Diagnosis and subclassification of follicle center and mantle cell lymphomas on fine-needle aspirates: A cytologic and immunocytochemical approach based on the Revised European-American Lymphoma (REAL) classification.

BACKGROUND: Cytologic distinction between follicle center lymphoma (FCL) and mantle cell lymphoma (MCL) is difficult with cytomorphology alone and requires immunophenotyping. The current study describes the distinction between follicle center and mantle cell lymphoma made with fine-needle aspiration (FNA) material. METHODS: One hundred ten cases primarily diagnosed and classified on FNA material as centroblastic-centrocytic (CBCC) and centrocytic (CC) non-Hodgkin lymphomas (NHLs) (Kiel classification) were included in the study. An additional retrospective immunocytochemical analysis was performed on frozen cytospin preparations using the monoclonal antibodies Bcl-2, CD10, CD5, CD23, CD43, and immunoglobulin M. RESULTS: The initial diagnostic workup classified 106 cases as CBCC-NHL and 4 as CC-NHL. The immunophenotype Bcl-2(+), CD10(+/-), CD5(-), CD23(-/+), CD43(-) was observed in 93 of 106 previously reported CBCC NHLs. In 11 of 106 cytospin preparations, neoplastic B cells expressed the CD5 pan T marker and, as a group, showed the pattern Bcl (+/-), CD10(-/+), CD5(+), CD23(-), CD43(+), which is considered typical of MCL. Based on the additional immunocytochemical data, all but 2 of the tumors were reclassified as FCL (n = 93) and MCL (n = 15). The mean proliferation fraction measured by MIB-1 (Ki-67) immunoreactivity was 16.3% and 17.5% in FCL and MCL, respectively. The revised cytopathologic diagnosis correlated significantly (P < 10(-9)) with the histology of 65 patients who underwent surgical excision biopsy. CONCLUSIONS: Subclassification of follicle-derived low grade NHL can be established with high accuracy on FNA material if cytomorphology is corroborated by a complete immunophenotypic analysis, which can be performed on both fresh and frozen stored cytospin material. The currently used criteria can be applied to aspirated cells for a conclusive cytopathologic diagnosis of MCL, which is of great clinical importance. Cancer (Cancer Cytopathol)

Fine-needle aspiration cytology and immunocytochemistry of soft-tissue extramedullary plasma-cell neoplasms.

This study presents 19 patients with extramedullary plasma-cell tumors diagnosed by fine-needle aspiration (FNA) cytology together with immunocytochemistry. Eight patients had primary extramedullary plasmacytoma, while 11 patients had tumors secondary to myeloma. The most common localization was soft tissue (9 cases), followed by lymph nodes (5), scalp (3), and oral and nasal mucosa (2). All FNA smears were cellular, and 12 cases showed dissociated monomorphic plasma cells. Seven cases showed a dominance of immature bare nuclei, which made then difficult to diagnose conclusively using cytomorphology only. Immunocytochemistry demonstrated monoclonal expression of light immunoglobulin chains in all cases which, together with demonstration of CD 38 positivity and cytomorphology, allowed a conclusive diagnosis of plasmacytoma.

Addition of etoposide to CHOP chemotherapy in untreated patients with high-grade non-Hodgkin's lymphoma.

BACKGROUND: Second- and third-generation chemotherapy protocols for the treatment of aggressive non-Hodgkin's lymphomas (NHL) have considerable, and age-related, toxic effects. In addition, they do not seem to prolong overall survival in comparison to standard CHOP chemotherapy. In this phase II study we investigated the feasibility and efficacy of the addition of etoposide to the conventional CHOP regimen. PATIENTS AND METHODS: Toxicity and clinical efficacy were determined in 132 patients with previously untreated high-grade NHL. There were 51 patients in clinical stage I and II and 81 patients in stage III and IV, with a median age of 54 years (range 17-85). Patients received standard-dose CHOP plus etoposide 100 mg/m2 i.v. on day 1 and 200 mg/m2 p.o. on days 2-3. RESULTS: The overall response rate was 84%, with 70% complete and 14% partial responses. The predicted three- and five-year survivals for the group as a whole were 60% and 53%, respectively, and the corresponding disease-free survivals for patients achieving complete remissions were 65% and 56%, respectively. Outcome was not different from that of CHOP-treated patients in a recently completed Nordic study performed during the same time period. Myelosuppression (WHO grade 3-4), observed in 87% of patients and infectious complications (WHO grade 3-4) in 33%, dominated the toxicity profile of this regimen. Fifty-seven of 92 complete responders (62%) received 6-8 CHOP-E cycles with no reductions in planned dose intensity. LDH level higher than normal, extranodal sites = 2, stage III-IV at diagnosis were all indicators of a poor survival. CONCLUSIONS: We conclude that CHOP-E treatment is effective in high-grade NHL. However, mainly due to severe myelosuppression frequent schedule modifications were required and the results are not obviously superior to those of conventional CHOP.

