Induction by interleukin-2 of oligoclonal expansion of cultured tumor-infiltrating lymphocytes.

To better understand the modulatory effects of interleukin-2 (IL-2) on lymphocyte proliferation, we examined the clonality of the in vitro T-cell response by Southern blot hybridization. Tumor-infiltrating lymphocytes (TILs) grown in the presence of IL-2 for 15-26 days had detectable T-cell receptor beta-chain gene rearrangements, which indicated oligoclonal enhancement in culture in four of nine TIL samples. In contrast, none of 11 uncultured TIL samples had detectable gene rearrangements. Lack of detection in at least three of the five negative, cultured TIL samples could be explained by increased numbers of natural killer cells. We hypothesize that the oligoclonal expansion noted results from the enhanced response of immune-primed T cells to IL-2.

bcr rearrangement: potential false positive secondary to an Eco RI restriction fragment length polymorphism.

Molecular studies have demonstrated that the Philadelphia chromosome (Ph) translocation characteristic of chronic granulocytic leukemia (CGL) and 50% of the cases of Ph positive acute lymphocytic leukemia (ALL) involves a limited 5.8 Kb region on chromosome 22 termed the breakpoint cluster region (bcr). Detection of bcr rearrangement by Southern blot analysis has proven to be a sensitive diagnostic method and can identify this translocation in some cases which appear cytogenetically negative. Restriction fragment length polymorphisms (RFLP) which involve bcr have the potential to be misinterpreted as gene rearrangements since they result in alteration of the DNA fragment size detected by Southern blot hybridization. We have identified a RFLP involving bcr that is detectable with Eco RI digestion but not with Bam HI, BgI II, or Xba I. The polymorphic fragments generated indicate that this RFLP is the result of an Eco RI restriction site sequence polymorphism.

Comparison of cisternal and lumbar CSF examination in leptomeningeal metastasis.

We compared cisternal and lumbar CSF examination in 14 patients suspected of having leptomeningeal metastasis from cancer. Malignant cells were present in 12 patients--in both cisternal and lumbar CSF in nine patients and only in cisternal CSF in three. Cisternal CSF cytologic examination should be considered in patients suspected of having leptomeningeal metastasis if lumbar CSF is nondiagnostic.

Reliable and cost-effective paraffin section immunohistology of lymphoproliferative disorders.

We studied 110 neoplastic and reactive lymphoid proliferations with three monoclonal antibodies--CD20 (L26), CD43 (Leu22), and CD45RO (UCHL1)--on B5-fixed, paraffin-embedded tissue to evaluate the utility of this panel as an immunotypic screen of such lesions. All cases were initially immunotyped by conventional methods. Genotyping by Southern blot hybridization was also done in 54 cases. Seventy-four of 79 malignant lymphomas and both of two hairy cell leukemias were of B-cell origin; and five lymphomas were defined as T-cell lineage. Lineage assignment was identical for paraffin section immunohistology and conventional immunotyping in 73 of 76 B cell and all of five T-cell tumors. CD20 was reactive with 73 of 76 B-cell tumors. CD43 was reactive with 12 of 74 B-cell lymphomas, and CD20/CD43 coexpression was seen in 11 of these cases. CD43 and CD45RO marked all of five and three of five T-cell lymphomas, respectively. Lineage assignment was identical for paraffin immunohistology and genotyping in 48 of 50 cases with identifiable gene rearrangements. Twenty-four nonneoplastic and five Hodgkin's disease cases that were studied also showed similar immunoreactivity patterns by both paraffin and conventional immunotypic methods. This panel of three monoclonal antibodies is an efficient, cost-effective approach for immunotyping most lymphoid proliferations in paraffin sections. Nevertheless, the pathologist should always try to obtain fresh or frozen tissue to aid in resolving occasional discrepant cases, to establish clonality in morphologically ambiguous ones, and to profile prognostically important phenotypic deletions.

Absence of B-cell or T-cell clonal expansion in nodular, lymphocyte predominant Hodgkin's disease.

