High incidence and early onset of nivolumab-induced pneumonitis: four case reports and literature review.

BACKGROUND: Nivolumab, an anti-programmed cell death-1 (PD-1) monoclonal antibody used as an immune checkpoint inhibitor, is commonly employed for its anti-tumor effects against various types of malignant tumors. However, its administration is complicated by immune-related adverse events (irAEs), including pneumonitis. CASE PRESENTATION: We present a case series of four patients with malignant melanoma, non-small cell lung cancer, and hypopharyngeal carcinoma who demonstrated pneumonitis induced by nivolumab, and further review clinicopathological characteristics of these patients in comparison with those of previously reported patients with nivolumab-induced pneumonitis. In our series, 20% of patients who were treated with nivolumab developed pneumonitis, all of which occurred approximately 2 weeks after the initiation of nivolumab treatment. Prompt recognition of the nivolumab-induced pneumonitis allowed for successful resolution. Computed tomography scan images of the patients demonstrated predominantly cryptogenic organizing pneumonia patterns. All patients were males, who had been heavily treated with antitumor drugs prior to nivolumab. CONCLUSIONS: Our case series showed that nivolumab had a high incidence of drug-induced pneumonitis with early onset, supporting the need for renewed attention to nivolumab-induced pneumonitis, particularly in patients with a history of heavy antitumor treatments.

Cytotoxic lymphocytes in the peripheral blood of patients with B cell lymphomas.

In order to analyze systemic immune surveillance in patients with B cell non-Hodgkin's lymphomas (B-NHL), we investigated circulating lymphocytes using two-color flow cytometry. The proportions of CD3-CD56+ natural killer (NK) cells and CD8++(bright) S6F1++ killer-effector T cells corresponding to activated cytotoxic T lymphocytes (aCTL) were studied in the peripheral blood of 26 patients with indolent lymphoma (IL) and 24 with aggressive lymphoma (AL). The AL patients with both limited disease and advanced disease had an increased proportion of NK cells. However, this feature was not evident in IL patients with either limited or advanced disease. In contrast, an increased proportion of aCTL was observed only in IL patients with advanced disease. These findings indicate that IL may differ from AL in terms of immune surveillance against neoplastic B cells.

Spontaneous reduction of leukemic lymphoma cells possibly by anti-tumor antibody-mediated phagocytosis; a kappa lambda-dual-positive B cell lymphoma.

We have investigated the possible role of anti-tumor antibody detected in a case of follicular lymphoma which demonstrated the spontaneous reduction of leukemic tumor cells. The tumor cells genotypically had monoclonal rearrangements of the immunoglobulin J H and C kappa genes, but phenotypically exhibited surface IgG, A, kappa and lambda (kappa lambda dual positivity). The culture study revealed that IgGlambda, at least, was derived from the serum, and IgAkappa was expressed intrinsically. Furthermore, the positive correlation between the densities of both surface light chains on two-color flow cytometry, the rosette formation study and its inhibition test by the Fcgamma fragment suggested that the serum IgGlambda combined with some antigens on the tumor-cell surface via its Fab portion and with the Fcgamma receptor of macrophages via its Fc portion. From these findings, we regarded the present case as an anti-tumor antibody-coated lymphoma. In addition, the phagocytic study disclosed that the serum-derived IgGlambda, at least, might have induced the phagocytosis of circulating lymphoma cells by macrophages. In conclusion, the existence of the anti-tumor antibody-coated lymphoma may be helpful in clarifying the immunological mechanism of the spontaneous regression occasionally seen in lymphomas.

Elevated plasma level of differentiation inhibitory factor nm23-H1 protein correlates with risk factors for myelodysplastic syndrome.

