Improving bone marrow biopsy quality through peer discussion and data comparisons: A single institution experience.

INTRODUCTION: Bone marrow biopsy (BMB) is crucial for the diagnosis, staging, and monitoring of a variety of hematologic diseases. Obtaining an adequate BMB can be challenging given the need to balance patient comfort with acquisition of high quality specimens. We had observed variable BMB quality at our institution with poor quality specimens sometimes affecting diagnosis. We thus undertook this quality improvement (QI) project to improve the quality of diagnostic BMB specimens. METHODS: We used an A3 QI process to identify factors possibly influencing BMB quality. We collected baseline data on 211 BMB, with short and long-term follow-up data on a further 382 cases. We used clinical conferences to discuss data, perform peer comparisons and identify strategies to create a sustainable improvement in BMB quality. RESULTS: Baseline data showed that BMB length was influenced most by the individual performer, with some influence of needle gauge. Other factors such as sedation, BMB indication were noncontributory. BMB lengths improved following performer education and individual performer data comparisons (15.2 mm post vs 12.8 mm baseline, P < .0001) and with use of an 8- rather than 11-gauge needle (18.3 mm 8-gauge vs 13.3 mm 11-gauge P < .0001), and were sustained over the long term. CONCLUSIONS: Education on BMB standards, sharing of performer data, and changing needle gauge are relatively straightforward methods to improve BMB quality, leading to easier pathology diagnosis.

Loss of wild-type Kras promotes activation of all Ras isoforms in oncogenic Kras-induced leukemogenesis.

Despite the well-established role of oncogenic RAS in promoting tumor formation, whether and how wild-type (WT) Ras inhibits tumorigenesis under physiological conditions remains controversial. Here, we show that in a fraction of endogenous oncogenic Kras-induced hematopoietic malignancies, including acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) and myeloproliferative neoplasm (MPN), WT Kras expression is lost through epigenetic or genetic mechanisms. Using conditional Kras(G12D/-) mice, we find that WT Kras deficiency promotes oncogenic Kras-induced MPN, but not T-ALL, in a cell-autonomous manner. Loss of WT Kras rescues oncogenic Kras-mediated hematopoietic stem cell depletion and further enhances granulocyte-macrophage colony-stimulating factor signaling in myeloid cells expressing oncogenic Kras. Quantitative signaling studies reveal that oncogenic Kras but not oncogenic Nras leads to cross-activation of WT Ras, whereas loss of WT Kras further promotes the activation of all Ras isoforms. Our results demonstrate the tumor suppressor function of WT Kras in oncogenic Kras-induced leukemogenesis and elucidate its underlying cellular and signaling mechanisms.

Loss of Dnmt3a and endogenous Kras(G12D/+) cooperate to regulate hematopoietic stem and progenitor cell functions in leukemogenesis.

Oncogenic NRAS and KRAS mutations are prevalent in human juvenile and chronic myelomonocytic leukemia (JMML/CMML). However, additional genetic mutations cooperating with oncogenic RAS in JMML/ CMML progression and/or their transformation to acute myeloid leukemia (AML) remain largely unknown. Here we tested the potential genetic interaction of DNMT3A mutations and oncogenic RAS mutations in leukemogenesis. We found that Dnmt3a(-/-) induces multiple hematopoietic phenotypes after a prolonged latency, including T-cell expansion in the peripheral blood, stress erythropoiesis in the spleen and myeloid malignancies in the liver. Dnmt3a(-/-) significantly promoted JMML/CMML progression and shortened the survival of Kras(G12D/+) mice in a cell-autonomous manner. Similarly, downregulating Dnmt3a also promoted myeloid malignancies in Nras(G12D/+) mice. Further studies show that Dnmt3a deficiency rescues Kras(G12D/+)-mediated depletion of hematopoietic stem cells and increases self-renewal of Kras(G12D/+) myeloid progenitors (MPs). Moreover, ~33% of animals developed an AML-like disease, which is driven by Kras(G12D/+); Dnmt3a(-/-) MPs. Consistent with our result, COSMIC database mining demonstrates that the combination of oncogenic RAS and DNMT3A mutations exclusively occurred in patients with JMML, CMML or AML. Our results suggest that DNMT3A mutations and oncogenic RAS cooperate to regulate hematopoietic stem and progenitor cells and promote myeloid malignancies.

Juvenile syndecan-1 null mice are protected from carcinogen-induced tumor development.

