residual tumor load and increased risk of recurrence warranting further evaluation of this marker of apoptosis in a larger prospective clinical trial.

Primary tumor size is associated with ALDHbr breast cancer initiating stem cells in bone marrow aspirates of primary breast cancer patients.

Abstract Abstract #4031 Background&amp;#x2028; The presence of epithelial cells in bone marrow (BM), disseminated tumor cells (DTC), that are CD326+CD45- is a negative independent prognostic factor in primary breast cancer (PBC). Cancer-initiating stem cells (CSC) are purported to be CD44+CD24-, express aldehyde dehydrogenase 1 (ALDH), and possess self-renewal potential. We examined the expression of CD44, CD24, and ALDH on BM aspirates of patients with PBC.&amp;#x2028; Methods&amp;#x2028; As part of an ongoing prospective study, from September 2006 to May 2008, BM aspirates were collected from 47 PBC patients, median age of 52 yrs (range: 30-76 yrs). At time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells were analyzed for DTC and CSC phenotype and ALDH function (Aldefluor® assay kit) by 6-color FACS analysis and data correlated with primary lesion tumor size, tumor markers (ER and PR; over-expressed HER-2 by FISH), nodal status, and neoadjuvant chemotherapy.&amp;#x2028; Results&amp;#x2028; The mean (±SEM) percentages of DTC and ALDHbr cells were 1.07% ± 0.09% and 2.17% ± 0.21%, respectively. Within the DTC population, the mean proportion of CSC and ALDHbr cells were 79.6% ±1.69% and 43.8% ± 3.4%, respectively. There was a statistically significant positive correlation between size of the primary tumor and the percentages of DTC (rho =0.364, p = 0.016, n = 43), DTC/ALDHbr (rho = 0.533, p = 0.28, n = 17), and CSC within the DTC/ALDHbr population (rho = 0.790, p = 0.004, n = 11). Specifically, the BM from patients whose primary tumors were &amp;lt;2 cm (T1) had statistically significantly fewer DTC (0.81% vs. 1.26%, p = 0.013), DTC/ALDHbr cells (0.82% vs. 1.40%, p= 0.035), and CSC within the DTC/ALDHbr population (88% vs. 96%, p = 0.009) compared to tumors &amp;gt;2cm (T2). Addition of Aldeflour® to the immunophenotype analysis further enriched the proportion of CSC phenotype from 79.1% in the DTC population to 92.3% in the DTC/ALDHbr population (p = 0.047). Of particular clinical relevance, patients who received neoadjuvant therapy had a near statistically significantly higher percentage of DTC than untreated patients (adjuvant 0.99% vs. neoadjuvant 1.41 %, p = 0.066) without a significant decrease in the proportion of CSC within the DTC population (adjuvant 78.6% vs. neoadjuvant 84.6%, p = 0.312), suggesting CSC were not eliminated by neoadjuvant therapy.&amp;#x2028; Conclusion&amp;#x2028; Epithelial cells with cancer-initiating stem cell-like phenotypes can be identified in BM aspirates of patients with PBC. The DTC population contained a larger subset of CD44+CD24- cells that overlapped with a smaller subset of ALDHbr cells. The increased percentage of CSC within the ALDHbr population and persistence following therapy further suggests that the relevance of both phenotypes to the mechanism responsible for recurrent disease in breast cancer warrants further investigation and validation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4031.

Notch-1 expression is not related to the percentage of CD326+ cells in bone marrow aspirates of patients with primary breast cancer.