Recent studies based upon immunophenotypic data have provided strong evidence that nodular lymphocyte predominant Hodgkin's disease (NLPHD) represents an entity that is distinct from other subtypes of Hodgkin's disease (HD). In contract to other forms of HD, the predominance of B-lymphocytes in NLPHD has prompted the thesis that this lesion is actually an atypical B-cell hyperplasia or follicular center cell lymphoma. Three cases of NLPHD by restriction endonuclease analysis were studied in an attempt to identify a clonal B-cell or T-cell expansion in this disorder. DNA was extracted from these tumors and hybridized to probes for the immunoglobulin genes (C kappa, C lambda, JH) and the T-cell receptor beta chain gene. Gene rearrangements were not detectable in any of the cases. The results provide genotypic evidence that there is not a monoclonal or oligoclonal proliferation of small B-lymphocytes or T-lymphocytes in NLPHD. The possibility that the L&H Reed-Sternberg cells are monoclonal cannot be excluded because their small number is below the level of sensitivity of this technique.

Immunoglobulin and T-cell receptor genes in thymomas: genotypic evidence supporting the nonneoplastic nature of the lymphocytic component.

On the basis of morphologic and immunophenotypic studies, it is generally accepted that the lymphocyte population in thymomas is not neoplastic. We studied 10 thymomas with restriction endonuclease and Southern blot/DNA hybridization methods in an attempt to provide genotypic evidence in support of this hypothesis. The clinical, gross, and microscopic features of each case were reviewed and found to be entirely consistent with the diagnosis of thymoma. In addition to conventional histologic methods, we also studied each tumor by immunohistologic techniques. The lymphocytes generally had an immunotype characteristic of immature cortical thymocytes, and the epithelial cells were uniformly stained by antikeratin antibodies. DNA probes for the T-cell receptor beta-chain gene and immunoglobulin genes (C kappa, C lambda, and JH) were used in the genotypic studies. No gene rearrangements were detected in any of the thymomas. This study provides additional evidence that clonal proliferations of T or B lymphocytes are not present in thymomas; therefore, these cells are almost certainly not neoplastic. The results also provide a basis for the effective use of restriction endonuclease and Southern blot/DNA hybridization analysis in the differential diagnosis of non-Hodgkin's lymphoma and thymoma.

Immunoregulatory Leu-7+ and T8+ lymphocytes in B-cell follicular lymphomas.

Lymphocyte subpopulations were profiled in lymph nodes and tonsils showing follicular hyperplasia and in follicular lymphomas with monoclonal antibodies on frozen tissue sections. Immunoregulatory lymphocyte subsets identified with T8 and Leu-7 monoclonal antibodies were quantified within the follicular centers (FC) of the nonneoplastic tissue and neoplastic follicles of the lymphomas with an optical grid defining a unit surface area (USA) of 0.04 mm2. T8+ cells were essentially confined to the interfollicular areas, with a few cells occupying the FC of the nonneoplastic specimens (mean, two and five cells/USA for tonsils and benign lymph nodes, respectively). Although lymphomas exhibited a similar pattern of distribution of T8+ cells, 17 T8+ cells/USA were observed in the follicular small cleaved cell (FSCL) group and eight T8+ cells/USA within the follicular mixed small cleaved and large cell (FML) group. Leu-7+ cells were almost entirely confined to the FC of the nonneoplastic tissues and increased (mean, 17 and 19 cells/USA for tonsils and benign lymph nodes, respectively) compared with the T8+ population. Variable distributions of Leu-7+ cells were found in the FSCL group, with a mean of 16 cells/USA. Very few Leu-7+ cells were present in the FML group. Natural killer cells and/or cytotoxic/suppressor T lymphocytes may play an immunoregulatory role in modulating the growth of follicular lymphomas.

Incidence of mixed chimerism using busulfan/cyclophosphamide containing regimens in allogeneic bone marrow transplantation.

The incidence of mixed chimerism (MC) following allogeneic bone marrow transplantation (allo-BMT) is in part a measure of the marrow ablative effect of preparative regimens. Although the incidence of MC has been reported for many patients treated with total body irradiation (TBI), limited data for busulfan/cyclophosphamide (BU/CY) recipients have been examined. We performed restriction fragment length polymorphism (RFLP) analysis on 68 peripheral blood samples from 26 patients treated with BU/CY prior to allo-BMT for chronic myelogenous leukemia or acute myeloid leukemia. MC was detected in four of 26 patients for an overall incidence of 15.4%. Three of four MC patients are alive with no evidence of disease at 263 to 795 days post-transplantation. A fourth patient is alive at day 501 but developed CNS relapse at day 274. The level of recipient origin cells was less than 10% in all samples and detectable MC was transitory with an RFLP pattern that reverted to full chimerism. These results are comparable to those reported for TBI-containing regimens in patients receiving non-T cell-depleted bone marrow. The efficacy of BU/CY in conjunction with a T cell depletion still requires exploration.

