Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance.

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-ß hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3+ CD4+ CD25high CD127low Foxp3+ ) in blood that resulted from peripheral proliferation (Ki67+ ), possibly new thymic emigration (CD31+ ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4+ and CD8+ cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.

Acute renal endothelial injury during marrow recovery in a cohort of combined kidney and bone marrow allografts.

An idiopathic capillary leak syndrome ('engraftment syndrome') often occurs in recipients of hematopoietic cells, manifested clinically by transient azotemia and sometimes fever and fluid retention. Here, we report the renal pathology in 10 recipients of combined bone marrow and kidney allografts. Nine developed graft dysfunction on day 10-16 and renal biopsies showed marked acute tubular injury, with interstitial edema, hemorrhage and capillary congestion, with little or no interstitial infiltrate (≤10%) and marked glomerular and peritubular capillary (PTC) endothelial injury and loss by electron microscopy. Two had transient arterial endothelial inflammation; and 2 had C4d deposition. The cells in capillaries were primarily CD68(+) MPO(+) mononuclear cells and CD3(+) CD8(+) T cells, the latter with a high proliferative index (Ki67(+) ). B cells (CD20(+) ) and CD4(+) T cells were not detectable, and NK cells were rare. XY FISH showed that CD45(+) cells in PTCs were of recipient origin. Optimal treatment remains to be defined; two recovered without additional therapy, six were treated with anti-rejection regimens. Except for one patient, who later developed thrombotic microangiopathy and one with acute humoral rejection, all fully recovered within 2-4 weeks. Graft endothelium is the primary target of this process, attributable to as yet obscure mechanisms, arising during leukocyte recovery.

Suppressive role of B cells in chronic colitis of T cell receptor alpha mutant mice.

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.

Identification of pre-T cells in human peripheral blood. Extrathymic differentiation of CD7+CD3- cells into CD3+ gamma/delta+ or alpha/beta+ T cells.

CD7+CD3- cells purified (greater than 99.99%) by FACS from the peripheral blood of healthy adults include precursors for mature T cells that have the capacity to differentiate into TCR-1+ or TCR-2+ CD3+ cells. Extrathymic differentiation was demonstrable from all eight healthy donors in the presence of a high concentration of IL-2, mitogenic levels of PHA, and irradiated blood mononuclear feeder cells, after a lag of approximately 40 d in vitro. The extrathymic T (ET) cells were predominantly TCR-1+, although TCR-2+ cells were also derived. ET TCR-1+ cells were CD4-CD8-, CD4-CD8DIM+, and CD4+CD8-, and were distinguished from natural T TCR-2+ cells by a variety of cell surface markers. The ET cells had phenotypes generally displayed by normal mature T cells, although the CD5DIM+ on ET cells was more typical of thymocytes. Acquisition of CD3 on purified CD7+CD3- cells was not due to antigenic modulation or growth of contaminants, and ET cells could be demonstrated at the clonal level. Studies in athymic mice and bone marrow recipients support the view that extrathymic maturation does occur in vivo. Whether the CD7+CD3- cell population was unexposed to the thymus, or exposed but not processed, is unknown. In any case, unusual or "forbidden" autoreactive specificities are predicted since ET cells differentiate without thymic selection of the TCR.

Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants.

Purging of tumor cells and selection of stem cells are key technologies for enabling stem cell transplantation and stem cell gene therapy. Here we report a strategy for cell selection based on physical properties of the cells. Exposing cells to an external pulsed electric field (PEF) increases the natural potential difference across the cell membrane until a critical threshold is reached and pore formation occurs, resulting in fatal perturbation of cell physiology. Attaining this threshold is a function of the applied field intensity and cell size, with larger cells porated at lower field intensities than smaller cells. Since hematopoietic stem cells are smaller than other hematopoietic cells and tumor cells, we found that exposure of peripheral blood mononuclear cells (PBMCs) to PEFs caused stepwise elimination of monocytes without affecting the function of smaller lymphocyte populations. Mobilized peripheral blood exposed to PEFs was enriched for CD34+/CD38- cells and stem cell function was preserved. Furthermore, PEF treatment was able to selectively purge blood preparations of tumor cells and eradicate transplantable tumor.

Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS.

Six human ovarian cancer cell lines and samples of ascites cells isolated from 27 patients with stage III or IV ovarian papillary serous cystadenocarcinoma were studied individually to test whether recombinant human Mullerian inhibiting substance (rhMIS) acts via its receptor. To do these experiments, we scaled up production of rhMIS and labeled it successfully with biotin for binding studies, cloned the human MIS type II receptor for mRNA detection, and raised antibodies to an extracellular domain peptide for protein detection. These probes were first tested on the human ovarian cancer cell lines and then applied to primary ovarian ascites cells. rhMIS inhibited colony growth of five of six cell lines that expressed the human MIS type II receptor mRNA by Northern analysis while not inhibiting receptor-negative COS cells. Flow cytometry performed on MIS-sensitive ovarian cancer cell lines demonstrated specific and saturable binding of rhMIS (Kd = 10.2 nM). Ascites cells from 15 of 27 or 56% of patients tested bound biotinylated MIS (MIS-biotin) and, of the 11 that grew in soft agarose, 9 of 11 or 82% showed statistically significant inhibition of colony formation. Of the 15 patients who bound biotinylated MIS, mRNA was available for analysis from 9, and 8 of 9 expressed MIS type II receptor mRNA by reverse transcription-PCR, showing a statistically significant correlation, compared with binding, by chi2 analysis (P = 0.025). Solid ovarian cancers were positive for the MIS type II receptor protein by immunohistochemical staining, which colocalized with staining for antibody to CA-125 (OC-125). Thus, the detection of the MIS type I receptor by flow cytometry may be a useful predictor of therapeutic response to MIS and may be a modality to rapidly choose patients with late-stage ovarian cancer for treatment with MIS.

Flow-cytometric and ultrastructural analysis of alveolar macrophage maturation.

Alveolar macrophages (AM) from adult and newborn rats were studied by flow cytometry and ultrastructural morphometry. We observed that the laser scatter and autofluorescent properties of newborn macrophages were different from those of adult cells. Relative to the adult AM, the forward-angle laser scatter obtained with the newborn AM was reduced; this optical measurement appeared to correlate with the smaller mean size, as determined by ultrastructural and electronic volume measurements. The diminished right-angle laser scatter (90 degrees angle) correlated with the presence of fewer small, irregularly shaped lysosomal structures in the newborn AM, compared with AM from adult animals. AM from 1-2-day-old rats displayed large vacuoles containing multilamellar structures, which proved to be less effective at scattering light. Cells from newborn rats were less autofluorescent, a finding that appeared to correlate best with the numbers of secondary lysosomes. Flow cytometry may be used to discern structural alterations that occur during the maturation of AM. These changes correlate well with quantitative ultrastructural analyses of these cells.

A novel approach to measuring cell-mediated lympholysis using quantitative flow and imaging cytometry.

In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.

Müllerian-inhibiting substance type II receptor expression and function in purified rat Leydig cells.

Müllerian-inhibiting substance (MIS), a gonadal hormone in the transforming growth factor-beta superfamily, induces Müllerian duct involution during male sexual differentiation. Mice with null mutations of the MIS ligand or receptor develop Leydig cell hyperplasia and neoplasia in addition to retained Müllerian ducts, whereas MIS-overexpressing transgenic mice have decreased testosterone concentrations and Leydig cell numbers. We hypothesized that MIS directly modulates Leydig cell proliferation and differentiated function in the maturing testis. Therefore, highly purified rat Leydig and Sertoli cells were isolated to examine cell-specific expression, binding, and function of the MIS type II receptor. These studies revealed that this receptor is expressed abundantly in progenitor (21-day) and immature (35-day) Leydig cells as well as in Sertoli cells. Prepubertal progenitor Leydig cells exhibit high affinity (Kd = 15 nM), saturable binding of MIS. No binding, however, is detected with either peripubertal immature Leydig cells or Sertoli cells at either age. Moreover, progenitor, but not immature Leydig cells, respond to MIS by decreasing DNA synthesis. These data demonstrate that functional MIS type II receptors are expressed in progenitor Leydig cells and support the hypothesis that MIS has a direct role in the regulation of postnatal testicular development.