Abstract Abstract #4030 Background: The presence of epithelial cells (CD326+CD45dim) in the bone marrow (BM), i.e. disseminated tumor cells (DTC), are related to poor prognosis of primary breast cancer (PBC). Notch signaling pathway is involved in the development of the breast and aberrant activation of Notch signaling is an early event in breast cancer. Others have shown that breast cancer stem cells (BCSC) with the putative phenotype of CD44+CD24- can be identified within cell lines and primary tumor and have upregulated genes including Notch-1. However, it is unclear if Notch-1 is highly expressed by DTC and BCSC in BM of patients with PBC. We hypothesized that the Notch-1 expression is correlated with the level of BCSC within DTC. In this study, we enumerated BCSC in DTC-enriched populations in patients with PBC and assessed the expression of Notch-1 by RT-PCR.&amp;#x2028; Methods and Patients: As part of an ongoing prospective study, from September 2006 to May 2008, 121 BM aspirates were collected from as many patients with PBC. Thirty-seven patients, median age of 52 years (range, 30-76 years), provided BM aspirates under anesthesia at time of surgery. At the time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells (BMC) were enriched for DTC using anti-CD326 antibody-coated magnetic beads, and isolated using the AutoMACS-Pro cell separator system. DTC and BCSC were identified by multi-color FACS analysis. Thereafter, DTC-enriched cell preparations were analyzed for expression of ER, PR, HER-2, Notch-1, and Numb by RT-PCR; level of each transcript was normalized to a ratio to the house keeping gene GAPDH.&amp;#x2028; Results: The mean ± SEM DTC in BMC was 1.05 % ± 0.09% and the majority was BCSC (79.7% ± 1.87%). The proportion of DTC-enriched fractions that expressed Notch-1, Numb, ER, PR, and HER-2 transcripts was 27/37 (73.0%), 33/37 (89.2%), 24/37 (64.9%), 15/37 (40.5%), and 20/37 (54.1%), respectively. The expression of Notch-1, Numb, ER, PR, and HER-2 transcripts did not correlate with the percentage of DTC. Contrary to our hypothesis, Notch-1 and Numb mRNA expression in DTC-enriched cells negatively correlated with the percentage of BCSC (Spearman's rho = -0.384, p= 0.027; and rho= -0.347, p= 0.048, respectively).&amp;#x2028; Conclusions: Our results show no correlation between the level of Notch-1 expression and the percentage of BCSC in DTC-enriched populations in BM of patients with PBC. Since these studies were conducted in CD326-enriched populations, it underscores the necessity to further purify the CD44+CD24- cells and conduct studies in isolated single cells to definitively delineate the gene expression of BCSC from patients with PBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4030.

A vast-range speed control microchip for retention of all cell types

This Technical Note is the first description of a large-scale logarithmic flow-rate damping system designed to retain cells of different adherence, different suspensibility and different motility. The chamber, which can easily retain and cultivate many types of cells, including high-motility cells and swimming cells, via a series of "speed bumps", readily facilitates cell retention for complex heterogeneous cultures. Yeast cells, red blood cells, rabbit bone marrow aspirate and dinoflagellate swimming cells were introduced into the chip for multi-cell retention, multi-cell culture and observation. Here, we show that the chamber creates a flow field with a ratio of end/start speeds as low as 0.01. The logarithmic distribution of flow-rate within the chamber is controlled precisely by pressure, all of the cell types that we tested were retained easily within the chamber. Many cell-cell interactions were observed, predicting a high potential for the success of on-chip heterogeneous cell experiments.

Gaucher's Disease at a National Referral Hospital

To determine the prevalence and to characterise Gaucher's disease in terms of socio-demographic data, clinical presentation, and management as seen at Kenyatta National Hospital. A retrospective record based study. Kenyatta National Hospital, a referral and teaching hospital. Prevalence (number of cases seen a year), of Gaucher's disease, sociodemographic data, clinical presentation, mode of diagnosis and treatment modalities of Gauchers disease. Nine patients were studied, four males and five females giving a prevalence of 0.9 cases seen a year and a M:F ratio of about 1:1. The most common presentation was splenomegaly in nine (100%) cases and hepatomegaly in seven (78%) patients, neurological and bone symptoms were rare, in one (11%) cases and in two (22%) cases respectively. Diagnosis was mainly on basis of presence of Gaucher cells in bone marrow and splenic aspirate as enzyme assay was unavailable. Management was mainly supportive and enzyme therapy was only available for two (22%) patients. Anaemia was the most common complication with seven (78%) patients and one death occurred due to osteomyelitis. Only four (44%) patients were followed up for a period of four years. Gaucher's disease is a rare condition at the Kenyatta National Hospital (KNH). The presentation of most patients is organomegaly, (hepatosplenomegaly) and best fits the type 1 or non-neuronopathic Gaucher's disease. Neurological manifestations are rare. Management of this condition at the KNH is mainly supportive and enzyme therapy still remains out of reach for most patients.

