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Background: In diffuse large B-cell lymphoma (DLBCL), bone marrow involvement (BMI) has an important clinical implication as a component of staging and International Prognostic Index. This study aimed to determine whether molecular analysis of immunoglobulin heavy chain (IgH) genes and positron emission tomography-computed tomography (PET/CT) could overcome the limitation of defining morphologic BMI by trephination biopsy and could increase the diagnostic accuracy or prognostic prediction., Methods: A total of 94 de novo patients with DLBCL underwent PET/CT, polymerase chain reaction (PCR) test for detection of IgH gene rearrangement, and unilateral bone marrow (BM) trephination at diagnosis., Results: A total of 9 patients (9.6%) were confirmed to present morphologic BMI (mBMI) based on trephination biopsy. On the other hand, 21 patients (22.3%) were confirmed to have IgH clonality (IgH BMI), while 16 (17.0%) were classified with BMI based on the assessment of PET/CT (PET BMI). Each IgH rearrangement PCR and PET/CT showed the high negative predictive value of detecting the BMI. However, the combined assessment of IgH rearrangement and PET/CT could increase the diagnostic accuracy and specificity with 87.2% and 97.0%, respectively. The survival outcome of patients with double positive PET BMI and IgH BMI was significantly worse than that with either single positive PET BMI or IgH BMI, and even less than patients with neither PET BMI nor IgH BMI (3-year PFS: 50.0% vs. 75.4% vs. 97.9%, P = 0.007, 3-year OS: 50.0% vs. 75.6% vs. 80.1%, P = 0.035, respectively)., Conclusion: This study suggests that the combined evaluation of PET/CT and IgH rearrangement could give additional information for predicting therapeutic outcomes in patients with negative morphologic BMI as an important part of the prognosis., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2022 The Korean Academy of Medical Sciences.)

Many solid cancers metastasize to the bone and bone marrow (BM). This process may occur even before the diagnosis of primary tumors, as evidenced by the discovery of disseminated tumor cells (DTCs) in patients without occult malignancies. The cellular fates and metastatic progression of DTCs are determined by complicated interactions between cancer cells and BM niches. Not surprisingly, these niches also play important roles in normal biology, including homeostasis and turnover of skeletal and hematopoiesis systems. In this Review, we summarize recent findings on functions of BM niches in bone metastasis (BoMet), particularly during the early stage of colonization. In light of the rich knowledge of hematopoiesis and osteogenesis, we highlight how DTCs may progress into overt BoMet by taking advantage of niche cells and their activities in tissue turnover, especially those related to immunomodulation and bone repair.

Purpose: In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites., Experimental Design: Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed., Results: Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells., Conclusions: These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion., (©2019 American Association for Cancer Research.)

Bone marrow involvement in neuroblastoma indicates an advanced stage of the disease. In this study, we retrospectively analyzed the bone marrow infiltration status and hematological parameters of 111 cases of neuroblastoma. Among the 111 cases, 62 (55.9%) exhibited marrow infiltration. Erythrocytopenia, anemia, and thrombocytopenia were significantly ( p <0.05) associated with marrow infiltration of >10% malignant cells. The evaluation of blood cell count parameters with receiver operating characteristic curves indicated that erythrocytopenia can substantially improve the accurate prediction of the risk of bone marrow involvement with a Youden index of 0.60. The sensitivity and specificity were 90% and 70%, respectively, which correspond to a cut-off value of 4.015×10^12/L. Hematological aspects with diagnostic value are different among neuroblastoma patients with and without bone marrow infiltration., (© 2017 by the Association of Clinical Scientists, Inc.)

Metastatic cancer is the leading cause of all cancer related deaths. Prostate cancer (PCa) metastasizes preferentially to the bone marrow, specifically within the endosteal niche. Endosteal cells secrete homing molecules that may recruit PCa cells to the bone marrow. Once there, the biochemical signature of this niche regulates PCa fate including cellular dormancy or cell cycle arrest, reactivation and resistance to chemotherapeutics. Growth factors, interleukins, adhesion molecules, as well as extra-cellular matrix proteins can collectively change the phenotype of PCa cells. Understanding the biochemical signature of endosteal niche parasitism by PCa is imperative for the establishment of new and innovative therapeutic strategies. This review seeks to summarize these important niche signatures and the potential therapeutic approaches to target metastatic PCa within the bone marrow hematopoietic stem cell (HSC) niche. J. Cell. Biochem. 118: 1956-1964, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Despite the success in detecting breast cancer (BC) early and, with aggressive therapeutic intervention, BC remains a clinical problem. The bone marrow (BM) is a favorable metastatic site for breast cancer cells (BCCs). In BM, the survival of BCCs is partly achieved by the supporting microenvironment, including the presence of immune suppressive cells such as mesenchymal stem cells (MSCs). The heterogeneity of BCCs brings up the question of how each subset interacts with the BM microenvironment. The cancer stem cells (CSCs) survive in the BM as cycling quiescence cells and, forming gap junctional intercellular communication (GJIC) with the hematopoietic supporting stromal cells and MSCs. This type of communication has been identified close to the endosteum. Additionally, dormancy can occur by soluble mediators such as cytokines and also by the exchange of exosomes. These latter mechanisms are reviewed in the context of metastasis of BC to the BM for transition as dormant cells. The article also discusses how immune cells such as macrophages and regulatory T-cells facilitate BC dormancy. The challenges of studying BC dormancy in 2-dimensional (2-D) system are also incorporated by proposing 3-D system by engineering methods to recapitulate the BM microenvironment., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aggressive nature killer (NK)-cell neoplasm includes aggressive NK-cell leukemia (ANKL) and extranodal NK/T-cell lymphoma (ENKTL), nasal type. ANKL is rare and is characterized by a systemic neoplastic proliferation of NK-cells, usually with a leukemic presentation. ENKTL is a predominantly extranodal lymphoma, occurring mainly in the upper aerodigestive tract. Both are aggressive neoplasms strongly associated with Epstein-Barr virus (EBV). Here we report two patients with aggressive NK-cells neoplasms localized in the bone marrow (BM) who presented as prolonged fever, anemia, and thrombocytopenia. Both were treated initially as infectious disease. Imaging studies revealed splenomegaly without any nodular lesion or lymphadenopathy. BM examination revealed extensive involvement by EBV-positive NK-cells in both cases. Staging workup including nasal examination/biopsy was negative. Both patients passed away in a month. One case showed gains of chromosomes 4q and 9p by array comparative genomic hybridization. Both tumors were diagnostically challenging due to the unusual clinical presentation and absence of leukemic change, tumor mass or lymphadenopathy. Our cases demonstrate that lymphoma should be considered in patients with fever of unknown origin and bone marrow aspiration/biopsy should be performed as early diagnosis and novel therapeutic regimens may benefit these patients.