Cladribine for untreated or early low-grade non-Hodgkin's lymphoma.

PATIENTS AND METHODS: Forty-four patients, with low-grade non-Hodgkin's lymphoma (LG-NHL) were included in a phase II study between June 1993 and May 1995 and treated with cladribine (CdA) 0.12 mg/kg as a 2 h i.v. infusion daily x 5, repeated after 28 days for up to 6 courses. Thirty-four patients were previously untreated and 10 had progressive disease after initial response to limited chlorambucil treatment. Five patients had also received involved field radiotherapy. Eight patients had mantle cell lymphomas, 22 follicle centre lymphomas, 5 lymphoplasmacytoid lymphomas, 4 small cell lymphocytic lymphomas, 4 marginal zone B-cell lymphomas and I had unclassified low-grade NHL. The response rate was 64%, with 11 (25%) CR and 17 (39%) PR while 5 (11%) patients progressed during treatment. The response rate was similar in previously treated and untreated patients. The median number of CdA courses delivered was 3 (1-6) in non-responding patients and 6 (2-6) in responders. Median survival from inclusion was not reached with a median follow-up of 40 months. The median time to progression was 7 mo for all patients, 25+ mo for CR and 16 mo for PR patients. Toxicity was sometimes severe with 2 treatment related deaths, one infectious related and one due to a mucocutaneous syndrome and pulmonary microembolism. In addition, 5 grade 3 or 4 infectious episodes were seen. Seven patients experienced grade 3 or 4 thrombocytopenia and 20 had grade 3 or 4 neutropenia. We conclude that the majority of patients with low-grade non-Hodgkin's lymphoma respond to CdA but that the adverse effects may be severe.

Immunoglobulin G abnormalities in HIV-1 infected individuals with lymphoma.

BACKGROUND: Polyclonal B-cell activation precedes the occurrence of malignant B-cell clones. Several recent reports suggest a perturbed cytokine regulation in HIV-related lymphomagenesis and Epstein-Barr virus (EBV) involvement in approximately half of the cases with generalized lymphoma. OBJECTIVES: We investigated whether altered immunoglobulin properties would be detected by fine analysis of the immunoglobulin G (IgG) subclass patterns against HIV and EBV epitopes. STUDY DESIGN: HIV-1 infected patients in early stage, late stage and with lymphoma were analyzed by ELISA for anti HIV and EBV IgG class and subclass antibodies. Avidity and affinity of the antibodies were studied. The lymphoma patients were also studied by PCR for EBV DNA in serum. RESULTS: The total IgG reactivity to several HIV antigens was similar in the three patient groups. However, lymphoma patients had a more restricted subclass pattern with significantly lower IgG1 and IgG3 anti gp120 titers compared to other HIV-infected patients but good and persistent total IgG and IgG1 (excluding the gp120 antigen) reactivities in contradiction to their low CD4 counts. IgG4 reactivity was sparse, detectable to significant levels in the symptomatic group only. The observed relative affinity of the HIV-specific IgG and IgG1 of lymphoma patients was similar to that of asymptomatic and symptomatic patients. The subclass reactivity to the EBV peptide was similar in all groups but lymphoma patients with EBV DNA in serum exhibited significantly lower anti EBV peptide titers than those who were EBV DNA negative. CONCLUSION: These findings indicate that subclass analysis to defined viral antigens may be a means to detect immune dysregulation in tumor development.

Fine-needle aspiration cytology and immunocytochemistry of Hodgkin's disease, suppurative type.