Treatment of plasma cell dyscrasias by antibody-mediated immunotherapy.

The use of serotherapy to treat patients with plasma cell dyscrasias (PCDs) has been sought by us and others. Candidate antigens that have been targeted or proposed for targeting in PCDs include the immunoglobulin idiotype, CD19, CD38, CD54, CD126, HM1.24, and Muc-1 core protein. Unfortunately, many of these antigens are not ideal for use in serotherapy since they are not selectively expressed, are either shed or secreted, or have not been fully characterized. Serotherapy with an anti-CD19 monoclonal antibody (B4) conjugated to a blocked ricin toxin had no significant activity in patients with multiple myeloma (MM). Circulating CD20+ clonotypic B cells have been detected in the circulation of most MM and Waldenstrom's macroglobulinemia (WM) patients. Plasma cells from most WM patients express CD20, but most MM patient plasma cells either lack CD20 or express it weakly. In view of recent successes with anti-CD20-directed serotherapy in other B-cell malignancies, we initiated a phase II trial to study the anti-CD20 monoclonal antibody rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) in patients with MM. We describe two PCD patients (one with WM and one with MM) who responded to therapy. By flow cytometric analysis, CD20+ plasma cells and B cells present in the bone marrow and peripheral blood of a patient with MM disappeared with response to rituximab therapy. However, residual CD20- tumor cells remained in the bone marrow following rituximab therapy, and after 6 months this patient progressed with CD20- myeloma cells. As a potential strategy to overcome this limitation, we demonstrated that interferon-gamma at pharmacologically achievable levels induced CD20 expression on these CD20- plasma cells, consistent with our recent findings that interferon-gamma is a potent inducer of CD20 expression on MM patient plasma cells and B cells. We also characterize a response to rituximab with a decrease in paraprotein and resolution of anemia in a patient with WM whose response to rituximab is ongoing after 19+ months. This preliminary experience supports the potential use of serotherapy targeting CD20 in PCDs. Our studies further suggest that interferon-gamma may enhance CD20 expression on MM plasma cells, thereby increasing their susceptibility to anti-CD20 monoclonal antibody therapies.

Flow cytometric analysis of DNA ploidy, percent S-phase fraction, and total proliferative fraction as prognostic indicators of local control and survival following radiation therapy for prostate carcinoma.

PURPOSE: Treatment recommendations for localized prostate cancer may be improved by the identification of tumor factors prognostic for local control and survival. In this retrospective study, flow cytometric deoxyribonucleic acid (DNA) ploidy analysis and cell cycle analysis were performed on paraffin-embedded biopsy material to determine if additional prognostic factors could be identified in patients treated with radiation therapy. METHODS AND MATERIALS: Seventy patients with T1-4NxM0 tumors were identified in whom the primary treatment had been radical radiation therapy with no prior or concurrent endocrine therapy and in whom sufficient prostatic tissue was available for flow cytometric analysis. There were 40 diploid, 26 aneuploid, and 4 multiploid cases. Aneuploid and multiploid cases were combined for analysis. Cell cycle data were obtained on all diploid and 10 aneuploid cases. RESULTS: The histologic differentiation of the tumor (well or moderate vs. poor) was an independent predictor of overall survival and disease-free survival (p = 0.05 and 0.01, respectively). Local control was worse in the poorly differentiated patients, although this was not statistically significant in a multivariate analysis (p = 0.08). Neither T-stage, deoxyribonucleic acid ploidy (diploid vs. nondiploid), percent S-phase fraction, nor total proliferative fraction (S-phase fraction + G2M) significantly predicted for any of these endpoints. Within the diploid and well or moderately differentiated subgroup (n = 25), S-phase (< 4.2 vs. > or = 4.2) was a significant predictor of local control (100% vs. 51%, p = 0.03). A comparable distinction could be made using total proliferative fraction (< 10% vs. > or = 10%) with local control rates of 100% vs. 56% (p = 0.05). Among the poorly differentiated tumors, no similarly favorable subgroup was identified. CONCLUSIONS: This retrospective and multivariate analysis identifies both histology and percent S-phase or total proliferative fraction as predictors of local control following irradiation, and confirms that histology, but not DNA ploidy, is significant for overall survival. If these previously unreported findings are confirmed by prospective studies, S-phase should be added to histology as a parameter in the evaluation of clinical trials.