We measured plasma nm23-H1 level (nm23-H1), a differentiation inhibitory factor, by an enzyme-linked immunosorbent assay (ELISA) in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The nm23-H1 in AA was not significantly elevated when compared to normal subjects (6.66 +/- 1.20 ng/ml vs 5.13 +/- 0.81 ng/ml; P = 0.274). In contrast, MDS patients had significantly high levels of nm23-H1 compared not only to normal subjects (11.16 +/- 1.42 vs 5.13 +/- 0.81 ng/ml; P = 0.0004) but also to those of the AA group (11.16 +/- 1.42 ng/ml vs 6.66 +/- 1.20 ng/ml; P = 0.018). In the MDS group of patients, no significant difference was observed in the nm23-H1 levels between patients with refractory anemia (RA) and RA with excess blasts (RAEB)/RAEB in transformation (10.71 +/- 1.61 ng/ml vs 9.24 +/- 2.66 ng/ml; P = 0.672). Of the patients with RA, patients with low risk according to the International Prognostic Scoring System (IPSS) had significantly low levels of nm23-H1 compared to those of IPSS INT-1 level cases (6.40 +/- 1.36 ng/ml vs 13.05 +/- 2.50 ng/ml; P = 0.0028), suggesting that nm23-H1 may be useful as a prognostic marker for MDS, especially in low risk patients.

Vitamin K2 selectively induces apoptosis of blastic cells in myelodysplastic syndrome: flow cytometric detection of apoptotic cells using APO2.7 monoclonal antibody.

We have previously reported that vitamin K2 (VK2) but not VK1 has a potent apoptosis-inducing effect on freshly isolated leukemia cells from patients with various types of leukemia. By multi-color flow cytometric analysis using monoclonal antibody (mAb), APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of VK2 on minor populations of leukemic blast cells in bone marrow from patients with myelodysplastic syndrome (MDS) and overt myeloid leukemia (post-MDS AML). Limiting dilution of CD95 (anti-Fas) mAb-treated apoptotic Jurkat cells with nonapoptotic CTB-1 cells revealed that APO2.7-positive Jurkat cells were consistently detectable by flow cytometry when present at levels of at least 5% in the CTB-1 suspension. In patient samples the gating area for leukemic clone was determined using cell surface antigen-specific mAbs conjugated with either fluorescein isothionate (FITC) or phycoerythrin (PE) and subsequently the cells stained with phycoerythrin cyanine (PE-Cy5)-conjugated APO2.7 mAb were assessed within the gating area of the leukemic clone for monitoring apoptosis. Treatment of the bone marrow mononuclear cells with 3-10 microM of VK2 (menaquinone-3, -4 and -5) in vitro potently induced apoptosis of the leukemic blast cells as compared with the untreated control cells in all 15 MDS patients tested. This effect was more prominent on blastic cells than that on mature myeloid cells such as CD34-/CD33+ gated cells. In addition, VK2 performed much less effectively on CD3-positive lymphoid cells. In contrast to VK2, VK1 did not show apoptosis-inducing activity. These data suggest that VK2 may be used for treatment of patients with MDS in blastic transformation.

Comparative multi-color flow cytometric analysis of cell surface antigens in bone marrow hematopoietic progenitors between refractory anemia and aplastic anemia.

