Comparative analysis of prognostic factors in men undergoing radical prostatectomy for adenocarcinoma of the prostate, including DNA ploidy, surgical tumor stage, prostatic specific antigen, Gleason grade, and age.

One hundred consecutive men with adenocarcinoma of the prostate, treated by modified pelvic lymphadenectomy and radical retropubic prostatectomy, were evaluated, comparing DNA ploidy as determined by flow cytometry to surgical tumor stage (pT), preoperative prostatic specific antigen (PSA), Gleason grade, and age at presentation, in an effort to assess the prognostic ability of DNA ploidy. There were 71 (71%) men found to have diploid tumors and 29 (29%) with nondiploid tumors. There was no statistical difference in surgical pathologic stage between these two groups (P = 0.2369). There was no statistical difference when comparing preoperative PSA between these two groups (P = 0.0925). There was no statistical difference when comparing Gleason grade between these two groups (P = 0.5807). Age at presentation was similar in both groups. Based on these findings, it is apparent that longitudinal studies of patient outcome will be necessary to fully assess the prognostic ability of DNA ploidy determined by flow cytometry in men undergoing radical prostatectomy for treatment of adenocarcinoma of the prostate gland.

DNA heterogeneity determined by flow cytometry in prostatic adenocarcinoma--necessitating multiple site analysis.

Although DNA ploidy analysis of prostate cancer is generally associated with grade, stage, clinical outcome, and responsiveness to androgen therapy, one possible reason cited for contrary reports may be tumor heterogeneity. A preliminary report using flow cytometric analysis of punch biopsies demonstrated DNA heterogeneity in five of nine patients. We evaluated 75 patients by cutting whole mounts of formalin fixed prostatectomy tissue every 0.6 cm. All malignant areas and a selected normal area were circumscribed, excised, remounted, and 1-3 50 mu thick sections removed. The nuclei were extracted by a Hedley technique and the DNA stained with propidium iodide. Each whole mount had an average of 1 distinct malignant area (range of 1-6 areas per whole mount block). Nuclei were analyzed on a Becton Dickinson (San Jose, CA) FACScan flow cytometer equipped with RFIT DNA software program. After excluding histograms with CVs > 8.0% and/or "suspicious" diploid histograms having a right "shoulder," 75 or 87 patients still had > or = 2 malignant sites available for analysis (average 4, range 2-9 malignant sites/patient). The 322 histograms had an average CV of 4.4%. Thirty of 75 patients (40%) showed DNA heterogeneity in multiple samples taken from the same prostate. There were 37 prostates with only diploid (D), 1 with only tetraploid (T), 7 with only aneuploid (A), 20 with D plus A, 7 with D plus T, 2 with D plus T plus A, and 1 with a D plus suspected hypodiploid DNA content. Exclusion of the tetraploid and "near diploid aneuploid" cases still resulted in 16% (12/75) of the patients having a diploid versus aneuploid DNA content heterogeneity. Because 40% of the prostates contained a different ploidy depending on which area was sampled, this report suggests multiple sites of malignancy must be analyzed to more accurately assess the ploidy status of prostatic adenocarcinoma.

CD3+, CD56+ non-MHC restricted cytotoxic T lymphocytes in two fraternal AIDS patients: a case report.