Fc-receptor for mouse IgG1 (Fc gamma RII) and antibody-mediated cell clearance in patients treated with Leu2a antibody.

A pilot study was performed to explore the clinical potential of Leu2a antibody in reversing acute renal allograft rejection. Anti-Leu2a, a murine IgG1 monoclonal antibody (mIgG1 mAb), is specific for the CD8 molecule that is expressed in high density on class I reactive T cells. Of the 6 recipients treated with anti-Leu2a, two responded with a complete reversal of rejection with long-term allograft function maintained for over a year. In two other recipients, acute rejection was initially reversed, but later rejection episodes resulted in allograft failure at 2-3 months posttreatment. Rejection in the two other recipients showed no response to Leu2a mAb treatment. Specific depletion of peripheral blood CD8+ cells occurred in four of the six recipients. Even in this small series, it was evident that cell clearance was neither necessary nor sufficient for reversal of rejection. However, a complete correspondence between cell clearance and the ability of the recipients' mononuclear cells to undergo mitogenic response to the mIgG1 anti-CD3 (Leu4) mAb in vitro was noted. Binding of IgG1 mAb to the Fc-receptor (Fc gamma RII/CD32) expressed on blood monocytes is known to be essential for the T cell mitogenic response to soluble mIgG1 CD3 mAb in vitro. Our data suggest that binding to Fc gamma RII may be an essential step in the process of mIgG1 Leu2a mAb-mediated cell clearance in vivo.

The effects of OKT4A monoclonal antibody on cellular immunity of nonhuman primate renal allograft recipients.

Significant differences in cellular responses were found among allograft recipients treated with various OKT4A mAb protocols. Recipients of multiple infusion low-dose and 2-bolus OKT4A immunosuppressive regimens regularly showed potent donor-specific cytotoxic CD8+ and CD4+ intragraft T cells and donor-reactive PBMC in MLC tests. In contrast, PBMC isolated from recipients of high-dose OKT4A therapy generally showed very weak or no response to donor-antigens during the later posttransplant periods. Furthermore, an absence of IL2-responsive intragraft cells was found to correlate with stable graft function in these recipients. We conclude that OKT4A mAb, in high doses, can block allosensitization and induce donor-specific nonresponsiveness in vivo. An OKT4A-based therapy, therefore, may have the potential of inducing long-lasting donor-specific immunosuppression, or even tolerance.

A critical analysis of serum and urine interleukin-2 receptor assays in renal allograft recipients.

A component of the interleukin 2 receptor (IL-2R) is released in soluble form during T cell activation and can be detected in the blood during acute renal allograft rejection. This study evaluates the diagnostic utility of a sandwich enzyme immunoassay test for serum and urine IL-2R in renal allograft recipients. A rise in serum IL-2R during the week prior to the clinical diagnosis of rejection correlated better with rejection than did isolated serum IL-2R levels or urine values. For the diagnosis of acute rejection, a rise in serum IL-2R (sensitivity 73%, specificity 87%) was comparable in overall test performance to a rise in serum creatinine (sensitivity 70%, specificity 84%). Overall, the two tests had equivalent receiver operating characteristic curves. Because the etiology of false positives in creatinine and IL-2R assays differed (primarily cyclosporine toxicity and infection, respectively), the predictive value of the combined tests was superior to either alone.

A phase I trial of immunosuppression with anti-ICAM-1 (CD54) mAb in renal allograft recipients.