Refractory anemia (RA) in myelodysplastic syndrome (MDS) without prominent dysplasia closely resemble the mild type of aplastic anemia (AA) in their hematological features. This sometimes makes it difficult to distinguish clearly between the two diseases. Using the multi-color flow cytometric technique, we compared cell surface antigen expression patterns on bone marrow hematopoietic progenitor cells which were isolated as a CD34 positive- CD45 dull positive with low side scatter intensity (CD34(+)CD45(dull+)SSC(low)) population in flow cytogram between RA (n=12) and AA (n=11). The antigens analyzed in CD34(+)CD45(dull+)SSC(low) mononuclear cells were: CD38 and CD71 for cell growth-related antigens, CD 33 and CD13 for myeloid and monocytoid lineage-associated antigens, CD7 and CD19 for lymphoid lineage, and CD14 for a monocytic lineage specific antigen. The percentages of CD34(+)CD45(dull+)SSC(low) cells in bone marrow non-erythroid mononuclear cells, and the expression frequencies of CD38, CD71, CD33 and CD13 antigens in CD34(+)CD45(dull+)SSC(low) progenitors were all significantly decreased in AA compared to normal bone marrows (n=7) (P<0.005). In contrast, in RA bone marrows the percentages of CD34(+)CD45(dull+)SSC(low) cells showed wide distribution and the cell surface antigen expression patterns varied among each case: some cases showed low frequencies of CD38 and CD71 expression as well as AA, whereas the others showed high expression frequency of specific antigen(s) which may reflect the clonal expansion of an abnormal clone in bone marrow. An MDS patient who had progressed from RA to RAEB showed further projecting pattern of expression of CD38 and CD33 in CD34(+)CD45(dull+)SSC(low) population in accordance with the disease progression. These data suggest that analysis of cell surface antigen expression patterns of CD34(+)CD45(dull+)SSC(low) progenitor cells by multi-color flow cytometry appears to be a useful method for qualitative and quantitative assessment of marrow progenitor states in AA and RA, therefore this method could be helpful for early detection of clonal evolution in MDS.

A sole del(15q) anomaly in post-myelodysplasia acute myeloid leukemia.

We report the second case of post-myelodysplasia acute myeloid leukemia (post-MDS AML) with a sole chromosome change del(15q). This anomaly is rarely seen. To our knowledge, only seven cases so far have been reported in human neoplasias, including one case each of acute myeloid leukemia (AML), acute lymphoid leukemia, post myelodysplasia AML, myelodysplastic syndrome, myelofibrosis, macroglobulinemia, Hodgkin's lymphoma and uterine leiomyoma. This case suggests that del(15q) is related to lympho-myeloproliferative disorders. Moreover, we speculate that certain oncogene(s) located on 15q might have some role in the progression of the disease, since the del(15q) anomaly appeared only in the AML phase in this case.

Refractory anemia with ringed sideroblasts with a low IPSS score progressed rapidly with de novo appearance of multiple karyotypic abnormalities and into acute erythroleukemia (AML-M6A).

We report here a case of refractory anemia with ringed sideroblasts (RARS) with a low risk group by the International Prognostic Scoring System (IPSS) at the time of diagnosis but had a rapid disease progression. Although the patient showed a normal male karyotype at the time of RARS diagnosis, his marrow cells had del(5)(q14) and add(17)(p12) abnormalities 2 months after the diagnosis, and later the marrow cells had multiple abnormalities and the patient expired 6 months after the initial diagnosis of RARS. The patient was diagnosed as having RARS with a low risk group by the IPSS classification, however, one should keep in mind that some patients with myelodysplastic syndromes with low risks by either the French-American-British (FAB) classification or the IPSS classification may have progressive disease and subsequential cytogenetic analysis could predict the disease progression.

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro.

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Establishment of a novel B-lymphoma cell line, CTB-1, with strong Pas antigen expression having chromosomal translocation (14;22).

We established a new lymphoma cell line, designated CTB-1, from pericardial effusion of a patient with diffuse large B-cell lymphoma. This cell line showing vigorous growth ability has undergone 260 passages over a period of 34 months in suspension culture, and is heterotransplantable to nude mice. The cultured cells were positive for CD10, CD19, CD20, CD21, HLA-DR, and surface IgG kappa, and negative for T cell antigens. Chromosomal analysis revealed a t(14;22)(q32;q11) that is consistent with original lymphoma cells. CTB-1 cells show the high cell surface expression level of Fas antigen/APO-1. However, ligation of Fas antigen with anti-Fas monoclonal antibody (clone CH-11) did not induce apoptosis of CTB-1 cells. This suggests that Fas itself or the downstream signaling pathways of Fas may be impaired in this cell line. This new cell line may provide a useful in vitro system to study the biology and pathogenesis of B-cell lymphoma which is independent of Fas-mediated apoptosis.

Chronic lymphocytic leukemia complicated by plasmacytoma originating from different clones.

