Multiple myeloma is an incurable malignant disease of clonal plasma cells which infiltrate in the bone marrow. The introduction of several novel drugs including immunomodulatory drugs and proteasome inhibitors, and high-dose therapy followed by autologous stem cell transplantation has doubled overall survival times. During the last decade, considerable progress has been also made in understanding the biology of myeloma and molecular heterogeneity which influences the prognoses. Along with previously described prognostic factors, gene expression profiling allows the assessment of prospective risk stratification of myeloma patients. To choose the optimal treatment for each patient, risk-adapted strategies using well-defined prognostic factors are urgently needed. Many clinical studies about the efficacy of novel therapies showed the achievement of complete response (CR) is associated with prolonged progression-free survival. However, the long-term outcome of myeloma patients after therapy is still heterogenous. Some patients lose CR within three years while a small fraction of patients (5-15 %) remain recurrence-free for over 10 years. Thus, more sensitive techniques for assessment of response and tumor burden after treatment would be helpful. In this article, we review the utility of multiparametric flow cytometry for evaluation of prognostic markers and of monitoring minimal residual disease in multiple myeloma.
In childhood acute lymphoblastic leukemia (ALL), the clinical significance of minimal residual disease (MRD) has been demonstrated. Polymerase chain reaction assays based on the detection of clonal rearrangement of immunoglobulin and T-cell receptor genes are most established method for MRD analysis. However, the flow cytometric detection of MRD also has advantages in simplicity of the method as well as a low-cost, high-performance. Conventionally, 3 or 4 color analysis employing multiple test tubes of the panel of antibodies is used for the detection of leukemia-associated immunophenotypes, whereas we developed 10 color methods of MRD detection for both BCP-ALL and T-ALL. Our methods are able to take effect easily and exhibit high performance. To put it to practical use, further investigations are now underway.
Histopathological examination is considered as a gold standard for diagnosing of malignant lymphoma. However, it takes several days to make a histopathological diagnosis. Cell surface marker analysis using flow cytometry (FCM) is helpful for more rapid and accurate diagnosis. FCM has advantages, such as swiftness, objectivity, and multiple staining in the same cell. In particular, FCM in body cavity fluids is useful in diagnoses of pleural, peritoneal and central nervous system (CNS) infiltration of malignant lymphoma. CD45 gating FCM has several ingenuities in the measurement of tissues and body cavity fluids. Using CD45 gating FCM, we can remove debris and choose appropriate cell population refer to cell morphology of the stamp and cytospin smear. If the sample contains small amount cells, selecting the measurement items mainly B-cell associated antigen may help diagnose B-cell malignancies. The light chain restriction is one of the most important findings in diagnosis of B-cell lymphoma. FCM is helpful in differential diagnosis of plasma cell disorders. Furthermore, in differential diagnosis of cerebrospinal fluid pleocytosis, increased proportion of B-cells in the sample suggests CNS infiltration of B-cell malignancies. In addition to FCM, fluorescence in situ hybridization (FISH) and gene rearrangement may provide more information on diagnosis. In conclusion, CD45 gating FCM in tissues and body cavity fluids could increase the sensitivity for diagnosis of malignant lymphoma. The histopathological examination in combination with FCM, FISH, and gene rearrangement leads more accurate diagnosis.
Adult T-cell leukemia (ATL) cells carry the HTLV-1 provirus in the same genomic site in each case, indicating that provirus insertion, demonstrated by Southern blot hybridization (SBH), is an excellent biomarker for the cellular clonality. However, it is unavailable for samples including small clones with 5% or fewer monoclonal cell populations. In this study, we analyzed cell-surface markers of 313 ATL cases (smoldering type 74cases, chronic type 60 cases, lymphoma type 27cases, and acute type 152 cases) and 70 cases of HTLV-1 carrier at our hospital between March, 1997 and March, 2012. Typical ATL cells indicated significantly higher levels of CD4, CCR4 and CD25 and down-regulation of CD3, CD7 and CD26. These markers were useful for clinical diagnosis and classification for ATL sub-types. CD38 was also useful to the differential diagnosis of acute and chronic types. To better understand indeterminate HTLV-1 carriers and smoldering ATL, cell-surface markers, SBH, proviral load (PVL), and ATL-related biomarkers were examined focusing on carriers and smoldering cases. We found that the antigen modulation rates of CD26 and CD7 and the increasing rate of CD25 and CCR4 cells were closely correlated to clonal size. In particular, the ratio of CD26/CD25 had a predictive detection of clonal band. Conclusively, CD26 plays a central role in the evolution from early to overt smoldering ATL.
Accumulating evidence suggests that cancer stem cells—which make up only a small proportion of heterogeneous tumor cells—possess a greater ability to maintain tumor formation than other tumor cell types. Although the concept of cancer stem cells greatly impacts cancer biology and evokes a reconsideration of cancer treatment, the molecular mechanisms involved in the contribution of cancer stem cells to tumorigenesis remain to be obscure. We found that heregulin (HRG), a ligand for HER3/ErbB3, induced tumor sphere formation of a breast cancer stem cell (BCSC)-enriched population as well as in breast cancer cell lines. HRG-induced tumor sphere formation was reduced by treatment with inhibitors for phosphatidyl inositol 3-kinase (PI3K). Furthermore, we found that the expression of IL8, a regulator of self-renewal in BCSC-enriched populations, was induced by HRG through the activation of the PI3K/NF-κB pathway. These findings illustrate that HRG/ErbB3 signaling appears to maintain tumor sphere formation through a PI3K/NF-κB pathway in human breast cancer.
Severe combined immunodeficiency (SCID) is a fatal syndrome of diverse genetic causes characterized by profound deficiencies of T cell and B cell function. In the absence of therapy, the lack of adaptive immunity results in overwhelming infections and death within the first year of life. Hematopoietic stem cell transplantation (HSCT) is a curative treatment for SCID. Although SCID patients are deficient in T cells, graft failure and prolonged mixed chimerism have occurred in some cases with SCID after HSCT. To explore the mechanisms that underlie these pathogenic conditions, we analyzed the chimerism of each leukocyte subset in two cases with SCID after HLA-mismatched cord blood transplantation (CBT) using flow cytometry, HLA-Flow. We found that the mixed chimerism in myeloid cells persisted for several years after CBT in both cases. In addition, the chimerism and the colony-forming ability were recipient dominant in the hematopoietic stem/progenitor cells (HSPC) of the patient two months after CBT. It showed that the replacement of recipient-derived HSPC by donor-derived ones had not been sufficient. The development of recipient-derived memory B cells in SCID patient with T−B＋NK− phenotype was skewed compared with donor-derived ones. These data suggest that the necessity of long-term support of intravenous immunoglobulin in SCID after HSCT is concerned with the insufficient replacement of HSC. HLA-Flow was very useful to investigate the pathophysiology of the immunological disorders after HSCT in SCID.