Usefulness of FICTION Method for the Evaluation of Response after Treatment in Multiple Myeloma

Introduction: According to the new uniform response criteria of International Myeloma Working Group (IMWG), stringent complete response (sCR) is defined as a condition of normal free light chain (FLC) ratio and absence of clonal cells in the bone marrow (BM) by immunohistochemistry or immunofluorescence, in addition to the CR condition. The kappa/lambda ratio is assessed to identify clonal cells in the BM and a minimum of 100 plasma cells is required for analysis of clonal cells. However, the evaluation of kappa/lambda ratio by immunohistochemistry or immunofluorescence may be inaccurate, because it is difficult practically to countthe number of anti-kappa/lambda antibodies-stained cells in the BM section and in case of low percentage of residual plasma cells, flow cytometric evaluation is also difficult. To investigate whether FICTION (Fluorescence Immunophenotyping and interphase Cytogenetics as a Tool for the Investigation Of Neoplasms) technique can be used as a tools for evaluation of clonal cells after treatment in multiple myeloma, we performed FICTION on BM cells in follow-up patients with myeloma and compared the results of FICTION with other parameters.Method: 18 myeloma patients, whose BM examination and serum free light chain were checked at the same time after treatment, were enrolled in Seoul National University Hospital. We performed FICTION for the fluorescence in situ hybridization (FISH) items that were abnormal at initial BM examination of each patient. The selected probes were LSI 1q25/1p36/1p subtelomere probe (Vysis, Downers Grobe, IL, USA) for trisomy 1q25, LSI 13 (RB1) 13q14 probe (Vysis) for RB1 deletion, LSI IGH probe (Vysis) for IGH rearrangement and LSI p16 (9p21)/CEP 9 probe (Vysis) for p16 deletion. The FICTION results were reported by the percentage of plasma cells with abnormal FISH signals among plasma cells stained with anti-kappa and lambda antibodies labeled with fluorescein isothiocyanate (FITC). We compared FICTION results with response parameters, such as % plasma cells in BM aspirates, serum FLC ratio, M-component in serum and/or urine protein electrophoresis (PEP) and FISH results.Results: Among 18 patients in follow-up, 5 (28%) showed <5% plasma cells by light microscope based differential count in BM aspirates and normal FISH results below the cut-off level. However, these patients turned out to have clonal cells in BM by FICTION techniques. Among these 5 patients, 3 patients showed abnormal serum FLC ratio and the other 2 patients showed M-component in serum PEP with normal serum FLC ratio. One patient with plasma cells fewer than 5% in BM aspirates, no cytogenetic abnormality in FISH and no M-component in serum, showed abnormal serum FLC ratio and abnormal FICTION results; 2 (25%) of 8 plasma cells showed RB1 deletion in FICTION. After 2 years, this patient progressed to plasma cell leukemia.Conclusion: Results of FICTTON correlated with FLC ratio and/or M-component in serum, while the percentage of plasma cells in BM aspirates or FISH results did not. The assessment of percentage of plasma cells in bone marrow aspirates might be inaccurate due to poor aspiration technique including dilution and focal infiltration of plasma cells. Also, FISH results based on the percentage of abnormal cells among all bone marrow nucleated cells do not reflect clonal plasma cells. In clonclusion, we suggest that FICTION technique is more sensitive method for identification of residual malignant plasma cells with clonalityalong with the evaluation of response after treatment in multiple myeloma.

Elimination of CD20-Expressing Cells in Multiple Myeloma by Iodine I-131 Tositumomab (Bexxar®) Correlates with Response to Therapy