We describe two brothers with hemophilia and AIDS, with an unusually large percentage of CD3+, CD56+ lymphocytes. They experienced no major complications with opportunistic infections (OI) and infrequent secondary infections, even though they had nearly 0% CD4 lymphocytes for 3 years. Both patients died in 1991 of progressive cardiomyopathy. The patients' lymphocytes were immunophenotyped by flow cytometry and analyzed for functional cytolytic activity against K562 and HIV infected HUT 78 cell lines. Single color CD4 counts were 2-9% for 4 years. Dual color CD4 counts at our laboratory demonstrated 0-1% CD4 for the last 6 months. When CD3+ lymphocytes were examined, both patient 1 and patient 2 demonstrated a significantly higher proportion and absolute number of CD3"bright"+, CD56+ double-positive cells, 47% and 22%, respectively, compared to other HIV-positive children with hemophilia (< or = 2%). Functional studies with the K562 target cell line demonstrated the highest natural killer (NK) lymphocyte activity in patient 1 that could not be augmented by in vitro addition of IL-2, whereas patient 2 had no NK activity unless IL-2 was added. Functional studies with HIV-infected HUT-78 cells demonstrated patient 2 had cytolytic activity against HIV-infected cells and patient 1 had high nonspecific cytolytic activity even against uninfected HUT-78 cells, whereas controls had minimal activity to HUT-78 cells or HIV-infected HUT-78 cells. The case report raises a speculative question requiring a larger database, whether the anti-HIV activity and/or unusual clinical course without typical O.I. of some AIDS patients may be related to the presence of CD3"bright"+, CD56+ lymphocytes of their immune system.

Interleukin-6 response to laparoscopic and open colectomy.

PURPOSE: It has been postulated that cortisol and interleukin-6 play a significant role in the modulation of the early inflammatory response following surgical intervention. There are no available data on the normal responses of these mediators following major laparoscopic procedures. The purpose of this study was to assess changes in cortisol (by fluorescence polarization immunoassay), interleukin-6 (by enzyme-linked immunoassay), and interleukin-1 (by enzyme-linked immunoassay) after elective laparoscopic colon resections. METHODS: All patients undergoing colon resection between February 1, 1992 and April 30, 1992 were eligible for study. Selection of laparoscopic (N = 12) vs. open (N = 41) resection was determined by the attending surgeon. All patients received a standard general anesthetic with endotracheal intubation. Cortisol, interleukin-6, and interleukin-1 were measured at preinduction, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours after the induction. Interleukin-6 and interleukin-1 were additionally measured at 12 hours, 24 hours, and 72 hours after induction. Comparisons were made between the laparoscopic patients (N = 12) and age, sex, and operation-matched open patients (N = 12). RESULTS: Cortisol levels rose in the early postoperative period in both open and laparoscopic groups with no significant differences occurring between the cohorts at any of the measured time intervals. The interleukin-6 levels of the laparoscopic cohort (N = 12) were significantly lower than those of the open cohort (N = 12) between 3 and 24 hours postinduction (P < 0.05). Interleukin-1 levels remained undetectable in virtually all patients irrespective of operative technique or postoperative interval. There was no correlation between peak interleukin-6 levels and operative times (laparoscopic, r = 0.31; open, r = 0.36) or blood loss (laparoscopic, r = 0.10; open, r = 0.20). CONCLUSION: The results indicate that laparoscopic colon resections do not appear to alter cortisol or interleukin-1 responses when compared with open colon resection. There is, however, a significant blunting of the interleukin-6 response associated with the use of laparoscopic techniques for colectomy compared with standard laparotomy.

Low level CD20 expression on T cell malignancies.

Although initially thought to be a B lineage restricted antigen, low "density" or antibody binding capacity (ABC) CD20 has recently been detected on subset(s) of normal T lymphocytes (Hultin et al.: Cytometry 14:196-204, 1993). We report low ABC CD20 expression in three (two children, one adult) cases of T-acute lymphoblastic leukemia (T-ALL). CD20 and other pertinent antigens were detected using a direct dual color method with a Becton Dickinson FACScan flow cytometer and Simulset software. Only one cell population based on light scatter was noted in each case that immunophenotypically represented almost a pure population of malignant cells expressing T lymphocyte antigens (for example, CD7 98%, 92%, and 100%, respectively). A total of 95%, 87%, and 79% of the cells from the three cases expressed CD20 with an unusual low ABC compared to the customary "bright" CD20 expression on normal B lymphocytes. Other B lymphocyte associated antigens, such as CD19, CD22, Dr, and immunoglobulin light chains, were negative. Eleven other T lymphocytic malignancies from 1991 to 1993 were CD20 negative, including three other case of T-ALL (one adult and two children). One unusual case of intestinal small lymphocytic non-Hodgkin's lymphoma with a natural killer/T lymphocytic immunophenotype not described in this report appeared to be CD20"dim"+. Low ABC CD20 expression by T lymphocytic malignancies may provide a more unique immunophenotypic "fingerprint" to help support the diagnosis of T cell neoplasia vs. normal/reactive T cells (for example, low ABC CD20 cells represent only 2.4 +/- 1.5% of normal peripheral blood lymphocytes). This characteristic might also facilitate monitoring patients for residual or recurrent disease.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA analysis by flow cytometry of paraffin embedded core biopsies of the prostate.