We previously showed that mice with a null mutation in syndecan-1 (Sdc1; CD138) were resistant to Wnt1-induced mammary tumor initiation. The absence of Sdc1 inhibited the increase in the mammary stem cell fraction that is characteristic of preneoplasia in this model. As the tumor precursor cells are recruited from the stem/progenitor cell compartment, tumor development was also inhibited (Liu et al., 2004; PNAS 101, 4158). Although Sdc1-/- mice are grossly normal, they are systemically smaller, suggesting that developmental abnormalities may extend further than their mammary glands. We have therefore evaluated the multi-organ response of Sdc1-/- mice to carcinogen-induced tumor development (7,12-dimethylbenz[a]anthracene, DMBA), and find these mice to be resistant to tumorigenesis in all the predominant carcinogen-susceptible lineages. Thus, Sdc1-/- mice administered DMBA during juvenile development are resistant not only to epithelial tumors, including liver (60-80%) and lung tumors (C57BL6 mice, 60-80%), but also to lymphoma (over 70%, depending upon strain and carcinogen dose). We demonstrate that CD138 is expressed (heterogeneously) in the hematopoietic stem cell fraction (and not only in pre-B and plasma cells), and that tumors arise in both myeloid and lymphoid lineages. Furthermore, carcinogen-induced mammary tumors are bilineal, implying a bipotent precursor cell. Both observations imply that the DMBA-induced tumor precursor cells are drawn from the stem/progenitor fraction, and we suggest that pathogenic activation of these cells could be abnormal in Sdc1-/- mice.

Ectopic prostate: case report of a presacral mass presenting with obstructive symptoms.

We report the unusual case of a 78-year-old man who presented with obstructive bowel symptoms and a 2.5-cm presacral mass. The mass was excised and found on pathologic examination to be ectopic prostate tissue complete with a muscular stroma. Review of the literature revealed a number of case reports describing variably sized fragments of ectopic prostate tissue involving a variety of organs, including spleen, uterine cervix, bowel wall, pericolic fat, anal submucosa, seminal vesicle, testis, and urinary bladder. However, to our knowledge, this case is unique in that it presented as a relatively large, isolated presacral mass causing functional bowel impairment. The ectopic location can be related to the normal embryonic development of the prostate, rectum, and bladder.

Sensitive detection of clonal immunoglobulin rearrangements in frozen and paraffin embedded tissues by polymerase chain reaction heteroduplex analysis.

Molecular detection of a clonal population of B or T cells through analysis of rearranged antigen receptor genes is an essential adjunct to the morphologic, flow cytometric, and immunohistochemical evaluation of tissue specimens for the presence of leukemia or lymphoma. Combining polymerase chain reaction (PCR) with heteroduplex annealing and polyacrylamide gel electrophoresis (PAGE) has been used to detect clonal T-cell receptor rearrangements, particularly in skin biopsy specimens. The authors have developed a similar PCR heteroduplex assay for detection of clonal VDJ immunoglobulin gene rearrangements using two sets of primers based on relatively conserved consensus regions in the J(H) and framework I and 2 regions of the immunoglobulin heavy chain V region gene. This method is able to detect a clonal rearrangement when the clone comprises as little as 1% of the population in a polyclonal B-cell background. It may be used on fresh, frozen, or paraffin-embedded tissue and detects a clonal population in a majority of lymphoma subtypes. Compared with conventional PCR analysis, this method requires only a short additional cycle of denaturation and slow renaturation before PAGE. Interpretation is simplified as the clonal PCR product migrates away from the polyclonal background products.

Highly active antiretroviral therapy results in HIV type 1 suppression in lymph nodes, increased pools of naive T cells, decreased pools of activated T cells, and diminished frequencies of peripheral activated HIV type 1-specific CD8+ T cells.