It has been proposed that treatment failures in multiple myeloma (MM) are related to the chemoresistance of a subset of malignant myeloma cells with clonogenic potential to the anti-myeloma drugs (Matsui et al., Cancer Research 2008, 68:190). Studies suggest that these putative myeloma stem cells (MSC) are CD138neg and express CD20, which is usually absent in more differentiated malignant plasma cells (Matsui et al., Blood 2004, 15:2332). These findings provided a rationale for a treatment strategy to eliminate chemoresistant clonogenic MSC using anti-CD20 antibodies. To evaluate the clinical effects of anti-CD20 treatment, we developed a phase II trial with Bexxar as a consolidation treatment for MM. We hypothesized that Bexxar would be more efficacious than unlabeled antibody in the eradication of highly radiosensitive myeloma cells by both direct and cross-fire effects. Preclinical studies showed that tositumomab, the antibody used in Bexxar, inhibited colony formation of clonogenic myeloma cells. To be eligible for Bexxar treatment, patients must have completed at least 4 cycles of therapy (1st to 3rd line) and have measurable disease in a plateau of at least partial response (PR). To date, 10 of 30 patients have been enrolled, of which 5 were treated with Bexxar after completion of initial therapy prior to proceeding to autologous stem cell transplant (ASCT). Patients proceeding to ASCT required hematopoietic stem cells collection prior to Bexxar and 3 months after Bexxar. Eight patients are evaluable for response and toxicities. At 3 months post Bexxar, 1 patient achieved a partial response (PR), 4 patients had stable disease (SD), and 1 patient progressed (PD). At 1 year post Bexxar, 1 patient with initial PR achieved CR and remains in CR, 1 patient is in unconfirmed CR, 2 are in partial response (PR), and 3 remain in SD. After a median 20 months of followup (range 4–24), all patient are alive, 4 in continued response, 3 with SD. Hematological toxicities were mild to moderate (1 patient grade 3 and 4 patients grade 2 thrombocytopenia, 4 patients grade 2 neutropenia). Non-hematological toxicity was limited to HAMA (6 patients). Out of patients who received Bexxar prior to transplant, 3 collected stem cells post-Bexxar without problems, one requires re-collection. Three patients who proceeded to ASCT to date using the post-Bexxar stem cell collection, engrafted at 11–12 days, and had no unexpected toxicities with ASCT. We also analyzed CD20+ cells in bone marrow aspirates (BM) and stem cell collections (SCC) using samples collected from 4 patients before and after Bexxar. Bexxar treatment eliminated a median 80% of CD20+ cells (range 23–97). For a given patient, elimination of CD20+ cells from SCC correlated with elimination of CD20+ cells from BM. The most complete elimination of CD20+ cells from BM was observed in 2 patients who at 1 year achieved CR (94% and 97%), compared to patients who achieved PR (23% and 68%). We conclude that anti-CD20 consolidation treatment of myeloma patients with Bexxar used as targeted therapy against clonogenic myeloma cells is feasible and well tolerated. Clinical outcomes to date are encouraging considering that clonogenic MSC represent <5% of malignant plasma cells and delayed responses observed in this study could be expected. While early, clinical outcomes appear to correlate with the efficacy of the CD20+ cell elimination by Bexxar treatment in myeloma.

Bone marrow micrometastases in advanced stage non-small cell lung carcinoma patients

The clinical relevance of bone marrow micrometastases in non-small cell lung cancer (NSCLC) is undetermined, and the value of such analyses in advanced stage patients has not been assessed previously. Immunomagnetic selection with the MOC31 (anti-EpCam) antibody was performed to isolate and detect tumor cells in bone marrow aspirates obtained from 196 patients with NSCLC, and the patients were subjected to follow-up for the assessment of survival. Repeated bone marrow samples, 2-7 samples per patient, were obtained from 13 long-term survivors. MOC31 positive tumor cells were detected in 107 of 196 (55%) samples, a frequency similar to results reported in low-stage patients prior to curative surgical resection for NSCLC. No association was found between the presence of bone marrow micrometastases and disease stage or histological subgroup, and survival was similar in patients with and without detectable tumor cells in bone marrow. Repeated bone marrow analysis revealed, for the first time in this group of patients, the continued presence of tumor cells regardless of the given therapy and treatment response. The immunomagnetic method utilized is a feasible strategy for the detection of bone marrow micrometastases in NSCLC. In advanced stage patients, the presence of MOC31 positive cells in bone marrow does not predict survival. Repeated analyses of bone marrow samples from long-term survivors revealed tumor cells in bone marrow which may represent dormant cells of particular interest for further characterization and follow-up.