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of Vγ9Vδ2 T cells (γδ T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of γδ T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to γδ T-cell-mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to γδ T-cell-mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of γδ T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of γδ T cells. On the basis of these data, we conclude that ex vivo expanded γδ T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.

Objectives: Antigen expression by neoplastic cells is important because of its effects on the behavior and survival of patients. We sought to gather data on renal transplant recipients who had developed posttransplant lymphoproliferative disorders in their posttransplant era, and had a documented report on CD20 antigen testing., Materials and Methods: A comprehensive search of the literature was done for reports that indicate test results for the CD20 antigen in kidney recipients having lymphoproliferative disorders after transplant. Their demographics, disease characteristics, and prognoses were analyzed., Results: CD20-positive posttransplant lymphoproliferative disorder patients had a significantly shorter time from transplant to developing posttransplant lymphoproliferative disorder (P < .001). None of patients who had early onset posttransplant lymphoproliferative disorder was CD20 negative. Bone marrow involvement was significantly more prevalent among CD20-negative patients (P < .05) with no CD20-positive patient developing a bone marrow metastasis. Log-rank test showed a relatively worse survival for renal recipients expressing the CD20 antigen (P = .07)., Conclusions: CD20-positive posttransplant lymphoproliferative disorder lesions in kidney transplant patients are significantly more likely to develop early after transplant and represent an inferior outcome. We suggest that all renal transplant recipients who develop posttransplant lymphoproliferative disorder within their early time after surgery should be given anti-CD20 therapy. Future prospective studies are required to confirm our conclusions.

Bone marrow is thought to be a primary hematopoietic organ. However, accumulated evidences demonstrate that active function and trafficking of immune cells, including regulatory T cells, conventional T cells, B cells, dendritic cells, natural killer T (NKT) cells, neutrophils, myeloid-derived suppressor cells and mesenchymal stem cells, are observed in the bone marrow. Furthermore, bone marrow is a predetermined metastatic location for multiple human tumors. In this review, we discuss the immune network in the bone marrow. We suggest that bone marrow is an immune regulatory organ capable of fine tuning immunity and may be a potential therapeutic target for immunotherapy and immune vaccination.

We generated MHC-independent chimeric antigen receptors (CARs) directed to the GD2 antigen expressed by neuroblastoma tumor cells and treated patients with this disease. Two distinguishable forms of this CAR were expressed in EBV-specific cytotoxic T lymphocytes (EBV-CTLs) and activated T cells (ATCs). We have previously shown that EBV-CTLs expressing GD2-CARs (CAR-CTLs) circulated at higher levels than GD2-CAR ATCs (CAR-ATCs) early after infusion, but by 6 weeks, both subsets became low or undetectable. We now report the long-term clinical and immunologic consequences of infusions in 19 patients with high-risk neuroblastoma: 8 in remission at infusion and 11 with active disease. Three of 11 patients with active disease achieved complete remission, and persistence of either CAR-ATCs or CAR-CTLs beyond 6 weeks was associated with superior clinical outcome. We observed persistence for up to 192 weeks for CAR-ATCs and 96 weeks for CAR-CTLs, and duration of persistence was highly concordant with the percentage of CD4(+) cells and central memory cells (CD45RO(+)CD62L(+)) in the infused product. In conclusion, GD2-CAR T cells can induce complete tumor responses in patients with active neuroblastoma; these CAR T cells may have extended, low-level persistence in patients, and such persistence was associated with longer survival. This study is registered at www.clinialtrials.gov as #NCT00085930.

The mechanisms underlying tumor dormancy in human primary lesions and bone marrow metastases of nasopharyngeal carcinoma (NPC) are still not completely understood. The aim of this study was to determine differences in the fates of cultured primary NPC (P-NPC) cells, interferon-γ-transduced primary NPC (IFN-γ-P-NPC) cells, bone marrow metastatic NPC (BM-NPC), and IFN-γ-transduced BM-NPC (IFN-γ-BM-NPC) cells following xenotransplantation into these four groups of SCID mice through subcutaneous injection of 5×10(6) cells/site/animal (4 animals/group). The injected mice were monitored for tumor development at the sites of injection. In only the group injected with IFN-γ-P-NPC cells, the resulting nodules remained small throughout the 60-day observation period after injection, but gradually became palpably prickly. Histopathological examination revealed that these lesions invariably consisted of mostly structures of horny pearls and keratin bridges with occasional apoptotic and degenerative cells. In contrast, animals injected with nontransduced-P-NPC cells developed tumors progressively with occasional central necroses. In the two groups injected with IFN-γ-NPC-BM and NPC-BM cells, progressive growths of tumors were noted, with the latter being at slightly faster rates, whereas the xenografts of both groups showed a poorly differentiated phenotype with abundant vascularity. The study results highlight the high susceptibility of P-NPC but not BM-NPC following IFN-γ gene transfer to the induction of tumor dormancy, which is mediated via induced cell differentiation. Thus, induced cell differentiation could provide a new mechanism by which tumor dormancy is induced.

Background: Flow cytometry is the sole available technique for quantification of tumor plasma-cells in plasma-cell disorders, but so far, no consensus technique has been proposed. Here, we report on a standardized, simple, robust five color flow cytometry protocol developed to characterize and quantify bone marrow tumor plasma-cells, validated in a multicenter manner., Methods: CD36 was used to exclude red blood cell debris and erythroblasts, CD38 and CD138 to detect plasma-cells, immunoglobulin light chains, CD45, CD56, CD19, and CD117 + CD34 to simultaneously characterize abnormal plasma-cells and quantify bone marrow precursors. This approach was applied in nine centers to 229 cases, including 25 controls., Results: Tumor plasma-cells were detected in 96.8% of cases, all exhibiting an immunoglobulin peak over 1g/L. Calculation of a plasma-cells/precursors (PC/P) ratio allowed quantification of the plasma-cell burden independently from bone marrow hemodilution. The PC/P ratio yielded the best results in terms of sensitivity (81%) and specificity (84%) for differential diagnosis between MGUS and myeloma, when compared with other criteria. Combination of both the PC/P ratio and percentage of abnormal plasma-cells allowed the best differential diagnosis, but these criteria were discordant in 25% cases. Indirect calculation of CD19 negative PC/R ratio gave the best results in terms of sensitivity (87%)., Conclusion: This standardized multiparameter flow cytometric approach allows for the detection and quantification of bone marrow tumor plasma-cell infiltration in nearly all cases of MGUS and myeloma, independently of debris and hemodilution. This approach may also prove useful for the detection of minimal residual disease., (Copyright © 2010 International Clinical Cytometry Society.)