We describe 3 cases of Hodgkin's disease (HD) of unusual suppurative type, which were diagnosed on fine-needle aspirates. The smears were dominated by neutrophils, macrophages, and cellular debris. Only a few large, atypical cells of Hodgkin and Reed-Sternberg type were observed. The differential diagnoses of such smears include infectious mononucleosis, tuberculosis, metastatic lymph node involvement, non-Hodgkin's large-cell anaplastic Ki-1-positive lymphomas, T-cell-rich B-cell lymphomas, and peripheral T-cell lymphomas of mixed type. Immunocytochemistry identified the large atypical cells as CD 30 (BerH2)-positive and negative for CD 45 (LCA) in cytospin material from 2 patients, which allowed a conclusive diagnosis of HD.

Fixed versus response-adapted MOPP/ABVD chemotherapy in Hodgkin's disease. A prospective randomized trial.

BACKGROUND: The optimal number of chemotherapy courses in responding patients with advanced-stage Hodgkin's disease (HD) is unknown. PATIENTS AND METHODS: With minimizing chemotherapy and thereby reducing late complications as the objective, patients with advanced HD were randomized to receive either 4 full MOPP/ABVD courses or treatment up to complete remission (CR). Forty-seven patients were given the fixed (FT) and 41 patients the individual treatment (IT). The two groups were balanced according to age, histopathology and sex, although stage IVB dominated in the IT group (20 vs. 8). RESULTS: Sixty-six of 88 patients (75%) achieved CR. No difference between the two treatment groups in the proportion of stage IVB patients was seen when those achieving CR, i.e., the efficacy population were compared. The mean number of single chemotherapy courses given was 3.7 of MOPP and 3.5 of ABVD in the FT group, compared to 2.6 of MOPP and 2.5 of ABVD in the IT group (p < 0.001). The predicted progression-free survival at 10 years was 81% in the FT and 68% on the IT arm, respectively (p < 0.05). No statistically significant difference in cause-specific 10 year survival was observed (82% and 83%, respectively; p = 0.18). Long-standing CRs were achieved following minimal chemotherapy. CONCLUSIONS: Since there are no available methods to identify long-term disease-free survivors among CR patients following a limited induction treatment, we suggest that the policy of giving 3-4 full MOPP/ABVD courses should continue. The price for such an approach is the overtreatment of a subset of already cured patients.

Growth fraction in non-Hodgkin's lymphomas and reactive lymphadenitis determined by Ki-67 monoclonal antibody in fine-needle aspirates.

The fraction of proliferation cells was analysed in fine needle aspirates from a series of 448 non-Hodgkin's lymphomas and 199 reactive hyperplasias using an immunoperoxidase staining with monoclonal antibody Ki-67. There was a good correlation between proliferation fraction and cytologic assignment to high and low grade lymphomas. Thus high grade lymphomas had a high median percentage of Ki-67 positive cells with a figure of 82.1 for lymphoblastic, 60.0 for immunoblastic, and 59.7 for centroblastic lymphomas. For low grade lymphomas the figures were 17.1 and 11.1 percent for centroblastic/centrocytic and CLL/immunocytoma, respectively. The fraction of proliferation cells in reactive lymphadenitis varied between 1-50% with a median of 11.5%. Analysis of Ki-67 positivity can accordingly not be used to differentiate benign from neoplastic proliferations. Within all lymphoma subgroups but lymphoblastic lymphoma, there was a marked variation in fraction of Ki-67 positive cells, which resulted in a certain overlap between high and low grade lymphomas. The results show that cells procured through fine-needle aspiration can be used to analyse the fraction of proliferating cells which contributes information about the growth rate of the individual tumours that can not be obtained through cytologic classification.

Diagnosis of lymphoma, leukemia, and metastatic tumor involvement of the cerebrospinal fluid by cytology and immunocytochemistry.

