Implementing multiplexed genotyping of non-small-cell lung cancers into routine clinical practice.

BACKGROUND: Personalizing non-small-cell lung cancer (NSCLC) therapy toward oncogene addicted pathway inhibition is effective. Hence, the ability to determine a more comprehensive genotype for each case is becoming essential to optimal cancer care. METHODS: We developed a multiplexed PCR-based assay (SNaPshot) to simultaneously identify >50 mutations in several key NSCLC genes. SNaPshot and FISH for ALK translocations were integrated into routine practice as Clinical Laboratory Improvement Amendments-certified tests. Here, we present analyses of the first 589 patients referred for genotyping. RESULTS: Pathologic prescreening identified 552 (95%) tumors with sufficient tissue for SNaPshot; 51% had ≥1 mutation identified, most commonly in KRAS (24%), EGFR (13%), PIK3CA (4%) and translocations involving ALK (5%). Unanticipated mutations were observed at lower frequencies in IDH and ß-catenin. We observed several associations between genotypes and clinical characteristics, including increased PIK3CA mutations in squamous cell cancers. Genotyping distinguished multiple primary cancers from metastatic disease and steered 78 (22%) of the 353 patients with advanced disease toward a genotype-directed targeted therapy. CONCLUSIONS: Broad genotyping can be efficiently incorporated into an NSCLC clinic and has great utility in influencing treatment decisions and directing patients toward relevant clinical trials. As more targeted therapies are developed, such multiplexed molecular testing will become a standard part of practice.

Selective deficiency of immunoglobulin A2.

A case of familial selective IgA2 deficiency is described. The mother had no detectable IgA2, but a low level of IgA1. She had anti-alpha 2 antibodies of the IgG class. One of her daughters also lacked IgA2 with a normal level of IgA1. The analysis of the immunoglobulin haplotypes of the family suggested the deletion of the alpha 2-gene. In addition, the analysis of B lymphocytes of mother and daughter showed the absence of IgA2-bearing cells. Upon stimulation with pokeweed mitogen, the B cells differentiated into IgA1-containing plasma cells, but IgA2-containing cells were not found. The results suggest a defect in the generation of intraclonal B cell isotype diversity. The molecular basis of this phenomenon is unknown.

Radioresistant pseudo-colony formation in the PHA-leukocyte feeder colony assay.

In the PHA-leukocyte feeder colony assay--a fluid assay on top of an agar underlayer--colonies might not be the product of clonogenic cells but rather from aggregates, as was already shown for hairy cell leukemia (Leukemia Res. 11, 911 (1987)). To study the role of aggregation in this colony assay in other B-cell malignancies, we irradiated cells from B-chronic lymphocytic leukemia, B-non-Hodgkin's lymphoma and multiple myeloma. In nearly all cases, viable "colonies" were seen after irradiation, albeit in lower numbers. These data indicate that in the PHA-leukocyte feeder colony assay, a considerable percentage of colonies from a large variety of B-cell malignancies originate from aggregating rather than from proliferating cells.

Monoclonal gammopathy as a model for B-cell differentiation: from "paraprotein" to artificial antibodies.

During the past fifty years new biochemical and cell biological techniques have been introduced in clinico-pathological practice. Studies on the bone-marrow related malignancy multiple myeloma (MM) proved particularly rewarding for the elucidation of basic concepts in immunology. In fact, by studying the characteristic monoclonal serum components, hallmarking this disease, the basic structure of immunoglobulins and their various types could be described. Two examples of the impact of using MM as a model system in studies on immunological differentiation will be presented: the involvement of precursor cells and the homing of malignant plasma cells in MM. Conversely, two examples of the growing impact of immunology on the clinical handling of MM will be given: new differential diagnostic procedures as well as novel developments in immunotherapy of MM.

Cost-effective detection of non-antidouble-stranded DNA antinuclear antibody specificities in daily clinical practice.