Purpose: Pain can hinder immunotherapy with anti-G(D2) monoclonal antibodies (MoAbs) like 3F8. Heat-modified 3F8 (HM3F8) lacks effector functions and could mask G(D2) or cross-reactive epitopes on nerves, thereby preventing a subsequent dose of unmodified 3F8 from activating pain fibers. We hypothesized that 3F8 dose escalation is possible without increased analgesic requirements in patients pretreated with HM3F8., Patients and Methods: Thirty patients with resistant neuroblastoma (NB) received one to two cycles of 3F8 plus granulocyte-macrophage colony-stimulating factor. 3F8 dosing began at 20 mg/m(2)/d and increased by 20 mg/m(2)/d in the absence of dose-limiting toxicity (DLT). Premedication included analgesics, antihistamines, and 5-minute infusions of HM3F8. On the basis of experience with 3F8 10 mg/m(2)/d in prior protocols, the DLT of pain was defined as more than seven doses of opioids administered within 2 hours. Opioid use was compared with a contemporary control group treated with 3F8 20 mg/m(2)/d but no HM3F8. Disease response was assessed., Results: Treatment was administered in the outpatient setting. Dose escalation stopped at 160 mg/m(2)/d because of drug supply limitations; even through this dosage level, analgesic requirements were similar to historical controls, and there were no DLTs. Analgesic requirements at 3F8 dosage levels through 80 mg/m(2)/d were significantly less compared with controls. Anti-NB activity occurred at all dosages., Conclusion: Multifold dose escalation of 3F8 is feasible. The findings can be interpreted as compatible with the possibility that HM3F8 can modify toxicity without blunting anti-NB activity. This pain control strategy may help achieve dose escalation with other anti-G(D2) MoAbs.

The aim of this retrospective study was to analyze the outcome and identify risk factors associated with progression-free survival (PFS) in 36 children with high-risk neuroblastoma who underwent autologous peripheral blood progenitor cell (PBPC) transplantation between 1994 and 2010. The conditioning regimen used in all cases consisted of high-dose of busulfan and melphalan. Median age at transplantation was 3 years (range: 0.7-14 years). The median times to neutrophil and platelet engraftment were 11 days (range: 9.16 days) and 13 days (range: 9.33), respectively. Twenty-one patients developed nonhematologic toxicity: 15 patients had mucositis, 4 patients developed an engraftment syndrome, and there were 2 cases of liver toxicity. No toxic deaths were observed. There were 15 patients who relapsed. The median time to relapse was 6 months after the transplant (range: 3-13 months). With a median follow-up of 55 months (range: 4-180 months), the PFS was 57% ± 8.5% for the whole group. In multivariate analysis, age below 3 years (P < .005), complete remission (CR) pretransplantation (P < .07) and 1p germline status (P < .01) were variables associated with better outcomes. Patients who were or achieved early CR following transplantation (3 months posttransplantation) had a probability of PFS of 91% ± 6% as compared to patients who did not (PFS 9% ± 8%) (P < .0001). This retrospective study shows that high dose of busulfan and melphalan as conditioning regimen in children with high-risk neuroblastoma is associated with very low morbidity and no mortality in the authors' hands. Younger patients with no 1p deletions and in first CR at transplantation had the better outcome.

Background: Understanding of immunobiology of bone marrow metastases (designated BM-NPC) versus primary tumors (P-NPC) of the nasopharynx is far from complete. The aim of this study was to determine if there would be differences between cultured P-NPCs and BM-NPCs with respect to (i) constitutive IL-6 and the IL-6 receptor gp80 subunit (IL-6Ralpha) levels in the spent media of nontransduced cells, and (ii) IL-6 and IL-6Ralpha levels in the spent media of cells transduced with a retroviral vector containing the IFN-gamma gene., Methods: A panel of NPC cell lines were transduced with the IFN-gamma gene through a retroviral vector. Four clonal sublines were isolated via limiting dilution methods. Cytofluorometric analysis was performed for the detection of cell surface antigens of HLA class I, HLA class II and ICAM-1. ELISA was used to assay for IFN-gamma, IL-6 and IL-6Ralpha in the spent media of cultured cell lines., Results: Our results showed that in day 3 culture supernatants, low levels of soluble IL-6 were detected in 5/5 cultured tumors derived from P-NPCs, while much higher constitutive levels of IL-6 were detected in 3/3 metastasis-derived NPC cell lines including one originated from ascites; the difference was significant (p = 0.025). An inverse relationship was found between IL-6Ralpha and IL-6 in their release levels in cultured P-NPCs and metastasis-derived NPCs. In IFN-gamma-transduced-P-NPCs, IL-6 production increased and yet IL-6Ralpha decreased substantially, as compared to nontransduced counterparts. At variance with P-NPC cells, the respective ongoing IL-6 and IL-6Ralpha release patterns of BM-NPC cells were not impeded as much following IFN-gamma transduction. These observations were confirmed by extended kinetic studies with representative NPC cell lines and clonal sublines. The latter observation with the clonal sublines also indicates that selection for high IL-6 or low IL-6Ralpha producing subpopulations did not occur as a result of IFN-gamma-transduction process. P-NPCs, which secreted constitutively only marginal levels of IFN-gamma (8.4 ~ 10.5 pg/ml), could be enhanced to produce higher levels of IFN-gamma (6.8- to 10.3-fold increase) after IFN-gamma transduction. Unlike P-NPCs, BM-NPCs spontaneously released IFN-gamma at moderate levels (83.8 ~ 100.7 pg/ml), which were enhanced by 1.3- to 2.2-fold in the spent media of their IFN-gamma-transduced counterparts., Conclusion: Our results showed that cultured P-NPCs and BM-NPCs could be distinguished from one another on the basis of their differential baseline secretion pattern of IFN-gamma, IL-6 and IL-6Ralpha, and their differential response profiles to IFN-gamma gene transfer of the production of these three soluble molecules. These results suggest that the IL-6 and IFN-gamma pathways in a background of genetic instability be involved in the acquisition of metastatic behaviour in BM-NPCs.