The majority of literature concerning DNA analysis of prostate cancer involves testing formalin-fixed prostatectomy tissue, fresh or formalin-fixed transurethral resections of the prostate (TURP), or fresh core biopsies. We were interested if flow cytometry could analyze the DNA of formalin-fixed, paraffin-embedded, core biopsies separated into normal versus malignant segments. Of the 50 potentially available samples for analysis representing 11 controls of normal core tissue and 39 core biopsies from the 11 patients, one patient had no normal tissue, one core had no malignancy, and three cores had no tissue visibly remaining in the paraffin blocks for analysis. Therefore, of 45 actual samples available for processing, sometimes representing segments as small as 0.2 cm, separate segments containing malignant glands or normal glands were excised from the blocks, and processed separately by the Hedley technique. Forty-four of the 45 available samples produced interpretable DNA histograms as defined by discernible G0/G1 peaks, a calculable cell cycle analysis, and the qualitative appearance of a "smooth" histogram appearance, reflecting sufficient nuclei were analyzed. This is the first report to our knowledge where flow cytometry has successfully been used to analyze paraffin blocks of core biopsies which were, in addition, separated into malignant versus normal enriched segments.

Cell-mediated immunity in allopurinol-induced hypersensitivity.

Allopurinol may induce severe hypersensitivity characterized by hepatitis, interstitial nephritis, and skin rash. The mechanisms for this hypersensitivity syndrome are incompletely elucidated. Immunologic studies were performed on tissue and peripheral blood lymphocytes from a patient with allopurinol hypersensitivity. Immunohistochemistry was performed on sections of the liver biopsy utilizing monoclonal antibodies for T and B lymphocytes. Peripheral blood lymphocyte immunophenotyping by flow cytometry and peripheral blood lymphocyte stimulation studies with either allopurinol or oxypurinol measured as tritiated thymidine uptake were performed in the hypersensitive patient and compared to a group of six patients treated with allopurinol without hypersensitivity and eight normal control patients. Additional single- and dual-color immunophenotyping by flow cytometry of oxypurinol-stimulated lymphocytes was performed in the hypersensitive patient and compared to normal controls. The liver biopsy demonstrated predominantly a T lymphocyte infiltrate. The number of peripheral blood lymphocytes expressing activation antigens was significantly greater in the hypersensitive patient compared to that of both control groups. Lymphocytes from the hypersensitive patient were moderately stimulated by allopurinol and markedly stimulated by oxypurinol compared to both control groups. Oxypurinol-stimulated lymphocytes from the hypersensitive patient demonstrated enhanced expression of activation antigens compared to unstimulated lymphocytes from this patient and normal controls. These studies suggest that cell-mediated immunity directed toward allopurinol and more importantly to its oxypurinol metabolite is involved in the pathogenesis of allopurinol-induced hypersensitivity.

Orbital lymphoma associated with acquired immune deficiency syndrome (AIDS).