This study examines sequential lymph nodes from 13 drug-naive patients before and after 24 weeks of highly active antiretroviral therapy (HAART). A multipronged approach was used to study changes in HIV-1 RNA in each paired lymph node in relation to tissue architecture and frequency of naive T cells. After 24 weeks, all patients showed significant suppression of plasma viral load and 12 of 13 showed concordant viral suppression in the lymph node (p = 0.001). Using in situ hybridization and quantitative image analysis, we showed that HIV-1 RNA was reduced to below detectable levels (two copies per cell) in follicular dendritic cell (FDC) and mononuclear cell pools. Independent immunohistochemical analysis of lymph node sections revealed that 5 of 13 patients displayed increased FDC networks and 6 of 13 showed no change and all patients showed increases in tissue-resident CD4+ cells. All lymph node biopsies at 24 weeks showed increased proportions of CD4+ and CD8+ cells coexpressing the naive markers CD45RA and CD62L when compared with baseline values. Significant correlations existed between viral load suppression and loss of activated CD8+ T cells after 24 weeks in both lymph node and blood, which was mirrored by significantly lowered frequencies of activated peripheral Gag peptide/MHC tetramer+ CD8+ cells. Overall, these data show that a potent and successful treatment strategy that significantly suppresses and removes FDC-resident HIV-1 results in improvements in lymphoid architecture and by so doing provides the structures available for increased numbers of naive cells to interact with cognate antigen. In addition, our article shows that suppression of HIV-1 replication results in diminished frequencies of peripherally activated antigen-specific CD8+ cells.

Spontaneously relapsing clonal, mucosal cytotoxic T-cell lymphoproliferative disorder: case report and review of the literature.

Primary T-cell lymphoma of the gastrointestinal tract is a rare and usually aggressive disorder that may be associated with celiac disease. The authors describe a unique case of a clonal proliferation of CD8+ T cells involving the oral mucosa, ileum, and colon of a 35-year-old man that has regressed spontaneously and recurred numerous times over a 9-year period without treatment. The patient's symptoms were limited to occasional rectal bleeding and recurring painful oral ulcers. Within the intestine, these collections of small T cells induced minimal architectural distortions and did not show extensive epitheliotrophism. Polymerase chain reaction and sequencing analyses revealed that the identical T-cell clone has been present for more than 9 years and in different mucosal locations in this patient. This may represent a unique T-cell lymphoproliferative process akin to a mucosal counterpart of lymphomatoid papulosis of the skin.

Disseminated peritoneal plasmacytoma following cardiac transplantation.

Posttransplant lymphoproliferative disorders (PTLD) are an important comorbidity in immunosuppressed transplant patients. We describe a unique case of PTLD, a disseminated peritoneal plasmacytoma in a 56-year-old cardiac transplant patient presenting with ascites. The associated computed tomographic findings include omental thickening, mesenteric stranding, and ascites.

Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells.

Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T-cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL-4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40-signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL.

Tumor suppressor genes and clonal evolution in B-CLL.

This review highlights the genetic alterations that have been detailed in the malignant B-cell clones of patients with B-chronic lymphocytic leukemia (CLL). In particular, the alterations seen in p53 and the retinoblastoma (Rb) genes are reviewed. In addition, the multiplicity of cytogenetic alterations observed at baseline and on sequential analysis are summarized. The cytogenetic and molecular biologic analysis of B-CLL clones has revealed that there is a dynamic array of genetic events which occur within a B-cell clone. This latter data strongly suggests that clonal evolution may occur in B-CLL patients. However the relationship of the clonal instability to the patient's clinical course is still unclear. The relatively frequent detection of multiple tumor suppressor gene alterations in the B-CLL clones offer several interesting clues regarding the transformation event within B-CLL. A model is proposed which attempts to explain the potential contribution and interaction of p53 and Rb gene alterations in a malignant B-cell transformation.

Tumor necrosis factor-alpha facilitates induction of CD80 (B7-1) and CD54 on human B cells by activated T cells: complex regulation by IL-4, IL-10, and CD40L.

Expression of immune accessory molecules, such as CD80 (B7-1), on antigen-presenting cells governs whether such cells can activate antigen-specific T cells. As such, the factors that regulate the expression of these accessory molecules may determine whether presentation of antigen leads to immune activation or anergy. We previously reported that anti-CD3-activated T cells (Ta) can induce expression of CD80 and CD54 (ICAM-1) on human B cells through a contact-dependent signal delivered to the CD40 molecule via the CD40 ligand. Here, we demonstrate that another molecule in the CD40-ligand family, namely tumor necrosis factor-alpha (TNF-alpha), also plays a role in the Ta-mediated induction of CD80 or CD54 on human B cells. Neutralizing mAbs specific for TNF-alpha can inhibit B cell expression of CD80 or CD54 that is induced when B cells are cultured with Ta cells or in Ta-cell conditioned media. Moreover, soluble, recombinant TNF-alpha or TNF-beta can induce significant increases in B cell expression of CD80 and CD54. The phenotypic changes effected by TNF-alpha can be recapitulated by crosslinking CD120b (p75 TNF-receptor), but not CD120a (p55 TNF-receptor), with mAbs presented on Fc gamma RII (CD32)-expressing L cells. IL-4 augments the expression of CD80 induced by crosslinking either CD40 or CD120b. However, although IL-10 augments CD40-induced expression of CD80, this cytokine inhibits the expression of CD80 that is induced by crosslinking CD120b. Further regulation of TNF-mediated CD80 expression may occur at the level of CD120b expression itself. We find that stimulation with exogenous IL-4 or CD40-cross-linking induces B cell expression of CD120b, but not CD120a. This study identifies an ancillary, TNF-mediated pathway, whereby activated T cells can induce B cells to express enhanced levels of the important costimulatory molecules, CD80 and CD54.