Lymphoplasmacytic lymphoma involving the bone marrow can be difficult to diagnose, and pathological features that predict the presence of associated Waldenström's macroglobulinemia have yet to be identified. To address these issues, marrow histology, immunohistochemistry, and flow cytometry were studied from 35 lymphoplasmacytic lymphoma cases that had comprehensive clinical assessment for Waldenström's macroglobulinemia. In all cases, the plasma cells were analyzed by a novel 6-color flow method. Both immunohistochemistry and flow cytometry were useful in identifying the lymphoid and plasmacytic disease components. In 19 cases, immunohistochemistry revealed an earlier unrecognized pattern of plasma cell infiltration in which they were physically separate from the lymphoid infiltrates. B-cell flow cytometry revealed monotypic cells in 96% of the cases. Approximately half of these were CD5 and/or CD23 positive, although none had features of chronic lymphocytic leukemia, and none of the B cells had flow cytometric features suggesting plasmacytic differentiation. In contrast, highly sensitive 6-color plasma cell flow cytometry revealed monotypic cells in 32 of the 35 cases; in 20 cases, the pattern of CD38 and CD138 coexpression detected was identical to that seen in plasma cell malignancies such as multiple myeloma. In 18 of these 20 lymphoplasmacytic lymphoma cases, these plasma cells were CD19 positive, distinguishing them from those of true plasma cell neoplasms, which are CD19 negative. It is interesting that the two lymphoplasmacytic lymphoma cases with CD19-negative plasma cells had an IgG isotype serum paraprotein. Apart from this, no other pathological correlates of the clinical or laboratory features of symptomatic Waldenström's macroglobulinemia were identified.

Purpose of Review: Cancer-associated fibroblasts/myofibroblasts and inflammatory cells produce a vast array of growth factors, chemokines and extracellular matrix (ECM) components that facilitate cancer progression, invasion/metastasis and neovascularization. This review highlights some surprisingly novel mechanisms of this paracrine relationship., Recent Findings: Mesenchymal stem/stromal cells (MSCs) are known for their tropism towards certain tumours, but now we find that cross-talk between tumours and MSCs leads to greater tumour motility and metastasis. Two closely related populations of immature myeloid cells, so-called 'cap cells' and myeloid-derived suppressor cells (MDSCs) also cross-talk with tumour cells, promoting invasion and metastasis through matrix metalloproteinase (MMP) secretion, as well as contributing to neovascularization and T-cell tolerance. The contribution of bone marrow-derived cells (BMDCs) to tumour neovascularization is controversial, but BMD--endothelial progenitor cells (EPCs)--are strongly implicated in the angiogenic switch in a mouse model. BMDCs are also credited with the creation of premetastatic niches to which metastatic cells adhere via integrins., Summary: There is no doubt that BMDCs are not simply bystanders in the tumour battleground. The mechanisms through which they aid tumour progression are numerous; effective treatments that combat BMDC-tumour cross-talk are surely on the way.

Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1beta, IL-6, and PGE(2) induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1(high)CD11b(high)F4/80(-)CD80(+)IL4Ralpha(+/-)Arginase(+) MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-kappaB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-kappaB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.

Allogeneic stem cell transplantation (allo-SCT) is used as curative therapy for malignant lymphoma, and reduced-intensity hematopoietic stem cell transplantation (RIST) is sometimes performed to avoid the toxicity and mortality associated with myeloablative allo-SCT. RIST is generally preferred for elderly patients with malignant lymphoma. A 62-year-old woman with follicular lymphoma (FL) involving bone marrow (BM) suffered relapse after autologous SCT. RIST was performed; cells were from an unrelated, fully human leukocyte antigen-matched donor. To study the hematopoietic reconstitution, BM biopsy specimens that were obtained at different times after RIST, were evaluated. Engraftment of donor cells was observed on days 19 and 48 after RIST, and residual FL in BM had completely disappeared by day 73 after RIST. This is the first report to document histological BM regeneration after RIST and disappearance of FL involving the BM.

We present a case of a 60-year-old male patient with primary bone marrow anaplastic large cell lymphoma. He was admitted to the hospital with the symptoms of anemia and fever. There was no evidence of lymphadenopathy or splenomegaly. Immunoelectrophoresis showed the presence of a triple M gradient (double IgM and an IgG), with the IgG and one of the IgM paraproteins functioning as a cryoglobulin. The patient had no hepatitis C virus infection. Bone marrow biopsy showed massive CD30-positive, ALK-negative large lymphoid cell infiltration of T-cell origin with anaplastic morphology. PCR analysis of lymphoid cells separated from the bone marrow demonstrated the presence of a B/T hybrid genotype disorder with no evidence of the t(2;5), nor t(1;2) translocations. The patient entered a period of remission following CHOP chemotherapy. The patient subsequently died of sepsis as a consequence of serious humoral immunodeficiency.

A panel of stable cell hybrids was generated by fusing a range of marrow-derived and solid tumour-derived human cell lines with the B-lymphoblastoid cell lines, HMy2 or KR4, and expression of immunologically relevant accessory and co-stimulatory molecules, and ability to stimulate allogeneic T-cell responses in vitro was investigated. Hybrid cell lines generated from three marrow-derived tumour cells consistently expressed both MHC class I and class II molecules, a range of accessory and T-cell co-stimulatory ligand molecules, including CD80 and CD86, and directly stimulated markedly enhanced T-cell proliferative responses in vitro, as compared with the parent tumour cell lines. The responses were blocked by addition of CTLA4-Ig fusion protein to the cultures, indicating a role of CD28/B7 interaction in induction of T-cell activation. By contrast, hybrid cells derived from three solid tumours only expressed MHC class II when the parent tumour cell line expressed MHC class II and consistently failed to express CD80 or CD86. These hybrid cells also stimulated greater T-cell proliferative responses in vitro than the parent tumour cell lines, although effective co-stimulation depended on the presence of responder non-T cells in the cultures. The expression of co-stimulatory ligand molecules and ability to directly stimulate strong allogeneic T-cell responses correlated with the EBV latency type of the hybrid cells. These data suggest that phenotypic and functional differences in fusion cells of professional antigen- presenting cells and tumour cells arise as a result of the parent tumour cell type.

There is general agreement that bone marrow (BM) examination for staging in patients with retinoblastoma should be limited to cases with advanced disease. However, there are limited data about the yield of sampling multiple sites with aspirations and biopsies and immunocytology. Our policy for BM examination included: 2 aspirates and 2 biopsies at the posterior iliac crest scheduled only for cases with postlaminar optic nerve extension (n=56), scleral invasion (n=10) or orbital (n=5) or metastatic disease at diagnosis (n=7) or at extraocular relapse (n=18). Immunocytology with the antibodies 3A7 or 3F8 for the ganglioside GD2 was performed. From 1/1994 to 3/2005, 277 newly diagnosed patients and 5 at extraocular relapse were included. BM invasion was not found in any of the 66 patients enucleated with disease confined to the globe, but was found in 11/27 of those with overt extraocular disease. There were 2/11 cases with at least 1 negative aspirate with positive biopsy and/or immunocytology for GD2. GD2 positivity was found in 9/9 cases. A more aggressive BM evaluation has a low yield in enucleated patients with high-risk features but disease limited to the globe. However, in cases with overt extraocular dissemination, the use of BM biopsy and immunocytology for GD2 allowed for the detection of cases that would have been missed by aspirations alone. GD2 was intensively expressed and it may also be used to monitor disease response and the presence of minimal residual disease.