The expression of CD22 (Leu 14) and CD11c (LeuM5) in chronic lymphoproliferative disorders using two-color flow cytometric analysis.

The monoclonal antibodies (MoAbs) CD22 and CD11c recognize B-lymphocyte- and monocyte-associated antigens, respectively. Reports indicate that when these two MoAbs co-express, they represent a unique marker for hairy cell leukemia (HCL) although neither is specific for that disease. The authors evaluated the expression and diagnostic utility of CD22 and CD11C in specimens from 26 normal subjects, 29 patients, with various nonlymphoproliferative disorders (NLPDs), and 75 patients with different types of chronic lymphoproliferative disorders (CLDs) using two-color flow cytometric analysis of peripheral blood lymphocytes. Lymphocytes co-expressed CD22 and CD11c in less than or equal to 3% of the normal subjects and in less than or equal to 6% of the patients with NLPDs. These markers were expressed in greater than 10% of the lymphocytes of 46% (32/69) of the patients with B-cell CLDs: B-cell chronic-lymphocytic leukemia, 9/41; B-cell non-Hodgkin's lymphoma, 8/14; HCL, 11/11; B-cell lymphoproliferative disorder (NOS), 1/2; and B-cell prolymphocytic leukemia, 1/1. None (0/6) of the lymphocytes of patients with T-cell CLDs expressed greater than 10% CD22-positive (CD22+) or CD11c-positive (CD11c+) cells. The HCL cases demonstrated a unique CD22+CD11c+ fluorescence histogram pattern, distinct from other lymphoproliferative disorders, that was characterized by uniformly intense CD11c and CD22 fluorescence. Differences in the expression of the CD22+CD11C- and CD22+CD11C+ phenotypes between diagnostic groups were found, most notable was a paucity of CD22+CD11c+ cells in lymphocytes of patients with HCL. CD22 also had more variable expression than CD19 and HLA-DR in the cases of B-cell CLD. This study demonstrates that the CD22+CD11c+ phenotype is not unique to HCL but is a consistent feature of that disorder and that the immunofluorescence pattern of co-expression in HCL is diagnostically useful.

Frozen sections of cellular lymphoid proliferations provide adequate DNA for routine gene rearrangement analysis.

Optimal use of frozen tissue procured as part of a thorough diagnostic workup of suspected lymphoma is important, and conservation of similar samples is a prerequisite for maintaining a large and varied frozen archive repository. The authors have evaluated a simple tissue-conserving method for the preparation of cellular lymphoid specimens for immunoglobulin and T-cell receptor gene rearrangement analysis. Initially, 16-microns-thick frozen tonsil sections were examined to determine adequacy for DNA extraction. Specimens containing three, six, and nine sections each were evaluated separately. DNA quantitation disclosed yields ranging from 84 to 204 micrograms (mean, 156 micrograms). The authors have used this technique on 24 cellular lymphoid proliferations from their frozen archives. Six to ten 16-microns sections were used, depending on tissue size. DNA quantitation ranged from 0 to 520 micrograms (mean, 135 micrograms). Twenty-one of 24 cases yielded adequate DNA for analysis; each showed appropriate germline or rear-ranged bands with respect to the particular morphologic diagnosis. Attempts to obtain adequate DNA with the use of this technique on skin biopsy specimens with lymphoid infiltrates resulted in overall poor yields; this may be because of dermal collagen or small sample size. This method of sample preparation provides adequate DNA for routine Southern blot hybridization analysis of cellular lymphoid tissues and offers the additional advantage of allowing preservation of frozen tissue for future study.

Immunotypic and genotypic characterization of non-Hodgkin's lymphomas of the ovary.

Ovarian non-Hodgkin's lymphomas (NHLs) are rare, and accurate diagnosis is frequently problematic. Previous studies have not provided either complete immunotypic or genotypic analyses. The authors report immunotyping and genotyping of three cases of ovarian NHL, including both primary and secondary types. Immunotyping disclosed all three were B-cell lymphomas composed of secretory blast stage lymphocytes showing kappa immunoglobulin (Ig) light chain clonal excess. DNA extracted from frozen tissue of each tumor was subjected to restriction endonuclease digestion and hybridized to probes for Ig genes, C kappa, C lambda, JH, and the T-cell receptor beta-chain gene. Rearrangements of the heavy chain and light chain Ig genes were observed in all three cases, confirming the monoclonal B-cell origin of the neoplastic population. No detectable rearrangements were observed in DNA extracted from three nonlymphoid ovarian tumors (dysgerminoma, granulosa cell tumor, and fibrothecoma). This study documents the potential value of immunotyping and genotypic analysis in the study of ovarian tumors.