Several adhesion molecules contribute to the interaction between T cells and antigen presenting cells or target cells. Leukocyte function-associated molecule-1 (LFA-1[CD11a/CD18]) and intercellular adhesion molecule-1 (ICAM-1 [CD54]) are one such critical adhesive receptor-counter-receptor combination. The importance of ICAM-1 dependent adhesion in the rejection response was initially demonstrated in cynomolgus renal allograft recipients treated with the anti-ICAM-1 murine monoclonal antibody BIRR1. BIRR1 also appeared to limit ischemic damage in these animals. A Phase I clinical trial has subsequently been completed in 18 patients who received cadaver donor renal allografts at high risk for delayed graft function (prolonged preservation time, highly-sensitized recipient). An adequate BIRR1 serum level was associated with significantly less delayed graft function (P < .01) and rejection (P < .01). In 1-hr biopsies, mouse IgG was detected along the endothelium of the vessels and glomeruli in the graft. There were no instances of primary non-function (PNF), and current allograft survival (followup: 16-30 months) in these "high-risk" mAb-treated patients is 78%. There were 3 instances of PNF and a graft survival rate of 56% in the recipients of the contralateral kidney allografts treated with conventional immunosuppression. No significant "first-dose" effect was associated with BIRR1 administration. These results establish a dosing schedule and the clinical safety of BIRR1. They also suggest that inhibition of leukocyte adhesion by mAb therapy may be useful in controlling allograft rejection and possibly in limiting reperfusion injury. Thus, these observations support the clinical importance of accessory molecules in T cell function. We hypothesize that anti-CD54 mAb acts by blocking leukocyte adhesion to the endothelium, thereby interfering with sensitization or target cell interaction.

Humanized IgG1 and IgG4 anti-CD4 monoclonal antibodies: effects on lymphocytes in the blood, lymph nodes, and renal allografts in cynomolgus monkeys.

BACKGROUND: Optimizing therapeutic monoclonal antibody (mAb) depends on the incorporation of the necessary effector functions and the development of hypoantigenic "humanized" antibodies by genetic engineering, which then need to be tested in appropriate preclinical trials. METHODS: Constructs of humanized OKT4A containing the complementarity-determining region (CDR) of murine OKT4A and the framework and constant regions of human light (kappa) and heavy chains (IgG1 and IgG4) were prepared and tested in cynomolgus monkeys who received a renal allograft. A prophylactic course of CDR-OKT4A/human (h) IgG1 or CDR-OKT4A/ hIgG4, either as high-dose single bolus (10 mg/kg) or as low-dose multiple infusion (1 mg/kg for 12 days) was given, and the effects on graft survival, immunohistology, circulating cells, and lymph node cells were assessed. RESULTS: The IgG1 isotype induced coating of T cells, modulation of surface CD4 molecules, and profound depletion of CD4+ lymphocytes in peripheral blood, which persisted as long as the animals were followed (up to 7 weeks). The IgG4 isotype induced only cell coating without cell clearance or modulation. In lymph nodes, coating of lymphocytes (approximately 60%) was seen with both isotypes in the earliest sample (6 hr). After 2 days, significant depletion of lymph node CD4 cells was evident, with a decrease in the CD4 to CD8 ratio in the IgG1-treated group; no depletion occurred in the IgG4 group. The emigration of CD4+ cells into the allograft was significantly delayed in the CDR-OKT4A/hIgG1-treated animals when compared with the CDR-OKT4A/hIgG4 group as judged by immunocytochemistry (23.8+/-13.2 days vs. 7.4+/-1.5 days, P<0.001) or interleukin-2-promoted T-cell outgrowth from allograft biopsies (22.2+/-11.0 days vs. 6.3+/-0.5 days, P<0.01). CONCLUSIONS: This study demonstrates that the in vivo effects of CDR-grafted OKT4A are dependent on its isotype. The depleting mAb CDR-OKT4A/hIgG1 significantly delays the entry of CD4+ cells into the graft, inhibiting the early phase of rejection. However, graft rejection occurs when CD4+ cells eventually infiltrate the graft, even in the presence of depressed levels of circulating CD4+ cells. Both isotypes demonstrated therapeutic efficacy: graft survival was prolonged over controls. In the case of CDR-OKT4A/hIgG4, neither lymphocyte depletion, antigenic modulation, nor prevention of infiltration is necessary for a beneficial effect, which indicates that this mAb blocks CD4 function or renders the CD4+ cell less responsive. The lack of depletion is a feature of potential clinical advantage in minimizing the risk of excessive immunosuppression.

A comparative study of flow cytometry and histopathologic findings in thyroid follicular carcinomas and adenomas.