In a woman with chronic lymphocytic leukemia (CLL), a plasmacytoma developed on the back region after four years. CLL cases complicated with plasmacytoma are rare. In the present case, the plasmacytoma showed kappa cytoplasmic immunoglobulin (Ig), and the CLL showed gamma lambda surface Ig. To reveal the clonal origin of CLL and plasmacytoma, we analyzed Ig gene rearrangements in the patient's peripheral blood and plasmacytoma. Ig gene DNA analysis confirmed the presence of different rearrangements in the heavy and light chain genes of CLL and plasmacytoma. These findings suggest that in this patient, the two B cell malignancies arose from expansion of two phenotypically and genotypically distinct clones.

Significance of VLA-4 and LFA-1 expressions in neoplastic follicle formation and its deterioration in B-cell non-Hodgkin's lymphomas.

In an attempt to determine the roles of adhesion molecules in the formation and deterioration of neoplastic follicles, we used flow cytometry to investigate how strongly neoplastic B-cells express VLA-4 alpha and LFA-1 alpha on their surfaces. Neoplastic and normal B-cells were taken from 24 patients with B-cell non-Hodgkin's lymphomas (B-NHL) and 6 with B-cell chronic lymphocytic leukemia (B-CLL). The expression intensities of the adhesion molecules were graded as follows: (-), (+), (+2) and (+3). Normal B-cells expressed those molecules with an intensity of (+2). The data for VLA-4 alpha expression were as follows: follicular B-NHL [10/11; (+2) and 1/11; (+)], partially follicular [5/5; (+)], diffuse [8/8; (+)] and B-CLL [6/6; (-)]. Those for LFA-1 alpha were as follows: follicular B-NHL [7/11; (+2), 4/11; (+)], partially follicular [3/5; (+2), 2/5; (+)], diffuse [3/8; (+2), 5/8; (+)] and B-CLL [3/6; (+), 3/6; (-)]. These results suggest that VLA-4 molecules expressed on neoplastic B-cells may be involved closely in the formation and deterioration of neoplastic follicles, although the expression of LFA-1 molecules seems to play only a minor part in such events.

Stimulatory effects of substance P on CD34 positive cell proliferation and differentiation in vitro are mediated by the modulation of stromal cell function.

Substance P (SP) is a neuropeptide widely distributed in the nervous system. Extensive study has shown SP stimulates production of various cytokines by bone marrow stromal cells, although, the role of SP in hematopoietic phenomena is still unclear. Recently, we established a human cloned stromal cell line, HAS303, which can support hematopoietic stem cell proliferation and differentiation in vitro. We used this culture system to examine the effects of SP. Expression of the mRNAs of neurokinin (NK)-1R, NK-2R and NK-3R, specific SP receptors, on HAS303 cells was demonstrated by the RT-PCR. CD34+ cells isolated from bone marrow were co-cultivated with HAS303 cells in the presence and absence of SP and the total hematopoietic cells and progenitors were counted every 5 days. Introducing SP (10(-8) M) to the co-cultures significantly increased the number of total cells and progenitors compared with control cultures. SP showed no enhancing activity on CD34+ cells cultured alone. SP also stimulated IL-3-dependent colony formation of whole bone marrow MNCs in a soft agar culture system, but showed no such activity on isolated CD34+ cells in this system. These observations suggest that SP stimulated HAS303 cells, activated HAS303 cells, and stimulated the proliferation and differentiation of CD34+ cells. Treating HAS303 cells with SP increased the intracellular Ca2+ concentration and stimulated production of G-CSF, GM-CSF, SCF and IL-6, but not IL-1alpha, IL-1beta and TNF-alpha, but did not enhance proliferation. All these findings suggest that SP mediates hematopoietic cell proliferation and differentiation in vitro by activating stromal cell function.

[Immunocompetent cells in lymph nodes of B-cell lymphomas].