Aim: To correlate the immunophenotype of metastatic cells in the bone marrow of patients with neuroblastoma with early treatment failure., Methods: The studies were performed on bone marrow material obtained from children treated in the department of paediatric oncology, haematology, and transplantology, Poznan University of Medical Sciences, Poland from 1996 to 2003. Immunocytochemical analysis of nervous tissue markers (using the immunomax technique) was performed on 108 bone marrow preparations obtained from 36 children diagnosed with neuroblastoma (stage IV with bone marrow metastases). The analysis included expression of PGP 9.5 protein, substance P, chromogranin A, bombesin, galanin, neuropeptide Y (NPY), and vasoactive intestinal peptide in neuroblastoma metastatic cells defined by the expression of neurone specific enolase., Results: Nineteen relapses occurred within 12 months of the end of treatment. Correlation between the various markers studied and early treatment failure, using Fisher's exact test, revealed that chromogranin A and NPY are strong indicators of an unfavourable prognosis in patients with stage IV neuroblastoma (p < 0.001 and p < 0.0002, respectively)., Conclusion: Determination of metastatic cell immunophenotypes in bone marrow (particularly chromogranin A and NPY) may help establish the short term prognosis in children with neuroblastoma.

Hairy-cell leukemia-variant (HCL-V) is a rare B-cell disorder which accountsfor 10% of HCL cases. The main features are splenomegaly, lymphocytosis and cytopenias without monocytopenia. The circulating cells have a morphology intermediate between prolymphocytes and hairy cells. The immunophenotype shows a mature B-cell phenotype with expression of B-cell antigens CD11c and CD103 but unlike typical hairy cell the cells are negative for CD25. The histology of bone marrow and spleen shows a pattern of infiltration similar to that in HCL. We present a case of HCL-V in a 66-year-old male. The bone marrow findings, immunophenotypic profile and electron microscopic features are described. The patient underwent splenectomy which also revealed infiltration by leukemia. Patients are resistant to alkylating agents and alpha-interferon (á-IFN). Splenectomy may be beneficial for long-lasting partial responses in some of the patients and is a good palliative treatment.

A comparison of flow cytometry (FC) and bone marrow biopsy (BMB) to evaluate bone marrow infiltration was made in 114 patients suffering from B-cell non-Hodgkin's lymphomas (NHLs; 51 at diagnosis, 63 during post-therapy follow-up). The following parameters were indicative of bone marrow infiltration: altered surface k/l ratio; specific immunophenotypic pattern in particular NHLs (CLL, mantle cell lymphoma, hairy cell leukemia). FC and BMB agreed in 89.5% of cases (i.e. both showed 48 positive and 54 negative cases). In discordant cases (7.9%) and in cases not evaluable by FC (2.6%) IgH rearrangement and bcl-1 gene expression, both evaluated by PCR methods, were used to detect bone marrow infiltration with higher precision. These results show that a more complex analysis of bone marrow is needed to diagnose bone marrow infiltration, particularly in samples with minimal residual disease.

Cytokines produced by T lymphocytes are critical to the efficacy of a given immune response and dysregulation of immune responses may play a role in cancer progression. We assessed the intracellular cytokine profiles of T cells in the peripheral blood of women with breast cancer and explored the relationship of these responses with the presence of cancer in lymph nodes and bone marrow. Peripheral blood lymphocytes from 84 patients and 26 healthy volunteers were analyzed by 4-color flow cytometry for surface markers and for intracellular cytokines. Bone marrow samples from some of these patients were also collected and analyzed for the presence of epithelial cells (micrometastases) by flow cytometry. The percentages of both CD4(+) and CD8(+) cells producing type1 (IL-2, IFN-gamma or TNF-alpha) and type 2 (IL-4) were significantly lower in patients with breast cancer compared to healthy controls. These results indicate a general immune dysfunction in these patients as opposed to a shift in the balance of type1 and type2 cells. These dysregulated T cell responses did not correlate with age, stage of disease, or nodal status. However, we did observe a correlation between number of micrometastases in the bone marrow and T cell responsiveness.

Natural killer (NK) cell lymphomas are rare, and atypical features might lead to diagnostic pitfalls. This report describes an unusual patient in whom lymphoma occurred initially as isolated lymph node involvement, an exceptional presentation of an almost exclusively extranodal disease. Furthermore, during the terminal haemophagocytosis in the bone marrow, lymphoma cells lost the expression of the NK cell marker, CD56, making the histopathological diagnosis of bone marrow involvement difficult. This was resolved by in situ hybridisation for Epstein-Barr virus encoded small RNA, which detected occult bone marrow infiltration.

The role of natural killer (NK) cells in multiple myeloma is not fully understood. Here, NK susceptibility of myeloma cells derived from distinct disease stages was evaluated in relation to major histocompatibility complex (MHC) class I, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), and UL16 binding protein (ULBP) expression. MHC class I molecules were hardly detectable on bone marrow cells of early-stage myeloma, while late-stage pleural effusion-derived cell lines showed a strong MHC class I expression. Conversely, a high MICA level was found on bone marrow myeloma cells, while it was low or not measurable on pleural effusion myeloma cells. The reciprocal surface expression of these molecules on bone marrow- and pleural effusion-derived cell was confirmed at mRNA levels. While bone marrow-derived myeloma cells were readily recognized by NK cells, pleural effusion-derived lines were resistant. NK protection of pleural effusion cells was MHC class I dependent. Receptor blocking experiments demonstrated that natural cytotoxicity receptor (NCR) and NK receptor member D of the lectin-like receptor family (NKG2D) were the key NK activating receptors for bone marrow-derived myeloma cell recognition. In ex vivo experiments patient's autologous fresh NK cells recognized bone marrow-derived myeloma cells. Our data support the hypothesis that NK cell cytotoxicity could sculpture myeloma and represents an important immune effector mechanism in controlling its intramedullary stages.

The aim of this study was to describe the presenting features, the frequency and outcome of myeloid sarcoma (MS) diagnosed in our Institution from January 1995 to December 2000. Twelve MS were seen and the frequency account for only 2% of all acute myeloid leukemia (AML) patients observed in our department in the same period. Median age was 45 years (range: 4-84). All had been initially misdiagnosed as malignant lymphoma (ML) and a median of 2.9 months (range: 1-6) elapsed between the misdiagnosis and the correct of MS, effectuated in our department. At that time, a bone marrow examination revealed a myelodysplastic condition in seven patients, an infiltration by blast cells >30% in two patients, and normal features in the other three. In the non-leukemic patients a median of 5 months (range: 2-44 months) elapsed between the diagnosis of MS and acute leukemia. In all, 10 patients received intensive treatment. A total of seven patients (70%) achieved MS complete remission (CR). Patients who presented isolated skin localization and received only radiotherapy, obtained a MS-CR, but subsequently developed AML. Only in patients who were treated within 4 months from the initial ML diagnosis we achieved complete remission of both MS and leukemia, whereas in patients who were treated after this time, we obtained a complete disappearance of MS without response at the bone-marrow level, irrespectively of the specific therapy regimen. Median survival time from MS diagnosis was 7 months (range: 1-49 months), and only one patient is still alive, 49 months after bone marrow transplantation. Our data stress the importance of an accurate and prompt identification of this rare form of AML, and suggest that, even in patients with isolated MS, the early administration of AML-like intensive chemotherapy followed by bone marrow transplantation might reduce the risk of subsequently developing systemic disease.