CD5- B-cell lymphoproliferative disorders presenting in blood and bone marrow. A clinicopathologic study of 40 patients.

We studied 40 patients with CD5- B-cell lymphoproliferative disorders (B-LPDs) presenting in blood or bone marrow and 28 control patients with CD5+ B-cell chronic lymphocytic leukemia (CLL). Fifteen study patients had morphologic features typical of CLL. The 15 patients with CD5- CLL were older and had lower absolute lymphocyte counts and more advanced-stage disease at diagnosis than controls. Ten study patients had morphologic features suggesting mantle cell lymphoma (MCL); 3 were later given a diagnosis of MCL based on lymph node biopsy results. The 10 patients with CD5- MCL were older and at a more advanced stage than CLL control patients. The remaining 15 study patients were given the following diagnoses: circulating non-Hodgkin lymphoma, 5; splenic lymphoma with villous lymphocytes, 5; lymphoplasmacytoid lymphoma, 3; and CLL/pro-lymphocytic leukemia, 2. For the patients with CD5- B-LPDs with morphologic features and manifestations resembling CLL, we prefer the term CD5- CLL variant because of clinical and immunophenotypic differences. Patients with CD5- B-LPDs with atypical nuclear morphologic features may represent the leukemic phase of MCL. Since CD23 is expressed in most patients with CD5- B-LPD, its use in subclassifying these disorders seems limited.

Cytoplasmic inclusions in leukocytes. An unusual manifestation of cryoglobulinemia.

Cryoglobulins are circulating immunoglobulins characterized by reversible, cold-induced precipitation. A variety of laboratory abnormalities, including hypocomplementemia, elevated erythrocyte sedimentation rate, rheumatoid factor activity, pseudoleukocytosis, and pseudothrombocytosis, are associated with cryoglobulinemia. Extracellular, faintly basophilic, amorphous deposits of cryoglobulins occasionally have been described in blood smears. In the present study, smears prepared from blood collected at room temperature from 6 patients with cryoglobulinemia exhibited neutrophil and, occasionally, monocyte inclusions containing clear, light pink, or faintly basophilic amorphous material. The inclusions were absent in smears from blood collected and maintained at 37 degrees C. Ultrastructural examination revealed that the material within the leukocyte inclusions was consistent with phagocytosed immunoglobulins. The identification of characteristic cytoplasmic inclusions in leukocytes may be an important clue in the early recognition of cryoglobulinemia.

Immunophenotypic characterization of acute leukemia by immunocytology.

The potential for specific immunophenotypic characterization of the acute leukemias has been enhanced greatly by the development of monoclonal antibodies. Currently, this immunologic data is obtained most commonly by flow cytometric analysis or cellular cytotoxicity assays. The former is an expensive technic that lacks morphologic evaluation unless cell sorting is performed. The latter precludes morphologic assessment by the nature of the assay. The authors have developed an immunostaining procedure utilizing cytospin preparations and immunoperoxidase methods that are relatively inexpensive and allow simultaneous assessment of the immunologic markers and cellular morphology. Although a comparison of flow cytometry and immunocytology revealed quantitative differences for individual cell surface markers, the "qualitative" immunologic phenotype of the leukemic population was virtually identical by the two technics.

In situ hybridization analysis of lymphoproliferative disorders. Assessment of clonality by immunoglobulin light-chain messenger RNA expression.