In non-Hodgkin's lymphoma (NHL), the precise analysis of non-neoplastic immunocompetent cells in lymph nodes may be important to understand the pathophysiology of anti-tumor immunity. We have investigated such immunocompetent cells of 14 patients with B-cell type NHL (B-NHL) by flow cytometry, and compared them with the data obtained from 10 patients with reactive lymphadenopathy. The results on B-NHLs were as follows; CD3+ (T lymphocyte) = 45.0 +/- 19.7%, CD4+/CD3+ = 62.7 +/- 14.2%, CD4+CD45RA-/CD4+ = 82.9 +/- 8.1% (Control 62.9 +/- 14.5%, p < 0.01), CD4+CD29++/CD4+ = 29.2 +/- 7.0% (Control 42.6 +/- 12.9%, p < 0.01), CD8+/CD3+ = 36.0 +/- 11.3%, CD8++S6F1++/CD3+ = 23.2 +/- 10.6% (Control 9.1 +/- 4.3%, p < 0.01), CD8++S6F1++/CD8++ = 75.3 +/- 16.7% (Control 41.5 +/- 19.6%, p < 0.01), CD3-CD56+ cells = 1.0 +/- 0.7% (Control 2.2 +/- 1.6%, p < 0.05). These findings suggest that CD4+ T lymphocytes in lymph nodes of B-NHL may change to memory cells (CD45RA- cells), but such memory cells could only weakly express CD29 molecules which are thought to play an important role in the manifestation of helper function. This phenotypic discordance of CD4+ T lymphocytes may produce incomplete anti-tumor immunity in B-NHL.

[The successful use of natural alpha-interferon single therapy in multiple myeloma of IgD (lambda)-type].

We have successfully treated multiple myeloma of IgD (lambda) type [IgD (lambda) -MM] by natural alpha-interferon (alpha-IFN) single therapy. A 45 year-old man was admitted to Tokyo Medical College Hospital because of general fatigue in August, 1989. Immunoelectrophoresis, bone marrow biopsy and systemic bone survey revealed IgD (lambda) -MM with Bence Jones (BJ) proteinuria and slightly osteolytic lesions. We started treating him with natural alpha-IFN single therapy. Three months later, serum IgD markedly decreased and BJ proteinuria disappeared. Bone marrow, which had been packed with myeloma cells at the admission, was almost replaced by normal hematopoietic cells. In April 1990, he is still free of disease with only alpha-IFN single therapy. This result might suggest that alpha-IFN single therapy is effective for IgD-MM.

[Immunoregulatory cells in patients with monoclonal gammopathies].

The authors detected immunoregulatory cells by two-color cytometric analysis in the peripheral blood of 21 patients with multiple myeloma (MM) and 10 patients with monoclonal gammopathy of undetermined significance (MGUS). CD3-CD56+ cells increased in both MGUS and IgG, IgA type MM in clinical stage (CS) I & II, whereas CD3+ cells decreased. On the other hand, the CD4/8 ratio, the proportions of CD45RA+ cells and CD29++ cells in CD4+ cells, and S6F1++ cells in CD8++ cells remained normal. In CS III MM of IgG and IgA type, CD3-CD56+ cells did not increase, although CD3+ cells decreased. Additionally, both CD4/8 and CD4+ CD45RA+/CD4+ ratios were low, while the proportions of CD29++ cells in CD4 cells and S6F1++ cells in CD8++ cells were high. However, this type of change in the T-cell subset balance was not observed in CS III MM of the Bence-Jones type, which showed no significant elevation of serum M-protein. These findings suggest that serum monoclonal idiotypes could affect immunoregulatory cells, especially T cells.

[Macrocytic anemia with anisocytosis due to alcohol abuse and vitamin B6 deficiency].