OBJECTIVES: To compare the utility of indirect immunofluorescence for the detection of antinuclear antibodies (ANA-IIF) and a fully automated test (ELiA Symphony) that detects antibodies against a mixture of nuclear and cytoplasmic antigens (ENA), to select sera that should be tested for non-antidouble-stranded DNA (dsDNA) antinuclear antibodies in a relatively expensive automated line immunoassay (INNO-LIA ANA update, Lineblot). METHODS: All 328 sera sent to the laboratory for ANA or anti-ENA tests, over a 4 month period were evaluated in all three assays. Results were related to signs and symptoms of systemic autoimmune disease (AID) that patients had before or at the time of blood sampling. RESULTS: Overall, 72 (22%) sera were Lineblot positive. Of 198 patients without clinical manifestations of AID, 7% were Lineblot positive. Limiting Lineblot to sera positive in either ANA-IIF or Symphony tests failed to detect 26 (ANA-IIF) and 22 (Symphony) Lineblot-reactive sera, with 15 sera being negative in both assays. From a clinical point of view, failure to detect these reactivities was not important in most cases. CONCLUSIONS: Restriction of performance of Lineblot to patients with at least one criterion for AID is an ideal and cost-effective strategy. In ignorance of clinical signs and symptoms, screening of sera by ANA-IIF or Symphony strongly reduces the costs of anti-ENA detection, with minimal loss in diagnostic capacity. Based on small differences, including the fact that anti-dsDNA antibodies give a positive ANA-IIF, we prefer screening with ANA-IIF over Symphony.

Utilization of nitrogen compounds and ammonia assimilation by Chromatiaceae.

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH4Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina. Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrated in the above organisms grown on NH4Cl.

Associated autoimmunity in Addison's disease.

As the last extensive series of patients with Addison's disease and coincident autoimmune phenomena were published approximately two decades ago, we studied the cause of the disease, the prevalence of autoimmune disorders and the frequency of occurrence of autoantibodies in 91 patients (31 men and 60 women, mean age 45.3-years-old, range 12-77) with Addison's disease. The cause of Addison's disease in six patients was tuberculosis (6.6%), and autoimmune adrenalitis was considered to be the cause in 83 patients (91.2%). In two patients (2.2%) other causes were responsible for Addison's disease. In 47% of the patients with autoimmune Addison's disease at least one other autoimmune disorder was present. Primary hypothyroidism had the highest prevalence (20.5%), followed by vitiligo (9.6%), non-toxic goiter (8.4%), premature menopause (7.3% of the women), Graves' disease (6%), pernicious anaemia (4.8%), Sjögren's disease (2.4%), hypoparathyroidism (1.2%), type 1 diabetes mellitus (1.2%) and coeliac disease (1.2%). The frequency of autoantibodies in the patients with autoimmune Addison's disease was: adrenal antibodies (82.7%), antibodies against microsomal antigens (58%), thyroglobulin antibodies (23.4%), parietal cell antibodies (19.8%), pancreatic islet cell antibodies (6.2%) and ovary antibodies (3.7% of the women). In comparison with other extensive series of patients with Addison's disease, we found the highest prevalence of autoimmune adrenalitis as the cause of Addison's disease, the highest prevalence of hypothyroidism and vitiligo as concomitant autoimmune disorders and the lowest prevalence of type 1 diabetes mellitus.

T cells in B cell chronic lymphocytic leukaemia. I. Decreased frequency of T lymphocytes secreting suppressor factor.

A reverse T cell plaque assay was employed to study the ability of purified T cells isolated from the blood of seven patients with B cell chronic lymphocytic leukaemia (B-CLL) to secrete antigen-specific helper or suppressor factors (ThF and TsF respectively) after activation in vitro. It was found that in spite of the phenotypical presence of CD8+ cells, the frequency of TsF-secreting cells was strongly decreased as compared to normal values. T cells secreting ThF could be generated in all B-CLL patients tested in about normal frequencies. These results may indicate a tumour induced change in the distribution of cellular subsets within the CD8+ cell compartment.

Idiotypic lymphocytes in the rabbit: occurrence and nature of idiotypic lymphocytes in normal and hyperimmunized rabbits.

Rabbits were hyperimmunized with Micrococcus Pysodeicticus, leading to homogeneous antibody responses. Peripheral blood lymphocytes were taken from the rabbits before and monthly (during 3 months) after the start of the immunization. The cells were stored frozen. Lymphocytes were tested with anti-idiotypic conjugates for the presence of surface idiotypic structures. The nature of the idiotype-positive cells was determined with respect to the presence of IgM or T-cell antigenic determinants on their surface. A sharp rise and fall in the percentage of idiotypic lymphocytes was found, ranging between 1/40,000 and 1/1,000. Initially almost all idiotypic lymphocytes were IgM-positive. In the blood taken 2 months after the start of the immunization 20% of the idiotypic cells belonged to the T-cell population and 10% were negative for both IgM and T-cell antigenic determinants.