Determination of clonality in B-cell lymphomas is a useful diagnostic adjunct. In situ hybridization (ISH) for the detection of kappa and lambda mRNAs has the potential to overcome some common specimen-related limitations in clonal assessment. Tritium-labeled antisense cRNA probes directed at conserved segments of the constant regions of the kappa and lambda mRNAs were used in an autoradiographic method to detect B-cell clonality. Using these probes, we analyzed 103 formalin-fixed, paraffin-embedded biopsy samples, and the results were subsequently compared to available immunophenotypic (all cases) and genotypic (50 cases) data. Of 103 samples, 82 (80%) had adequate RNA preservation as determined by actin RNA signals, and 73 (89%) of the 82 cases demonstrated concordant clonality assignment by both ISH and immunophenotyping. The remaining nine cases showed a specific form of discordance in that each exhibited no protein (Ig) expression but had evidence of mRNA immunoglobulin light-chain expression. Forty-five (90%) of 50 cases evaluated for immunoglobulin and T-cell receptor beta-gene rearrangements demonstrated concordant results with respect to clonality assignment by ISH. Thus, ISH demonstrates adequate sensitivity with respect to traditional methods of clonality assessment. However, its practical utility awaits the development of nonradioactive detection methods with adequate sensitivity to improve turnaround time.

Educating residents for managed care: report on a multidisciplinary conference.

A growing number of residency programs are preparing their graduates for the realities of managed care practice. In 1996, The Cleveland Clinic Foundation, a private, nonprofit academic medical center, hosted a two-day conference on managed care education to develop innovative instructional and evaluative approaches that, where appropriate, would build on existing expertise. The conference was attended by invited national experts who had a stake in residents' education: clinical faculty, residents, medical educators, executives of managed care organizations, and representatives of other interested organizations. Participants spent much of their time in four small break out groups, each focusing on one of the following topics that were judged particularly relevant to managed care: preventive and population-based medicine, appropriate utilization of resources, clinician-patient communication, and interdisciplinary team practice. Participants shared existing materials, discussed teaching goals and objectives, and generated ideas for teaching methods, teaching materials, and evaluative methods for their respective topics. The authors summarize the recommendations from the four groups, with an overview of the issues that emerged during the conference concerning curriculum development, integration of managed care topics into existing curricula, staging of the curriculum, experiential teaching methods, negative attitudes and resistance, evaluation of trainees and profiling, program assessment, faculty development, and cooperation between academic medical centers and managed care organizations.

Immunoglobulin and T-cell receptor gene rearrangement. Implications for the diagnosis of lymphoproliferative disorders.

Recent studies have significantly broadened understanding of immunoglobulin production and T-cell receptor formation at the gene level. A limited number of genes can be rearranged during the course of B-cell or T-cell development to yield unique DNA sequences that will code for specific antibodies and T-cell receptor proteins. Southern blot hybridization analysis allows sensitive examination of lymphocyte DNA for the presence or absence of gene rearrangements. General mechanisms that underlie immunoglobulin and T-cell receptor rearrangement are reviewed, along with the diagnostic applications of detection of gene rearrangement by Southern blot hybridization technique.

Oncogenes and cancer: clinical applications.

Oncogenes are aberrant forms of proto-oncogenes, which are normal cellular genes that participate in cell growth and development; proto-oncogenes contribute to tumor formation when mutations or chromosomal translocation cause them to escape normal controls. Anti-oncogenes, also involved in neoplasm development, normally participate in inhibition of cell growth and proliferation; they become tumorigenic when mutations alter their function. Oncogene or anti-oncogene abnormalities have been characterized for a variety of tumors, with resulting clinical applications. In some forms of leukemia, for example, determining the presence or absence of the bcr-abl gene rearrangement has both diagnostic and prognostic value. The best-studied anti-oncogene is that found in retinoblastoma. Molecular techniques can differentiate the hereditary from the nonhereditary form of this disease and, with hereditary retinoblastoma, predict disease likelihood in family members.

Lambda light chain myeloma with pleural involvement.

A case is reported of lambda light chain multiple myeloma complicated by a myelomatous pleural effusion. Pleural effusions are uncommon in multiple myeloma, and most are secondary to nonmalignant causes. The clinical characteristics, natural history and pathophysiology of myelomatous pleural effusions are reviewed.

Lymphocytic alveolitis in primary HIV infection.

Primary infection with the human immunodeficiency virus (HIV-1) has been associated with a self-limited illness resembling acute infectious mononucleosis. Pulmonary manifestations have been notably absent in published reports. The authors describe a 28-year-old homosexual male who presented with primary HIV-1 infection associated with CD8+ lymphocytic alveolitis. Diagnosis was delayed because HIV antibody was not detected by the Abbott ELISA, although the same and subsequent specimens were later found to be positive by Genetic Systems' ELISA and Western blot analysis. Lymphocytic alveolitis must be added to the expanding clinical spectrum of acute HIV-1 infection. The time to detection of seroconversion may vary with different immunoassays.