A new case of AIDS-associated, small noncleaved cell (Burkitt's) lymphoma that presented as an orbital mass is described. Extraocular muscle involvement was documented by computed tomography and confirmed by orbital biopsy. Extensive abdominal involvement was subsequently diagnosed and this caused the patient's death only 15 days after the initial consultation and orbital biopsy. A literature review discloses only eight previous case reports of AIDS-associated non-Hodgkin's lymphoma (NHL) involving the orbit: two cases of Burkitt's type and six cases of large cell immunoblastic type. Three additional cases of orbital NHL can be found among larger series of AIDS cases. Affected patients are uniformly young adult males with a history of homosexuality and/or IV drug abuse. Variations in clinical presentation are discussed. Although the morphology of the histopathologic specimen in our case was consistent with a high-grade, small noncleaved cell NHL, flow cytometry revealed an atypical immunophenotype for Burkitt's lymphoma in that CD20 and immunoglobulin light chain antigens were not expressed. Morphologic classification and immunophenotypic characteristics of AIDS-associated NHLs are discussed.

An acute form of T gamma lymphoproliferative disease presenting with massive splenomegaly--importance of immunophenotyping for diagnosis.

We describe a case of T gamma lymphoproliferative disease (T gamma LPD) which presented in an uncustomary acute onset in an adult with massive splenomegaly. Morphologically the cells represented monocytic leukemia. Karyotyping and equivocol special stain results suggested hairy cell leukemia. Gene rearrangement indicated a T lymphocytic malignancy. Immunocytochemistry stains were not definitive. Immunophenotyping by flow cytometry defined the cells as consistent with T gamma LPD (CD45+, CD56+, CD2+, CD3+, CD11b+, and CD38+; some cells CD8+; and CD57-). Although the cells did not have spontaneous activity, which is often the situation for most cases of T gamma LPD, the cells could be partially induced with exogenous interleukin 2 to exhibit in vitro cytotoxicity against a natural killer lymphocyte-susceptible target cell line (K562) but not a lymphocyte-activated killer target cell line (HEPG2). This report hopefully continues to increase the awareness of T gamma LPD as well as demonstrates an unusual acute form which could have been misdiagnosed unless a multidisciplinary approach, especially including flow cytometric immunophenotyping, was used to evaluate the patient.

MY4 expression on B-lymphocyte malignancies may be associated with a more adverse prognosis.

We have noted what other preliminary reports have also described as a new specificity of the MY4 monoclonal antibody from Coulter Immunology which previously was designated to only have myelogenous CD14 specificity. The MY4 marker appears to characterize a subpopulation of some B-lymphocytic malignancies that are CD19, CD20, surface immunoglobulin as well as MY4 positive. The results occurred when other myelogenous markers such as CD11b, CD13 and CD33 were unreactive. Another monoclonal antibody marker of CD14 specificity, MO2, did not demonstrate a similar reactivity. Various other monoclonal antibodies of the same IgGI subclass as MY4 were also not reactive and thereby excludes non-specific adsorption as an explanation of the results. The six patients described in this report represented five non-Hodgkin's lymphoma cases and one chronic lymphocytic leukemia case. Fifteen B-lymphocytic leukemias and 30 other B-lymphocytic non-Hodgkin's lymphomas analysed during the same period were not MY4 positive. In reviewing the clinical course of the six patients compared to the general behavior of these types of malignancies and making a speculative generalization from the small group of cases, the MY4 antigen appeared to be expressed by low to intermediate grade B-lymphocytic malignancies of a type that were more rapidly progressive and/or had a greater tendency to undergo a transformation of their malignancy. Two of the three transformed cases were proceeded by chronic lymphocytic leukemia.

An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry.

A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples. The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system. When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method. The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference. Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry. The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable. The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples. The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes. Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions. The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.

Natural killer lymphocyte blast crisis of chronic myelogenous leukemia.