Megaloblastic anemia due to folic acid deficiency and ringed sideroblastic anemia have been reported in alcohol abusers. It has also been reported that vitamin B6 deficiency causes ringed sideroblastic anemia as well as microcytic anemia that is not associated with ringed sideroblasts. We encountered a case of macrocytic anemia with anisocytosis in a 75-year-old alcohol abuser who suffered vitamin B6 deficiency. Neither megaloblastic changes nor ringed sideroblasts were observed in specimens of the patient's bone marrow. Analyses of porphyrin content and heme biosynthetic enzyme activity suggested a decline in ALA-synthase activity (an enzyme that depends on vitamin B6) as well as decreased ferrochelatase activity or abnormal iron metabolism. Abstention from alcohol led to a reduction in mean corpuscular volume and the disappearance of Pappenheimer bodies commonly observed in the red blood cells of drinkers. Follow-up supplements of vitamin B6 resolved the patient's anisocytosis and anemia.

[Analysis of T cell subsets in autoimmune hemolytic anemia patients].

To clarify a role of immunoregulatory T cells in the pathophysiology of autoimmune hemolytic anemia (AIHA), we investigated T cell subsets in the peripheral blood of 15 patients with AIHA by two color analysis using flow cytometry. Consequently both CD4+ cells and CD4+CD45RA+ cells decreased in proportion, irrespective of the disease activity (active or remission phase). CD4+CD45RA+ cells are regarded as naive T cells. Incidentally the ratio of CD45RA+ cells in CD4+ cells also fell in the low level in active phase, but it recovered to the normal ratio in remission. On the other hand, CD8+ cells and CD8+ +S6F1+ cells that may represent activated cytotoxic T lymphocytes increased in active phase and then both entered the normal range in remission. These findings suggest that AIHA could be caused partly by the alternative balance of CD4+CD45RA+ cells probably constituting a member of IRT and moreover by the activation of CTL.

[A two-dimensional analysis of B-lymphoma cells by flow cytometry--a comparative study with pathological classification].

In order to investigate the size variation of neoplastic cells from lymph nodes of 19 patients with B-cell non-Hodgkin's lymphomas (B-NHL), we have carried out the two-dimensional analysis by flow cytometry using the parameters of the forward light scatter (FLS) and the fluorescence intensity of surface immunoglobulin light chain (sIgL). Neoplastic B cells were identified as having the homogeneous characters of both FLS and sIgL (kappa or lambda) in comparison of normal B cells with the heterogeneity of counterpart sIgL (lambda or kappa). Then the size variation of neoplastic B cells was analyzed by regarding CD3+ T cells to a control scale. Consequently, the present procedures disclosed that B-NHL cases could be divided into five patterns from the view-point of the cell size and distribution. These results almost coincided with the cell types determined by the pathological diagnosis. Furthermore, it was also found that our data made it possible to classify small cleaved cell or large cell lymphomas into subtypes. The two-dimensional analysis by using the parameters of FLS and sIgL would be clinically useful for the rapid diagnosis of B-NHL and its malignant grade in addition to supporting pathological findings.

[The successful use of recombinant human erythropoietin therapy to anemia of granular lymphocyte proliferative disorder].

A 54-year-old man was admitted with fatigue. The peripheral blood count showed leukocytosis (9, 600/microliters), including 76% granular lymphocytes (GLs), which expressed CD2, 3, 8, 16 and HLA-DR, and anemia (hemoglobin 8.1 g/dl). He was diagnosed as having T cell type-granular lymphocyte proliferative disorder with anemia. Bone marrow examination revealed the involvement of 4.6% of GL and erythroblastopenia. A clonogenic assay of bone marrow cells revealed the decrease in erythroid colony formation in both CFU-E and BFU-E, but the number of erythroid colonies increased when CD8-positive cells were depleted from bone marrow cells and the number of erythroid colonies decreased again when CD8-positive GLs were added. The supernatant of cultured CD8-positive GLs had no inhibitory effect on CFU-E and BFU-E colony formation. These suggested that CD8-positive GLs suppressed the erythroid colony formation in this case. Treatment with 6,000 U/body of recombinant human erythropoietin (rh-Epo) subcutaneously three times a week was started and increased dose of 12,000 U/body of rh-Epo led to an increase in the hemoglobin level to 10.5 g/dl two months later. He has been treated with rh-Epo only.