Fifty-five cerebrospinal fluid (CSF) specimens from 42 patients with suspected meningeal tumor involvement were reviewed. Cytology in conjunction with immunocytochemistry identified 26 CSF specimens as malignant. There were fifteen cases of lymphoma, four cases of leukemia, two cases of carcinoma, and two cases of melanoma. A monoclonal light chain expression was demonstrated in nine out of eleven B cell lymphomas. The three T-cell lymphomas all expressed pan T markers (CD 3) and two the T-helper antigen (CD 4). One patient had meningeal involvement of a true histiocytic lymphoma which was identified by its large atypical cells which were positive for alpha-1-anti-trypsin and muramidase. In four patients with a primary diagnosis of acute lymphoblastic leukemia, CSF involvement was confirmed by the demonstration of blasts with CD 10 (cALLA) or light chain restriction. Epithelial or melanocytic markers were demonstrated on the tumor cells in CSF from the remaining four patients. In 29 CSF specimens a diagnosis of reactive lymphocytosis was made using cytomorphology which mostly was characterized by macrophages mixed with small mature lymphoid cells. Immunologic evaluation showed that these mature cells were CD 10 negative T-cells and only few specimens contained polyclonal B-cells. The subsequent clinical course of these patients showed no evidence of CNS malignancy. It is concluded that cytology should be used in conjunction with immunocytochemistry to accurately evaluate CSF specimens from patients with possible malignant meningitis.

Lymphocyte abnormalities predicting a poor prognosis in Hodgkin's disease. A long-term follow-up.

Two hundred sixty-two adult patients with Hodgkin's disease (HD) were studied. Incorporation of carbon-14-thymidine was measured in unstimulated monocyte-depleted lymphocyte cultures, and in cultures activated by concanavalin A (Con-A) before institution of therapy in all patients. Total blood lymphocytes and T-cell subsets were enumerated in the last 108 patients. Patients had significantly decreased total (CD3+, CD4+, CD8+) and relative (CD3+, CD4+) T-cell counts compared with healthy controls. Stage IV patients tended to have lower total lymphocyte and subset counts than remaining patients. However, significantly reduced total lymphocyte and CD8+ counts were only observed in comparison to patients in clinical stage II. Thirty-three percent of patients had an increased spontaneous and a decreased Con-A-induced blood lymphocyte DNA synthesis. Functional lymphocyte abnormalities were related to advanced clinical stage, high age, mixed cellularity, and lymphocyte depletion histopathology and presence of B symptoms. The 10-year survival of patients in this group was 36%, compared with 62% for the remainder. In a multivariate analysis of the whole series lymphocyte DNA synthesis was besides age the strongest predictor of prognosis. In univariate analyses of the second patient series total lymphocyte, T-cell and subset counts were related to prognosis. These relatively simple lymphocyte functional tests may help to identify young HD patients for whom intensive cytoreductive therapy with or without autologous stem cell support may be the best therapeutic option.

Changes of the blood lymphocyte population following 32P treatment for polycythemia vera.

Orally administrated Na2 32PO4 mainly accumulates in bone marrow where it emits beta-particles which may damage cells. Previously, we showed that 32P treatment for polycythemia vera (PVC) increased the phytohemagglutinin reactivity and proportions of T cells in the blood. Now we have examined the effects of 32P treatment for PCV on natural killer (NK) and B-lymphocyte subsets which are considered to undergo their maturation in bone marrow. A mean isotope dose of 240 MBq given to 14 patients reduced the peripheral lymphocyte counts to 60% at 6 weeks. B cells and NK cells were reduced to the highest relative extent followed by HNK-1 cells and T cells. Although the proportion of NK cells was reduced to 50% there was no concomitant reduction of NK activity against K562 cells. Pokeweed mitogen-triggered secretion of IgM was significantly reduced, but not that of IgG or IgA. It is suggested that lymphocytes which mature in bone marrow may be affected to the highest extent by 32P treatment in PCV.

Cytomorphology and immunocytochemistry of fine needle aspirates from blastic non-Hodgkin's lymphomas.