Mitogenic stimulation of malignant B cells. Chronic lymphocytic leukaemia: secretion of monoclonal IgM by in vitro-induced plasmablasts.

This study provides evidence that leukaemic B cells are able to proliferate and differentiate into plasmablasts. The Ig produced was monoclonal in nature and this was shown by gel electrophoresis.

VAD chemotherapy for refractory multiple myeloma.

Thirty-four patients with refractory multiple myeloma were treated with 4-d continuous infusions of vincristine and adriamycin in combination with 4-d pulsed high-dose dexamethasone (VAD). Of 31 evaluable patients, 16 entered a complete remission (50%) and three a partial remission (10%). No difference in response rate was observed between primary refractory and relapsed patients. The median response duration was 9 months and the median survival of the responding patients was 12 months versus 4 months for the non-responders. Ten patients have currently survived longer than 360 d, of which six are stable in complete remission without therapy. All responding patients showed a remarkable improvement of their performance status and 70% of these patients became pain-free. Bacterial infection was the major complication and was probably due to the intensive corticosteroid programme. Severe myelosuppression was rarely observed. Irrespective of the response to VAD, a high beta 2-microglobulin of 4 micrograms/ml or more was a bad prognostic parameter. As early relapses were seen especially in this group of patients, in the patients with a plasma-cell LI% of 3 or more, and in patients with previous anthracyclin treatment, early consolidation, with, for instance, high dose melphalan, might improve the prognosis for these patients.

In vitro and in vivo measurement of cell-mediated immunity in patients with HIV-1 infection.

We studied the usefulness of the in vitro lymphoproliferation assay and the in vivo skin test in HIV-1-infected patients by using Clostridium tetani and tuberculin as testing antigens. Moreover, the relationship between data obtained from both assays was studied. In 56 HIV-infected patients not receiving antiretroviral therapy CD4+ cell counting was performed. In addition, in vitro (lymphocyte proliferation assay) and in vivo (delayed type hypersensitivity skin test) measuring of the immune status was done using C. tetani and tuberculin as testing antigens. When using C. tetani a significant correlation between the results of both tests and the CD4+ cell count was found. In contrast to earlier reports from African countries, in vivo skin testing using tuberculin did not yield clinically significant information on the degree of immunodeficiency. We explain our findings by the fact that health care policy in The Netherlands encompasses vaccination with C. tetani, which enables the application of C. tetani as testing antigen for measuring immune function both in vitro and in vivo.

Phenotypical and functional characterization of the idiotype-positive blood B cells in multiple myeloma.

In this study the idiotype-positive B cells of one patient with smouldering multiple myeloma (IgG kappa) and of one patient with multiple myeloma (IgG lambda) were analysed phenotypically and functionally. As regards the expression of B cell-associated differentiation antigens and size distribution, the idiotype-positive B cells resembled normal IgG-bearing blood B cells. In functional studies the lymphocytes were cultured in vitro with Staphylococcus aureus, pokeweed mitogen, T-cell factors, or combinations of these. After culture, proliferation and differentiation of the idiotype-positive B cells were measured by autoradiography, an idiotype-specific ELISA, and a spot ELISA. The results show that idiotype-positive B cells of both patients are able to proliferate after stimulation in vitro. In contrast to their normal counterparts, however, almost no increase in the amount of secreted idiotype IgG could be induced. This suggests that the idiotype-positive blood B cells have lost some of their ability to respond to exogenous stimuli.

Release of cytokines and soluble cell surface molecules by PBMC after activation with the bispecific antibody CD3 x CD19.

Bispecific antibodies (BsAb) consist of two different heavy and light chains and may bind to two different antigens present on different cell types. With their dual specificity BsAb may recognize effector cells (e.g. T cells) on one hand and tumour cells (e.g. malignant B cells) on the other hand. The authors analysed whether T cell activation and subsequent killing of malignant B cells mediated by the bispecific antibody CD3 x CD19 was reflected by the release of cytokines. In addition, the authors investigated whether the in vitro cytokine release was similar to that observed in vivo in the patients treated with BsAb. The in vitro release of cytokines into the supernatant of cell cultures of peripheral blood mononuclear cells (PBMC) and malignant B cells was measured after incubation with either the bispecific antibody CD3 x CD19 or the monospecific anti-CD3 (aCD3) antibody in the presence or absence of interleukin (IL)-2. Release of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-6, IL-8, IL-10, soluble (s) CD4, sCD8 and sCD25 by PBMC was equal under both conditions and could be used as an indicator for T cell activation. However, the cytokine pattern and level did not correlate with the cytotoxic capacity, which was 4 logs higher with BsAb + IL-2 compared to aCD3 + IL-2. The in vitro pattern of cytokine release was similar to that observed in vivo in the serum of patients treated with BsAb and IL-2, indicating the possibility of predicting cytokine release in future patients with other therapeutic regimens.