Ultrastructural Characteristics of Nucleated Cells in Bone Marrow of Patients with Acquired Aplastic Anemia

The objective of this study was to investigate the ultrastructural characteristics of nucleated cells in the bone marrow of patients with aplastic anemia (AA). This was done by observing the morphology of nucleated cells in bone marrow aspirates from 20 patients with AA by transmission electron microscopy. Erythroblasts were decreased in all cases and not observed in 6 cases. Nuclear abnormalities, such as pyknosis, karyolysis, karyorrhexis, apoptosis, and “Swiss cheese”-like changes, were found in 10 cases. Focal cytoplasmic necrotic changes and cytolysis were found in 3 cases. There were more megaloblasts in 4 cases. Abnormalities of granulocytes were found in 12 out of 18 cases. Megakaryocytes showed focal cytoplasmic necrotic changes. Most monocytes had dendritic features, including excessive cytoplasm, processes, and large round nuclei in all cases. Other monocytes illustrated typical monocytic features with twisted nuclei, plentiful RER, vacuoles, lysosomes, and prominent Golgi apparatus. Macrophages and hemophagocytes occurred in all cases. The incidence of lymphocytes was high in 17 out of 20 cases and occasionally lymphocytes were enlarged in 8 cases. More plasmacytes and plasmacytoid lymphocytes were found in 5 and 3 cases, respectively. The observations suggest that (1) the universal nuclear injury of erythroblasts may be related to the pathogenetic pathway of AA development; (2) the dendritic cells and hemophagocytes from the mononuclear phagocyte system may play a more critical role in hematopoietic failure of AA, directly and/or indirectly; and (3) besides T lymphocytes, increasing numbers of plasmacytes or plasmacytoid lymphocytes are associated with AA in some cases.

Efficacy of Imatinib Mesylate Dose-Escalation in CML Patients Showing Suboptimal Response to Standard Dose.

Introduction: We report the results of our prospective single arm study performed to evaluate the efficacy of Imatinib mesylate (IMT) dose-escalation therapy and the influence of BCR/ABL mutational status on the treatment outcome.Methods: Patients with chronic phase(CP) chronic myelogenous leukemia (CML) who had not achieved a complete hematologic response (CHR) after 3 months, a major cytogenetic response (MCyR) after 6 months, nor a complete cytogenetic response (CCyR) after 6-12 months of standard IMT therapy (400mg per day) were included. Patients in accelerated phase (AP) or blast crisis (BC) who had failed to achieve CHR after 3 months of IMT (400 or 600mg per day) were also eligible. 600mg of IMT had been administered for patients with CP CML per day, and 600mg or 800mg for AP or BC CML for at least 12 months. For each patient, BCR/ABL gene mutation test was performed on 6 loci. Cytogenetic response (CyR) was evaluated with the percentage of bcr/abl positive cells in bone marrow aspirate by means of FISH per 6 months. Molecular responses (MR) were assessed per 3 months with a logarithmic decrease in BCR-ABL/ABL gene ratio (BA/A) of peripheral blood or bone marrow aspirate from a standardized median value of BA/A in 56 patients with newly diagnosed CML.Results: 77 patients were enrolled and 61 patients were assessable for the evaluation of MR. The median age was 50 years old (range: 20-70). Stages at the time of the enrollment were CP for 56, AP for 4, BC for 1 patient(s). The median time from the initiation of IMT therapy to dose-up was 20 months (range: 0-62). BCR/ABL gene mutational status could be obtained in 24 patients (wild:mutant=20:4). The median follow-up was 19.9 months (range: 6.5-24.3). For 34 patients whose 6th-month CyR is available, MCyR rate (MCyRR) was 26.5% and CCyR rate (CCyRR) was 23.5%. The 12th-month MCyRR was 61.1% and CCyRR was 50.0%. Patients with wild type showed a MCyRR of 20% and a CCyRR of 15% on the 12th month after dose-up, whereas no patient with mutant type showed a CCyR or a MCyR. CMR rate and MMR rate on the 12th-month were 16.7% and 26.7%. Among 20 patients with wild type, one patient achieved a 6th-month CMR, 2 patients MMR's, whereas no patient with mutant type could achieve a MMR during the entire treatment period. Hematologic and non-hematologic toxicities were tolerable.Conclusion: IMT dose-escalation could induce quite a number of durable CyR's and MR's without any serious toxicity in patients who had failed to achieve either a CHR or a CyR with a standard IMT therapy.