Elevated expression of CD80 (B7/BB1) and other accessory molecules on synovial fluid mononuclear cell subsets in rheumatoid arthritis.

OBJECTIVE: To assess the expression of important accessory molecules such as CD80 (B7/BB1) and CD54 (intercellular adhesion molecule 1) on potential antigen-presenting cells (APC) in the synovial fluid (SF) of patients with rheumatoid arthritis (RA). METHODS: The level of expression of various accessory molecules on T cells, monocytes, and CD5+ or CD5- B cells from RA SF was compared, by multiparameter flow cytometric analysis, with the expression on peripheral blood (PB) mononuclear cells from the same patients and from normal controls. The functional significance of increased expression of these accessory molecules was assessed by the ability of the different cell populations to stimulate T cells in the mixed lymphocyte reaction. RESULTS: Monocytes, T cells, and B cells isolated from the SF of inflamed joints of patients with RA expressed significantly higher levels of CD80 and CD54 than those from the PB of RA patients or normal controls. CD11b and CD11c expression also were increased on SF B cells, while SF monocytes exhibited enhanced levels of CD40. Further, we found that the elevated CD80 and CD54 levels in RA SF cells may enhance the capacity of these cells to act as stimulatory APC. CONCLUSION: This is the first demonstration that specific cell subsets constitutively express increased levels of CD80 at a site of autoimmune pathology, which could potentially contribute to the initiation or maintenance of autoimmune disease.

Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) B cells are hyporesponsive or refractory to mitogens and growth factors in vitro. This study examined whether transforming growth factor beta (TGF-beta), a potent inhibitor of lymphocyte proliferation may play a role in the growth regulation of CLL B cells. CLL B cells from all donors treated expressed detectable TGF-beta 1 mRNA. In vitro release of TGF-beta by unstimulated cultures, or cultures stimulated by antibody to cell surface immunoglobulin (anti-mu) plus phorbol 12-myristate 13-acetate (PMA) was higher in CLL than in normal B cells. High levels of TGF-beta activity were also detected in plasma samples of CLL patients. The role of TGF-beta in growth regulation of CLL B cells was tested in assays using different B cell activators. Purified neoplastic B cells from most CLL patients proliferated in response to anti-mu, or the combination of anti-mu plus PMA. Levels of CLL B cell proliferation were lower than observed in normal B cells. Some CLL were refractory to these stimuli. Antibody to CD40 induced proliferation of CLL B cells from all donors tested when presented on Fc gamma RII (CDw32)-expressing L cells. Neutralizing antibodies to TGF-beta increased CLL B cell proliferation in the absence or presence of additional stimuli. These effects were dose dependent and specific. Exogenous TGF-beta completely inhibited CLL B cell proliferation induced by anti-mu, PMA, and anti-TGF-beta. CLL B cell proliferation induced by anti-CD40 was reduced by exogenous TGF-beta. However, even at high doses, TGF-beta did not completely inhibit the anti-CD40 effect. In summary, TGF-beta is overexpressed in CLL. CLL B cells are sensitive to TGF-beta and this cytokine functions as an autocrine growth inhibitor accounting at least in part for reduced proliferative responses of these leukemic cells and for the slow progression of the malignant process in vivo.

Malignant B cell CD5 membrane phenotype and B cell colony growth in vivo and in vitro in patients with B-chronic lymphocytic leukemia: analysis with clinical parameters.