Background: The efficient detection and characterization of micrometastatic cells in the bone marrow of patients with breast carcinoma are of prognostic and therapeutic importance. The technique used must overcome the challenges that result from the small number of target cells (1 per 1 million hematopoietic cells) and the heterogeneous expression of micrometastatic cell markers. In this study, the authors assessed and improved the current methods for purifying and characterizing rare disseminated carcinoma cells., Methods: The authors developed a single-step assay that does not require density-gradient separation. This assay can be performed directly on crude human bone marrow aspirates and is based on the use of immunomagnetic beads coated with an antibody that recognizes an epithelial cell-surface epitope, the epithelial cell adhesion molecule (EpCAM). To determine the specificity of the assay, the authors evaluated bone marrow specimens from 46 control patients., Results: The novel method was highly reproducible and was capable of detecting as few as 10 carcinoma cells among 50 million hematopoietic cells. The yield was nearly 100%, with only 0.01% nonspecific cell draining. The authors found that 68 +/- 51 cells were trapped per 50 million cells in control crude aspirates and that density-gradient separation increased this number by 2-fold to 29-fold. These trapped cells expressed EpCAM, represented 1.4 x 10(-4) % of the sample, and were characterized as of hematopoietic cell origin (CD45 positive) or progenitor cell origin (CD34 positive)., Conclusions: The authors developed a highly efficient and reproducible, single-step immunomagnetic assay that may be performed directly on crude human bone marrow aspirates. The authors believe the current study is the first to demonstrate that some rare bone marrow cells (CD45-positive or CD34-positive cells) may express EpCAM and, to some extent, may contaminate the purified micrometastatic cell fraction. Thus, a universal marker for micrometastatic cells remains to be discovered., (Copyright 2004 American Cancer Society.)

Aims: To investigate CD27 expression in splenic marginal zone lymphoma (SMZL), an indolent low-grade B-cell lymphoma with constant involvement of the bone marrow, especially with an intrasinusoidal pattern. It is not clear if the neoplastic clone is composed of virgin or somatically mutated B cells. CD27 is reported to be a hallmark of memory B cells., Methods and Results: We evaluated 64 bone marrow biopsy specimens (BMBs) from 36 patients with SMZL for the expression of CD27. For comparison, splenectomy specimens of patients with traumatic splenic rupture or with SMZL were used. All BMBs showed lymphomatous infiltration. When located in the marrow sinusoids, neoplastic cells were CD27- in all cases and therefore corresponded to naive B cells. In nodular/interstitial infiltration, the cells were CD27+ and therefore corresponded to memory B cells. No difference in immunohistochemical expression of B and T antibodies was found between intrasinusoidal and interstitial/nodular infiltration. CD27 was constantly expressed in the splenic marginal zone of normal spleen, surgically removed for trauma, and in seven out of 10 spleens with SMZL., Conclusion: We propose the existence of two different phases of neoplastic progression with, first, expansion of a virgin B clone in the bone marrow and, following exposure to antigen, a re-colonization of the bone marrow.

Diffuse large B cell lymphoma (DLBL) comprises a heterogenous entity characterized by the presence of large cells, exhibiting a mature B cell phenotype. The high proliferation rate and aggressive disease remain a therapeutic challenge, but the apparent biological diversity permits a risk-stratification model for prognostic grouping through the International Prognostic Index (IPI). Empirical to this approach is the consideration of cytogenetic data, offering an insight into the pathogenetic events which may underlie neoplastic clonal evolution and disease progression. We describe three cases of DLBL presenting with isolated marrow disease, a rare primary finding in this lymphoma. All three cases showed involvement of blood and bone marrow without evidence of splenic or lymph node involvement on imaging studies. Histological and immunophenotypic findings were similar in all three cases, outlining the phenotypic maturity of this disease. Cytogenetic analysis revealed complex karyotypes in the two cases examined. M-FISH (multicolour fluorescent in situ hybridization) performed on bone marrow from case 1 showed several cryptic translocations not evident on G-banding, including a novel translocation between 2p and 9p, and an unbalanced translocation between 14q and 11q. Cytogenetic analysis in case 2 showed abnormalities involving 7q, 9p at the site of the INK4a gene, and the bcl-2 locus, findings confirmed by M-FISH. These cases serve to highlight the biological and cytogenetic heterogenity of DLBL and emphasize the need for complementary investigations in the characterization of this entity.

This is a case report of rosette formation in non-Hodgkin's lymphoma in the bone marrow. A 66 year old female on treatment for metastatic papillary carcinoma thyroid and non-Hodgkin's lymphoma of the nodular small cleaved cell type was found to have bone marrow infiltration by a low grade B cell lymphoma. Bone marrow aspirate, imprint and trephine biopsy showed rosettes of the abnormal lymphoid cells. The associations of papillary carcinoma thyroid, hyperglobulinemia and retroperitoneal fibrosis in this case which explain the histogenesis of rosette formation are discussed.

The bone marrow (BM) is a frequent site of involvement in non-Hodgkińs lymphomas (NHL) and evidence of an infiltrated BM may implicate different therapeutical regimens. Flow cytometric immunophenotyping of bone marrow aspirates now is included in the assessment of patients with NHL and used as an adjunct to morphologic evaluation in the staging of lymphoma. The aim of the study was to compare flow cytometric immunophenotyping of BM and paraffin section staining of BM biopsies in the marrow involvement of NHL. Cytometric immunophenotyping of bone marrow and immunohistochemical paraffin section staining of bone marrow biopsies in 53 B- and T-cell lymphoma patients were performed. We used the following fluorochrom conjugated monoclonal antibodies specific for: CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, CD22, CD23, CD79B, FMC7 and Ig kappagamma light chain. Unilateral BM trephine biopsies were obtained in all cases, fixed, decalcified and paraffin-embedded. Morphologic marrow involvement by lymphoma was found in 24 cases; flow immunophenotyping identified 26 cases with NHL: morphology-positive/flow-positive (n=21), morphology positive/flow-negative (n=3), morphology-negative/flow-positive (n=4), and morphology-negative/flow-negative (n=23). The concurrence rate of BM trephine biopsy and flow cytometric immunophenotyping in evaluation of NHL bone marrow infiltration was 88.7%. Immunophenotyping of the bone marrow of NHL patients by flow cytometry is helpful for assessment of bone marrow infiltration, especially in B-cell disorders. Both trephine biopsies and flow cytometry are better than single investigation for detection of infiltration in NHL.