Quantitation of plasma cells in bone marrow aspirates by flow cytometric analysis compared with morphologic assessment.

Abstract Context.—Accurate quantitation of bone marrow plasma cells is an important component in the diagnosis and posttreatment assessment of plasma cell dyscrasias. Although flow cytometry is sometimes used for this purpose and can rapidly evaluate many cells, the accuracy of flow-based plasma cell quantitation compared with morphologic assessment (currently the gold standard) is uncertain as direct comparison studies have not been previously reported. Objective.—To determine how percentages of plasma cells in diagnostic aspirate smears quantitated by morphologic assessment relate to percentages of plasma cells quantified by flow cytometry. Design.—Thirty bone marrow cases with 10% or more plasma cells and leukemia/lymphoma flow cytometry immunophenotyping studies were identified from our hematopathology database. The Wright-stained aspirate smears, marrow biopsy sections, and flow cytometry histograms were reviewed. Results.—Morphologically determined plasma cell percentages from the diagnostic aspirat...

Quantitation of Plasma Cells in Bone Marrow Aspirates by Flow Cytometric Analysis Compared With Morphologic Assessment

Accurate quantitation of bone marrow plasma cells is an important component in the diagnosis and posttreatment assessment of plasma cell dyscrasias. Although flow cytometry is sometimes used for this purpose and can rapidly evaluate many cells, the accuracy of flow-based plasma cell quantitation compared with morphologic assessment (currently the gold standard) is uncertain as direct comparison studies have not been previously reported. To determine how percentages of plasma cells in diagnostic aspirate smears quantitated by morphologic assessment relate to percentages of plasma cells quantified by flow cytometry. Thirty bone marrow cases with 10% or more plasma cells and leukemia/lymphoma flow cytometry immunophenotyping studies were identified from our hematopathology database. The Wright-stained aspirate smears, marrow biopsy sections, and flow cytometry histograms were reviewed. Morphologically determined plasma cell percentages from the diagnostic aspirate smears were consistently higher than those determined by flow cytometry. Much of this difference appeared to be related to differences in sample quality. However, the cellular processing involved in performing flow cytometry also appeared to reduce plasma cell percentages in many cases. This study helps define the limitations of flow cytometry for quantitating plasma cell loads in marrow aspirate specimens that may significantly affect the diagnosis or assessment of treatment response.

Incidence and Clinical Significance of Sex Chromosome Losses in Bone Marrow of Patients with Hematologic Diseases

Loss of sex chromosomes in bone marrow is observed both in elderly persons as an aging phenomenon and in patients with hematologic malignancies. The purpose of this study was to evaluate the incidence and clinical significance of sex chromosome losses in patients with hematologic diseases, comparing the characteristics between patients with sole and secondary sex chromosome losses in conjunction with other chromosomal abnormalities. Study group included 868 patients with hematologic diseases between June 1998 and May 2006. The cells of bone marrow aspirates were processed using unstimulated culture methods such as direct, 24-hr and/or 48-hr culture. Sex chromosome losses were included in the karyotype, when X or Y chromosome loss is observed in more than 2 metaphase cells. The sex chromosome losses in bone marrow were found in 5.1% of the patients and 1.8% showed sex chromosome losses as a sole chromosomal abnormality. According to the disease categories, the incidences of sex chromosome losses were as follows: acute myelogenous leukemia (AML), 9.5%; acute lymphoblastic leukemia, 0%; myelodysplastic syndrome, 6.0%; chronic myelogenous leukemia 3.6%; myeloproliferative disorders, 1.3%; multiple myeloma (MM), 13.0%; chronic lymphocytic leukemia, 0%; malignant lymphoma, 3.8%; and benign hematologic diseases 2.2%. The patients with sex chromosome losses as a sole chromosomal abnormality were all male and median age was higher than that of patients with sex chromosome losses as a secondary abnormality (64 vs. 58 yr, P=0.02). The proportion of metaphase cells with sex chromosome losses was significantly lower in patients with sex chromosome losses as a sole chromosomal abnormality (40% vs. 100%, P<0.0001). The changes of sex chromosome loss were correlated with the disease status of AML and MM. These results suggest that secondary sex chromosome losses in conjunction with other chromosomal abnormalities seem to be one of the clonal abnormalities, whereas sex chromosome losses as a sole change seem to be an aging phenomenon, but further studies are needed.