Chronic lymphocytic leukemia (CLL), despite an overall good prognosis, has a subgroup of patients with more rapid, aggressive disease. In an attempt to generate additional information about the B cell clones in B-CLL which could be used as predictive parameters, we analysed CD5 membrane phenotype and B cell colony growth in 29 B-CLL patients. CD5, a 67-kd glycoprotein, has been reported to be a consistent feature of the malignant B cell membrane phenotype in CLL. We used an in vitro B cell colony assay to study the in vitro growth, differentiation, and cell surface properties of CLL B cells. Finally, a variety of standard clinical parameters were collated for each patient. Monoclonal antibodies to both CD5 and CD19 (pan B cell marker) were used to perform 2-color flow cytometry on freshly purified CLL B cells and on CLL B cells harvested after 7 days of in vitro culture. We demonstrate here that CLL B cells are heterogeneous with respect to their expression of CD5, and that this expression is not fixed but may vary both in vivo and in vitro. In vitro growth potential, as measured by the B cell colony assay, was also heterogeneous with three subgroups defined as low growth (< 10 colonies), intermediate (10-100 colonies) and high growth (> 100 colonies). Furthermore a T cell conditioned medium was not found to be a requirement for in vitro colony growth in the majority of CLL B cells. In addition, we evaluated the potential correlation of B cell CD5 phenotype or B cell colony growth on standard clinical parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Confirmation of Rb gene defects in B-CLL clones and evidence for variable predominance of the Rb defective cells within the CLL clone.

In 70 B-CLL patients, deletion or translocation, at or near the retinoblastoma (Rb) site, was detected in 20 by cytogenetic analysis. Purified B cell clones from 13 of these B-CLL patients were isolated and studied for Rb gene status, Rb mRNA and the Rb protein product. Southern blot analysis of the Rb site detected internal deletions (N = 1) or a single allele loss (N = 2) in five patients. Northern blots detected reduced Rb mRNA in four patients. Immunoblot of whole cell lysate revealed reduced levels of unphosphorylated Rb protein in six CLL patients. No CLL B cell clone contained phosphorylated Rb species. These molecular studies have confirmed the cytogenetic alteration of 13q12-14 sites in B-CLL cells. In addition, cytogenetic and molecular biologic analysis suggest heterogeneity in the B cell clone for Rb gene abnormality. B-CLL patients with abnormalities in both cytogenetic and Rb DNA/RNA analysis will have a dominance of B cells with an Rb abnormality (N = 5). In patients whose Rb defective CLL cells constitute only a minor subpopulation of the total B cell clone, only cytogenetic defects would likely be detected (N = 7).

Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal.

Cognate interactions between antigen-presenting B and T cells play crucial roles in immunologic responses. T cells that have been activated via the crosslinking of CD3 are able to induce B cell proliferation and immunoglobulin secretion in a major histocompatibility complex-unrestricted and contact-dependent manner. We find that such activated human CD4+ T cells, but not control Ig-treated T cells, may induce normal or leukemic B cells to express B7/BB1 and significantly higher levels of CD54 intercellular adhesion molecule 1 via a process that also requires direct cell-cell contact. To discern what cell surface molecule(s) may be responsible for signalling B cells to express B7/BB1, we added various monoclonal antibodies (mAbs) specific for T or B cell accessory molecules or control mAbs to cocultures of alpha-CD3-activated T cells and resting B cells. We find that only alpha-CD40 mAbs can significantly inhibit the increased expression of B7/BB1, suggesting that the ligand for CD40 expressed on activated T cells may be an important inducer of B7/BB1 expression. Subsequent experiments in fact demonstrate that alpha-CD40 mAbs, but not control mAbs, induce changes in B cell phenotype similar to those induced by activated T cells when the mAbs are presented on Fc gamma RII (CDw32)-expressing L cells. These phenotypic changes have significant effects on B cell function. Whereas chronic lymphocytic leukemia (CLL) B cells normally are very poor stimulators in allogeneic mixed lymphocyte reactions (MLRs), CLL-B cells preactivated via CD40 crosslinking are significantly better presenters of alloantigen, affecting up to 30-fold-greater stimulation of T cell proliferation than that induced by control treated or nontreated CLL-B cells. Similarly, the MLR of T cells stimulated by allogeneic nonleukemic B cells can be enhanced significantly if the stimulator B cells are preactivated via CD40 crosslinking. The enhanced MLR generated by such preactivated B cells may be inhibited by blocking B7/BB1-CD28 interaction with CTLA4Ig. These studies demonstrate a novel, CD40-dependent pathway for inducing B cell expression of B7/BB1 and enhancing B cell antigen-presenting cell activity that can be initiated via cell-cell contact with alpha-CD3-stimulated CD4+ T cells.