Although squamous cell carcinoma of the head and neck region very rarely metastasize to the skeleton, epithelial cells have been found in bone marrow aspirates of these patients. This observation reflects the general spread of the disease, indicating a poor clinical prognosis with a much higher risk of developing local or distant recurrences. In a first attempt to characterize the phenotypic properties, the expression of the major histo-compatibility complex (MHC) class I antigens on bone marrow micrometastases was assessed. It has been shown that the down-regulation of these molecules is a potential mechanism to escape from HLA class I restricted lysis by cytotoxic T-cells. The significance of reduced MHC class I expression might be relevant for the survival of residual metastatic cells in the bone marrow of patients with squamous cell carcinoma of the head and neck region. Bone marrow aspirates were screened for individual disseminated epithelial cells using the immunoalkaline phosphatase technique with monoclonal antibodies to the epithelial differentiation marker cytokeratin 19 (CK19), as described previously. Specimens containing CK19-positive cells were colabelled with the monoclonal antibody W6/32. The loss of MHC expression is not related to the tumor stage but clearly to the degree of differentiation: 6 out of 7 patients with low-grade SCCHN, but only 3 out of 13 patients with medium-grade SCCHN showed a complete loss of MHC class I molecules. This finding could indicate the reduced prognosis of undifferentiated SCCHN. The lack of MHC class I expression could encourage the survival of residual tumor cells in the bone marrow of patients with SCCHN that evade immunosurveillance.

Aims: Thirty per cent of breast cancer patients with axillary lymph node negative at primary surgery will relapse within 10 years. This may be caused by disseminated tumour cells from the primary tumour. This study report the phenotypic profiles of disseminated tumour cells and microenvironmental characteristics in bone marrow of breast cancer., Methods: We detected the biologic markers on the disseminated tumour cells with immunocytochemical staining, analysed the immunological changes through flow-cytometry, and investigated the u- PA activity in the plasma of bone marrow., Results: With the immunocytochemical staining of EMA and CK19, we detected micrometastasis in thirty out of 72 (41.67%) breast cancer patients. Compared with the primary tumours, disseminated tumour cells expressed low protein cyclin D1, P53, Ki-67, EGFR, and high protein P21. The percentage of memory CD4+ T cells was significantly higher in the micrometastasis-positive group than in the micrometastasis-negative group. Tumour size and axillary lymph node status were found to be significantly correlated with the u- PA activity level., Conclusions: Immunophenotypic profiles of disseminated tumour cells could be measured by immunocytochemical staining and microenvironment can be analysed by flow cytometry., (Copyright 2002 Elsevier Science Ltd.)

We used immunohistochemical analysis to investigate 146 bone marrow (BM) clot specimens from patients with fever of unknown origin (FUO) (n = 124) or hemophagocytic syndrome (HPS) (n = 22). Intravascular lymphoma (IVL) primarily involving BM was detected in 12 (8.2%) of the 146 patients. Diagnosis was based on the presence of CD20+ and CD79a+ tumor cells confined within the lumina of sinuses and surrounded by CD34+ endothelial cells. Of the 12 IVL cases, 6 were CD5+; of these 6 CD5+ cases, 5 were positive for vimentin. The finding of a considerably high prevalence of IVL tumor cells in BM from patients with FUO or HPS suggests that immunohistochemical examination of BM may be helpful in the diagnosis of IVL in these patients. Vimentin coexpression in CD5+ IVL might be evidence of origin from a subset of prefollicular B-cells.

Mice deficient for the STAT6 gene (STAT6(-/-) mice) have enhanced immunosurveillance against primary and metastatic tumors. Because STAT6 is a downstream effector of the IL-4R, and IL-13 binds to the type 2 IL-4R, IL-13 has been proposed as an inhibitor that blocks differentiation of tumor-specific CD8(+) T cells. Immunity in STAT6(-/-) mice is unusually effective in that 45-80% of STAT6(-/-) mice with established, spontaneous metastatic 4T1 mammary carcinoma, whose primary tumors are surgically excised, survive indefinitely, as compared with <10% of STAT(+/+) (BALB/c) mice. Surprisingly, STAT6(-/-) and BALB/c reciprocal bone marrow chimeras do not have increased immunosurveillance, demonstrating that immunity requires STAT6(-/-) hemopoietic and nonhemopoietic components. Likewise, CD1(-/-) mice that are NKT deficient and therefore IL-13 deficient also have heightened tumor immunity. However, STAT6(-/-) and CD1(-/-) reciprocal bone marrow chimeras do not have increased survival, suggesting that immunity in STAT6(-/-) and CD1(-/-) mice is via noncomplementing mechanisms. Metastatic disease is not reduced in BALB/c mice treated with an IL-13 inhibitor, indicating that IL-13 alone is insufficient for negative regulation of 4T1 immunity. Likewise, in vivo depletion of CD4(+)CD25(+) T cells in BALB/c mice does not increase survival, demonstrating that CD4(+)CD25(+) cells do not regulate immunity. Cytokine production and tumor challenges into STAT6(-/-)IFN-gamma(-/-) mice indicate that IFN-gamma is essential for immunity. Therefore, immunosurveillance in STAT6(-/-) mice facilitates survival against metastatic cancer via an IFN-gamma-dependent mechanism involving hemopoietic and nonhemopoietic derived cells, and is not exclusively dependent on counteracting IL-13 or CD4(+)CD25(+) T cells.

Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5(-) "immature" plasma cells showed the highest levels of CD126 expression, but "mature" VLA-5(+) myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS. (Blood. 2000;96:3880-3886)

STAT4 and STAT6 are essential for the development of CD4(+) Th1 and Th2 development, respectively. Tumor immunologists have hypothesized that Th1 cells are critical in tumor immunity because they facilitate differentiation of CD8(+) T cells, which are potent anti-tumor effectors. We have used STAT4(-/-) and STAT6(-/-) mice to test this hypothesis. BALB/c and knockout mice were challenged in the mammary gland with the highly malignant and spontaneously metastatic BALB/c-derived 4T1 mammary carcinoma. Primary tumor growth and metastatic disease are reduced in STAT6(-/-) mice relative to BALB/c and STAT4(-/-) mice. Ab depletions demonstrate that the effect is mediated by CD8(+) T cells, and immunized STAT6(-/-) mice have higher levels of 4T1-specific CTL than BALB/c or STAT4(-/-) mice. Surprisingly, Th1 or Th2 cells are not involved, because CD4 depletion does not diminish the anti-tumor effect. Therefore, deletion of the STAT6 gene facilitates development of potent anti-tumor immunity via a CD4(+)-independent pathway.