Fine needle aspirates from 54 consecutive patients with primary or recurrent blastic (high-grade malignant) non-Hodgkin's lymphomas (NHLs) were analyzed by cytomorphology and immunocytochemistry. The cytologic diagnoses induced follicular center-cell-derived (centroblastic or anaplastic centrocytic) lymphoma (31 cases), immunoblastic lymphoma (11 cases), lymphoblastic lymphoma (9 cases) and histiocytic lymphoma (3 cases). Immunocytochemistry showed a B-cell phenotype of the neoplastic lymphocytes in all lymphoblastic lymphomas, 29 follicle center-cell lymphomas and 4 immunoblastic lymphomas. Four of the immunoblastic lymphomas were of T-cell origin while one case was not evaluable due to necrosis. A histiocytic origin was confirmed in two of the three cases that had a cytologic diagnosis of histiocytic lymphoma; the third case was shown by immunocytochemistry to be a true Ki-1-positive large cell lymphoma. Histologic and immunohistochemical analysis were performed on surgical biopsies from 18 patients. The results were in agreement with those on the fine needle aspiration (FNA) material in 14 cases. Three lymphomas could be phenotyped on aspirated material while marker studies on excised material were inconclusive. One lymph node aspirate contained mostly necrotic cells, which were unsatisfactory for adequate immunocytochemistry. However, sections from a removed tonsil from the same patient could be used for conclusive histology and phenotyping. In conclusion, the high diagnostic accuracy of combined cytomorphologic and immunocytochemical assessment of FNA samples validates the use of the technique in the diagnostic work-up of blastic (high-grade malignant) NHLs. In fact, the diagnostic accuracy seems so high that the technique can safely be used in the final diagnosis of blastic NHLs.

Fine-Needle Aspiration Cytology and Immunocytochemistry of Abdominal Non-Hodgkin's Lymphomas.

The cytomorphology and immunologic characteristics of cells obtained by fine-needle aspiration biopsy of 34 consecutive patients with abdominal lymphomas were analyzed. Nineteen patients had no previous diagnosis, while 15 had previously known or suspected lymphomas. On cytology 21 high-grade and 13 low-intermediate-grade lymphomas were diagnosed. Immunologic characterization of aspirated cells identified one T-cell and 33 B-cell neoplasms. A monoclonal light chain expression was detected in 27 of the B-cell lymphomas. The results were in good agreement with those from histologic (n = 19) and immunohistochemical (n = 5) evaluations. The value and accuracy of fine-needle aspiration cytology in conjunction with immunocytochemistry are detailed.

Changes of the blood lymphocyte subpopulations and their functions following 131I treatment for nodular goitre and 32P treatment for polycythemia vera.

The blood lymphocyte population was examined in 34 patients who were treated with 131I for toxic or atoxic nodular goitre. One to three doses of 300-550 MBq of 131I were administered at 1-week intervals. Lymphocyte counts were found to be significantly reduced at both 1 and 6 weeks after treatment. This decrease was accompanied by a changed composition of the lymphocyte subpopulations. The frequency of lymphocytes expressing membrane receptors for C'3 (EAC-rosette forming cells) was significantly reduced at 1 and 6 weeks following 131I administration. At 6 weeks there was a small but statistically significant increase of the frequency of T cells as identified by Leu 1 monoclonal antibodies. This was essentially due to an increased proportion of helper/inducer T cells as identified by Leu 3 monoclonals. 131I treatment also decreased the capacity of lymphocytes to secrete immunoglobulins (Ig) when stimulated with pokeweed mitogen (PWM). The greatest effect was observed for IgM. Secretion of IgG and IgA were less reduced. Mitogenic stimulations of lymphocytes with phytohemagglutinin (PHA) and concanavalin A were not significantly changed. It is concluded that these findings, with the exception of mitogen reactivity, are largely similar to those occurring following external radiation therapy for cancer. It is suggested that blood lymphocytes passing through the continuously irradiated gland are damaged mainly by beta-rays. The effect of 32P treatment on the blood lymphocyte population was examined in 16 patients with polycythemia vera. Before treatment the lymphocyte counts were within the normal range but the expression of certain membrane structures, as identified by monoclonal antibodies against total T cells (Leu 1 and 4), helper/inducer (Leu 3) and suppressor/cytotoxic T cells (Leu 2), were slightly decreased. Moreover, mitogenic responses of the lymphocytes to PHA and PWM-induced Ig secretion were impaired. Following a single oral dose of 32P (150-305 MBq), which normalized the production of erythrocytes and/or platelets, the blood lymphocyte counts were reduced by approximately 40 per cent 12 weeks after treatment. Examination of subsets demonstrated that the proportion of B-cells, as identified by B1 monoclonal antibodies, was decreased by the highest relative extent. On the other hand, lymphocytes expressing the above-mentioned T cell markers were somewhat increased. 32P treatment markedly increased PHA reactivity but it further reduced PWM-induced Ig secretion. The latter observation was in agreement with the finding that serum concentrations of Ig were reduced after treatment.