We describe for the first time a case report documenting a chronic myelogenous leukemia (CML) patient who developed a blast crisis of natural killer (NK) lymphocytes. Many of the blasts exhibited large granular lymphocytic (LGL) morphology. Single parameter immunophenotyping results determined that the granulated as well as the agranulated blast cells were NK lymphocytes (CD45, NKH1, CD2, LEU 17, and CD16 positive; CD3, CD8, and LEU 7 negative). Dual parameter flow cytometric testing also determined that some of the blasts expressed the CD11b and CD11c markers as reported for some types of NK lymphocytes. Approximately 10% of the cells were in the S phase of the cell cycle as determined by a modified Vindelov DNA content analysis test and may theoretically reflect some of those cells expressing CD11b and CD11c. The cells did not express in vitro NK lymphocyte functional activity against a K562 target and therefore similar to other reported cases of presumably immature NK lymphocytic leukemias. The NK lymphocyte blast crisis was successfully treated with vincristine and prednisone. The patient's disease eventually relapsed and transformed to a progenitor stem cell before she died (CD45, 13, CD38, and CD34 positive). The flow cytometric immunophenotyping results contributed significantly as an important adjunct in determining the appropriate diagnosis, helping to select the type of therapy, and monitoring the patient with this unusual type of blast crisis.

False negative results resulting from antigen excess by immunophenotyping a leukemia patient with a whole blood method.

A patient with prolymphocytic leukemia was immunophenotyped by whole blood and Ficoll-Hypaque methods. Discordance was noted between the two methods, especially for the HLE1, B1, I2, and T1B monoclonal markers. The disparity in results reflected the use of 15 X 10(6) cells versus 1 X 10(6) cells per reaction tube. This report represents a variable antigen excess problem with certain monoclonal antibodies while immunophenotyping a patient with leukemia by a whole blood flow cytometry method. The problem was resolved when both labeling methods used 1 X 10(6) cells per reaction tube. The results helped to encourage the vendor of the authors' commercial whole blood system to modify their procedure accordingly, but two other vendors have not yet made similar modifications. It suggests all commercial whole blood methods must be modified to accommodate for the leukocyte count and differential of the patient. Otherwise, the sensitivity of flow cytometers as used in traditional immunophenotyping protocols will not distinguish weakly stained cells from negative controls.

Function of the secretory immune system in bronchogenic carcinoma. Immunoglobulin A levels in respiratory secretions.

This study was designed to investigate the association between bronchogenic carcinoma of the lung and elevated immunoglobulin A (IgA) levels in respiratory secretions. Sixty-nine patients underwent bronchoscopic examination for evaluation of benign and malignant pulmonary diseases. The concentration of IgA in bronchoscopic washings was determined by a radioimmunoassay (RIA) procedure. The average IgA concentration in 20 washings from patients with benign disease was 171 micrograms/ml and agreed with reported IgA values in normal human volunteers undergoing bronchoscopic examination. In contrast, the average IgA concentration from patients with non--oat cell bronchogenic carcinoma was greater than 1,000 micrograms/ml, and 80% of these patients had values exceeding 300 micrograms/ml. Furthermore, in 16 of these patients, selectively collected washings had been obtained from the tumor-bearing lung and contralateral normal lung. In this group, elevated levels localized to the tumor-bearing side. In contrast, eight patients with extrathoracic cancer metastatic to the chest had an average IgA value of 160 micrograms/ml, and seven of eight (87%) had concentrations below 300 micrograms/ml. Aside from the association of cigarette smoking and pulmonary cancer, we found no clear relationship between smoking history and the concentration of IgA measured in secretions. Finally, biochemical analysis established that the changes in immunoglobulin levels were specific to the secretory immune system. In conclusion, measurement of secretory immune system components may be useful in the early diagnosis of malignant bronchogenic disease. Biological mechanisms and suggested clinical application are discussed.

Conjugation of triaziquinone to immunoglobulin G by a thiolation procedure catalyzed by 2-pyridinealdoxime methiodide.

Thiolation of immunoglobulin G (IgG) with DL-N-acetylhomocysteinethiolactone, catalyzed by 2-pyridinealdoxime methiodide, incorporated new sulfur groups into IgG. Triaziquinone was subsequently conjugated to the sulfur groups. Triaziquinone-IgG complex retained the alkylating activity of the drug and the immunological activity of the antibody. The conjugation procedure was inhibited by the thiol-blocking agent methyl methanethiolsulfonate.