Del(6)(p23) in two cases of de novo AML – a new recurrent primary chromosome abnormality

Previously, deletion 6p23 was generally reported in therapy-related secondary acute myeloid leukemia (AML) as part of complex karyotypes. In this report, we present two young adult patients with de novo AML-M2 and a terminal deletion 6p23 as a sole primary abnormality, confirmed by chromosome 6 specific subtelomeric probes. Two female patients 35 and 20 yr of age presented with anemia, but no bleeding, infections, lymphadenopathy or organomegaly. Morphological, immunophenotyping, chromosome and fluorescent in situ hybridization (FISH) analysis was performed on bone marrow aspirate cells. A diagnosis of AML-M2 was confirmed in both patients by morphological and immunophenotyping studies. Chromosome analysis in case no. 1 showed deletion 6p23 in 20% of metaphases whereas in case no. 2 the deletion 6p23 was present in 100% metaphases. FISH analysis confirmed the deletion as terminal in both cases. The DEK oncogene at 6p23 in both cases was found not to be deleted. To our knowledge, deletion 6p23 as a sole primary abnormality was reported in only one case. The common morphological, immunophenotypic, and cytogenetic features in our two patients strongly support a separate new entity of de novo AML with deletion 6p23.

The use and correlation with flow cytometry of fluorescent molecular beacons in real time PCR of IgH gene rearrangements for evaluation of minimal residual disease in multiple myeloma

17552 Background: At present, the prognostic value of the amount of residual tumor cells in PB, BM or stem cell harvests and its changes over time is stil not clear. Also the advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by using flow cytometry. The aim of the present study was to evaluate a real time PCR test for the IgH gene using alellespecific molecular beacons as fluorescence probes to quantify residual disease and also correlate flow cytometric detection of plasma cells in MM patients during followup after treatment with high dose of chemotherapy or standart chemotherapy. Methods: After clinical diagnosis of 17 MM patients, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. We have also examined the co-expression of CD19, CD38, CD45, CD56, and CD138 molecules in cells of bone marrow aspirates in patients with multiple myeloma by flow cytometry. Results: The active disease had been accepted of whom plasma cell infiltration ratio was over 10% in bone marrow and also of whom labeled by CD38 and CD138 by FCM. The detection of the MRD was positive in 13 patients by RT-PCR, respectively. The infiltration ratio was correlated with CD138 expression (p = 0.009) and RT-PCR detection of plasma cells (p = 0.006) and also significant correlation had been found between RT-PCR detection and CD138 expression respectively. No any correlaton was found between other surface antigens (CD38, CD45, CD56). Conclusion: Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM. By FCM only CD138 expression may have been used as disease marker in addition of the RT-PCR detection. No significant financial relationships to disclose.

A complex, four-way variant t(15;17) in acute promyelocytic leukemia

Complex variant 15;17 translocations are increasingly recognized in acute promyelocytic leukemia (APL). We report the case of a 47-year-old woman with APL harboring a novel four-way translocation. She presented with persistent bleeding after a tooth extraction. Blood cell counts at admission were hemoglobin at 9.0 g/dL, platelets at 15 × 10 9/L, and white blood cells at 0.460 × 10 9/L with abnormal promyelocytes. Most nucleated cells of bone marrow aspirates were abnormal promyelocytes with Auer rods. Chromosome analysis of unstimulated bone marrow cell cultures revealed a variant t(15;17) in the form of t(10;17;15;22)(q22;q21;q22;q11.2). Fluorescence in situ hybridization with a PML/RARA dual-color DNA probe showed the fusion signals on der(15) and the residual PML signals on der(22). RT-PCR showed long-form PML/RARA fusion transcripts. A complete remission was attained with a course of conventional chemotherapy including ATRA. A literature review revealed that our case is one of the very rare four-way translocations and the first report of the involvement of chromosomes 10 and 22 in a variant t(15;17).