Purpose: To improve autologous leukapheresis strategies in high-risk neuroblastoma (NB) patients with extensive bone marrow involvement at diagnosis., Patients and Methods: Anti-G(D2) immunocytochemistry (sensitivity, 1 in 10(5) to 10(6) leukocytes) was used to evaluate blood and bone marrow disease at diagnosis and during the recovery phase of the first six chemotherapy cycles in 57 patients with stage 4 NB and bone marrow disease at diagnosis. A total of 42 leukapheresis samples from the same patients were evaluated with immunocytology, and in 24 of these patients, an anti-G(D2) immunomagnetic enrichment step was used to enhance tumor-cell detection., Results: Tumor cytoreduction was much faster in blood compared with bone marrow (3.2 logs after the first cycle and 2.1 logs after the first two cycles, respectively). Bone marrow disease was often detectable throughout induction, with a trend to plateau after the fourth cycle. By direct anti-G(D2) immunocytology, a positive leukapheresis sample was obtained in 7% of patients after either the fifth or sixth cycle; when NB cell immunomagnetic enrichment was applied, 25% of patients had a positive leukapheresis sample (sensitivity, 1 in 10(7) to 10(8) leukocytes)., Conclusion: Standard chemotherapy seems to deliver most of its in vivo purging effect within the first four cycles. In patients with overt marrow disease at diagnosis, postponing hematopoietic stem-cell collection beyond this point may not be justified. Tumor-cell clearance in blood seems to be quite rapid, and earlier collections via peripheral-blood leukapheresis might be feasible. Immunomagnetically enhanced NB cell detection can be highly sensitive and can indicate whether ex vivo purging should be considered.

In view of the high incidence of early distant tumor relapses in apparently completely resected (R0, M0) non-small cell lung cancer (NSCLC), there is a need for an adjuvant therapy. Considering the low tumor burden in these patients, an adjuvant therapy with monoclonal antibodies (i.e., the 17-1A MAb) might be appropriate. The purpose of our study was to test whether the 17-1A antigen is expressed on primary and metastatic NSCLC carcinoma cells. Using immunohistochemistry, the expression of 17-1A was analysed in primary tumors (n = 60) and in lymph node metastases (n = 7) of patients with NSCLC. Additionally, we investigated in 6 patients the expression of 17-1A on disseminated tumor cells in the bone marrow, which were detected by the pan-cytokeratin MAb A45-B/B3 using a double-labeling technique. The 17-1A antigen was homogeneously expressed in 47 (78.3%) out of 60 primary NSCLCs. The expression of 17-1A was independent from the tumor histology, the grade of differentiation, and other clinicopathological parameters (ploidy status, TNM-stage). Lymph node metastases were positive in 4 (57.4%) out of 7 cases. The double-labeling experiments demonstrated that 17-1A is coexpressed on disseminated tumor cells in the bone marrow in 5 (83%) out of 6 patients. The 17-1A antigen is expressed on the majority of primary, metastatic, and disseminated NSCLC cells. Patients with 17-1A-positive tumors might benefit from an adjuvant therapy with MAb 17-1A after completely resected NSCLC., (Copyright 2000 Wiley-Liss, Inc.)

We describe herein a case of CD56+ natural killer cell lymphoma (NKCL) that showed unusual clinical manifestations and a fulminant course of disease. A 70-year-old male patient presented with rapidly growing skin lesions and fever; a nasal lymphoma was subsequently detected and bone marrow involvement developed within a few weeks. Although the time relationship is not clear, bone marrow involvement could be dissemination from localized disease of nasal and nasal type NKCL. As seen in our case, the course can be excessively aggressive and fulminant even when it first appeared as a localized disease, and CD56 positivity, which is a specific characteristic of NKCL, may serve as a factor showing a poor prognosis for a malignant lymphoma.

Intravascular lymphoma is a rare, often fatal disease characterized by a widespread intravascular proliferation of neoplastic lymphoid cells. Dermatological and bizarre neurological manifestations usually predominate. We report a case of intravascular lymphomatosis with an exceptional clinical presentation showing splenomegaly combined with early bone marrow involvement. The diagnosis was made on bone marrow biopsy examination using both immunohistochemistry and molecular biology analysis. We stress the histopathological features of bone marrow involvement by intravascular lymphoma which allow the prompt recognition of this disease. Early systemic chemotherapy, which represents the only chance of remission in such an aggressive disease, can then be initiated.

The immunocytological detection of disseminated epithelial cells in bone marrow in patients with breast cancer has been performed at many hospitals and institutes since the early 1980s. Despite numerous publications in this field, it has not been possible to standardize the method and establish the 'ideal' antibody, either nationally or internationally. Molecular biological methods using PCR technology could extend the diagnostic spectrum. However, one of the major problems in breast cancer is the lack of a disease-specific marker gene. As a result, immunocytology is still the standard procedure for tumour cell detection. The detection of disseminated single cells in bone marrow in primary breast cancer (also known as minimal residual disease) is a new prognostic factor for disease-free and overall survival. This has been demonstrated in two large (N > 300) groups and several small to medium groups (N = 50-300). As a marker of dissemination in a target organ for metastasis this prognostic factor corresponds much more closely to the tendency of breast cancer to early haematogenic spread. Tumour cell detection may predict the course of the disease better than the axillary lymph node status. Bone marrow aspiration and detection of disseminated cells might replace lymph node dissection, at least in those patients with small tumours and no clinical signs of lymph node involvement. This strategy will soon be investigated in appropriate studies. Another possible clinical use might be in deciding on whether or not to give adjuvant systemic therapy to node-negative patients. Patients with positive tumour cell detection are at a higher risk of subsequent metastasis, even if the axillary nodes are histologically normal. The immunohistological or molecular biological detection of tumour cells in axillary lymph nodes might also be very useful, now that it has been shown that a considerable subset of patients determined to be node-negative by means of conventional methods, are positive according to these new techniques. These methods could be a useful supplement to sentinel node biopsy. A further potential use of this method is in monitoring therapy with new treatment modalities such as gene therapy and immunotherapy. Repeated bone marrow aspiration can provide information on the success of therapy in minimal residual disease (cytoreduction). Immunocytochemical investigation of individual cells may be useful in studying the pathogenesis of metastasis, in particular in the skeleton. Phenotyping of cells might allow statements to be made on the metastatic potential of cells and the question of cell dormancy. It remains to be hoped that this aspect of minimal residual disease will be granted more attention in future.