Effect of 32P treatment for polycythaemia vera on blood lymphocyte subpopulations and their functions.

The influence of 32P treatment on the blood lymphocyte population was examined in 16 patients with polycythaemia vera who had not previously been treated with cytotoxic drugs or irradiation. Before treatment the lymphocyte counts were within the normal range but the expression of certain membrane structures, as detected by monoclonal antibodies directed against total T cells (CD 3 and 5), helper/inducer (CD 4) and suppressor/cytotoxic T cells (CD 8), were slightly reduced. In addition, mitogenic responses of the lymphocytes to PHA and PWM-induced Ig secretion were severely impaired. Following a single oral dose of 32P (150-305 MBq), which was shown to normalize the production of erythrocytes and/or platelets, the blood lymphocyte counts were reduced by approximately 40% 12 wk after treatment. Subset analysis showed that the proportion of B cells, as identified by monoclonal antibodies (CD 20), was reduced to the highest relative extent. On the other hand, lymphocytes expressing the above T cell markers were somewhat increased. 32P treatment sharply increased PHA reactivity but it further reduced PWM-induced Ig secretion. The latter observation was in line with the finding that serum concentrations of Ig were reduced following treatment.

Virologic, immunologic, and clinical observations on a patient during the incubation, acute, and convalescent phases of infectious mononucleosis.

One patient with infectious mononucleosis (IM) was studied from the probable time of Epstein-Barr virus (EBV) infection (38 days before the onset of clinically overt disease), during the incubation and acute phases, until 6 months after clinical remission. Analysis of spontaneous outgrowth of EBV-carrying lymphoblastoid cells, by limiting dilution on feeder layer cultures, showed that virus containing B lymphocytes are already present early during the incubation period. Also low interferon serum levels were detected early after infection, and only before the onset of clinical disease. All other studied clinical laboratory and virus-associated variables were within normal range during the incubation phase, but changed to a pattern characteristic of IM in parallel to the clinical symptoms. During the acute disease EBV-associated nuclear antigen (EBNA)-positive cells could be directly detected among the lymphocytes, and antibodies to EBV antigens appeared. Lymphocytes stained by monoclonal antibodies, detecting Ia-like determinants (activated cells) and suppressor cells, increased dramatically, in parallel to a strong increase of functional suppressor cell activity, measured by inhibition of blastogenesis and PWM-induced immunoglobulin production. During the acute phase there was also a decrease of spontaneous cytotoxicity against the NK-sensitive cell line K562, while cytotoxicity (spontaneous) against an autologous EBV-positive lymphoblastoid cell line (LCL) was detected only during this phase. These reactions correlated to the presence of blasts, and the autologous reaction was exerted mainly by Fc-receptor-negative cells. Lymphokine production in response to EBV antigens was also initiated during the acute phase. During the convalescence period the serological and cellular immune parameters adjusted to the pattern of a normal EBV-seropositive person.

T-cell response to B-cells and Epstein-Barr virus antigens in infectious mononucleosis.

After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist indefinitely through life. These cells grow in vitro on explanation and can be established as immortal B-cell lines. To reconcile the unlimited growth potential in vitro with the maintenance of a low proportion of B-cells infected by EBV in vivo, a strict in vivo control mechanism has to be postulated. Certain aspects of this control are apparent when the primary infection is followed by infectious mononucleosis. This is characterized by lymphocytosis and the presence of activated T-cells. The T-cell proliferation is probably the manifestation of the immune response against EBV antigens. However, the reaction of T-cells upon encounter of B-blasts is also likely to contribute to the events. At present, it is difficult to detect an EBV-specific component in the action of the T-cells in the acute phase of mononucleosis exerted on B-cells. However, for the clinical course of the disease the activation of T-cells is important. The activated T-cells may control and also eliminate the B-cells infected by EBV. In addition to the immunity which develops during the disease, th immunoregulatory mechanism is likely to have a role in the inhibition of B-cell proliferation.