Large-scale production of natural cytokines during activation and expansion of human T lymphocytes in hollow fiber bioreactor cultures.

We studied the large-scale production of a variety of natural cytokines during the activation and expansion of human T lymphocytes in a hollow fiber bioreactor culture system. Peripheral blood mononuclear cells (PBMC) were activated using phytohemagglutinin plus recombinant interleukin-2 (IL-2). Phytohemagglutinin was either present in the hollow fiber bioreactor during the entire 15-16-day culture period or only during the 20-h preactivation of the PBMC in culture bags. The expanding T lymphocytes were mainly CD3+,8+ and exerted maximal natural, activated, bispecific monoclonal antibody-redirected and lectin-dependent cytolytic activities between days 9 and 13 of culture. IL-1 and IL-4 were only produced in low amounts. IL-8 and lymphotoxin were primarily produced during the first week of culture. Harvest of the hollow fiber bioreactor culture supernatant at the time of peak cytokine concentration would have yielded per 10(8) PBMC input between 3.7 and 4.9 micrograms of IL-8 (at days 2 or 3), and between 0.02 and 0.5 microgram of lymphotoxin (at days 6 or 7). Tumor necrosis factor-alpha and IL-6 were produced during the entire culture period of 15 or 16 days: per 10(8) PBMC input, between 0.1 and 0.4 microgram of tumor necrosis factor-alpha (at days 2 or 3) and between 0.03 and 0.5 microgram of IL-6 (at days 15 or 16). Production of interferon-gamma and granulocyte-macrophage colony-stimulating factor started from initiation of cultures onwards to reach peak levels at the end of the 15- or 16-day culture period, yielding at that time between 2.1 and 17.7 micrograms/ml of interferon-gamma and between 0.4 and 4.2 micrograms of granulocyte-macrophage colony-stimulating factor per 10(8) PBMC input. The production of tumor necrosis factor-alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor was proportional to the extent of lymphocyte multiplication. These results demonstrate the usefulness of hollow fiber bioreactor cultures to produce natural cytokines during the activation and expansion of predominantly CD3+,8+ T lymphocytes.

Mitogenic stimulation of malignant B cells. Chronic lymphocytic leukaemia: relationship between stimulation and surface phenotype.

Peripheral blood lymphocytes (PBL) from 14 patients with chronic lymphocytic leukaemia (CLL) were stimulated with pokeweed mitogen (PWM), formalinized Staphylococcus aureus (Sta) and a combination of both mitogens. The leukaemic B cells were characterized by rosetting techniques (using mouse erythrocytes and complement coated erythrocytes) and immunofluorescence for membrane bound immunoglobulin (mIg). No clear correlation between phenotype and the reactivity with PWM could be found. Results of stimulation with Sta however, indicate that lymphocytes carrying membrane bound IgM (mIgM) and IgD (mIgD) and the receptor of the third complement component (C3R) can be induced to differentiate into immunoglobulin (Ig) containing cells. Addition of PWM to these cultures often enhanced this response. Some leukaemic B cells are able to differentiate after challenge with the appropriate stimulus.

Differentiation between benign and malignant monoclonal gammopathy by the presence of the J chain.

Malignant monoclonal gammopathy (MMG) cannot easily be distinguished from the benign (BMG) form in a number of cases. Using two different anti-J chain reagents it could be shown in the present study that in MMG, J chain is present in most of the IgG-containing plasma cells in the bone marrow. In BMG, the frequency of J chain-containing IgG plasma cells is low and not different from that obtained in healthy individuals. Essentially similar findings regarding idiotype were obtained, using anti-idiotype conjugates, specific for the patients' monoclonal immunoglobulins. In one patient malignancy was indicated by the presence of a high percentage of J chain-containing IgG plasma cells in the bone marrow, prior to clinical expression of multiple myeloma.