As a possible diagnostic aid in the often difficult histopathologic distinction of thyroid follicular carcinomas from adenomas based on invasion most flow cytometry studies have indicated a higher aneuploidy incidence in carcinomas. However, these reports often are difficult to analyze mainly due to nonuniformity of pathologic diagnostic criteria. The present study compares the flow cytometry results of 65 follicular tumors with pathologic findings based on the World Health Organization's specific diagnostic and staging criteria. Aneuploidy was significantly higher in the 28 cancers than in the 27 hypercellular (fetal and embryonal) adenomas (57% v 22%; P = .02). There was a high percentage of aneuploidy (75%; nine of 12 cases) in the widely invasive follicular carcinomas, compared with 40% (six of 15 cases) in the minimally invasive carcinomas, 22% (six of 27 cases) in the hypercellular adenomas, and 10% (one of 10 cases) in the normofollicular or macrofollicular adenomas. However, aneuploidy was not significantly different between the most difficult differential histopathologic diagnoses of minimally invasive follicular carcinoma (40%; six of 15 cases) and hypercellular adenoma (22%; six of 27 cases) (P = .12). Other data included relatively high frequencies of aneuploidy in hypercellular adenomas (29%; six of 21 cases) and diploid status of carcinomas (36%; 12 of 33 cases). In summary, although the overall findings show a trend toward increasing aneuploidy from well-differentiated and hypercellular adenomas to minimally and widely invasive follicular carcinomas, the aneuploidy data are inconsistent and indicative of its nonspecificity and limited diagnostic usefulness.

Squamous cell carcinoma of the lung. Clinicopathologic study.

This study was undertaken to investigate the diagnostic, therapeutic, and prognostic features of squamous cell carcinoma of the lung. The overall five-year survival rate for 734 patients was 5 percent, with a median survival of 5.9 months. Surgical excision for early-stage disease was particularly effective therapy. In 151 of 201 patients having surgical excision, the resections were deemed curative. Other modes of therapy proved ineffective, although a combination of radiation and chemotherapy provided cure in one of 39 patients; that patient is alive today after 11 years and three months.

Myeloperoxidase detection by three-color flow cytometry and by enzyme cytochemistry in the classification of acute leukemia.

In the classification of acute leukemia, the presence of myeloperoxidase (MPO) within the leukemic blasts indicates myeloid leukemia. Previous studies compared enzyme cytochemistry (EC) or flow cytometry (FC) with immunocytochemistry, in detecting MPO. Our study is the first direct comparison of EC with 3-color FC in a large group of acute leukemias. We studied 26 cases of acute myeloid leukemia (AML) and 4 cases of B-precursor acute lymphoblastic leukemia (B-ALL). Classification was according to the French-American-British criteria. The cells were analyzed for MPO expression by 3-color FC after cell permeabilization followed by staining with anti-MPO antibody. For FC, the blasts were defined by a combination of light scatter characteristics and dim CD45 expression. Concordance between EC and FC was seen in 27 of 30 cases (23/26 AML and all B-ALL), including all AML cases of M1, M2, M3, and M4 subtypes. In 1 of 4 AML-MO and 2 of 5 AML-M5a cases, FC demonstrated the presence of MPO in 8%, 86%, and 94% blasts; EC detected none. Three-color FC may be more sensitive than routine EC in demonstrating the presence of MPO in AML cases and may offer the advantage of multiparametric analysis.

The AgNOR technique, PCNA immunohistochemistry, and DNA ploidy in the evaluation of choroid plexus biopsy specimens.

The histologic distinctions between normal choroid plexus and choroid plexus papilloma and between choroid plexus papilloma and choroid plexus carcinoma are sometimes difficult. The authors performed the silver nucleolar organizer region (AgNOR) technique, immunohistochemistry for proliferating cell nuclear antigen (PCNA), and DNA ploidy analysis by flow cytometry on 9 samples of normal choroid plexus, 8 papillomas, and 13 carcinomas to evaluate whether these techniques can aid in these differential diagnoses. Significant differences were found in the mean AgNOR count between normal choroid plexus (1.35 +/- 0.11) and choroid plexus papillomas (2.42 +/- 0.81) (P < 0.001), but not between choroid plexus papillomas and carcinomas. In the normal choroid plexus, AgNORs were smooth and round; in the papillomas and carcinomas, however, they varied in size and shape. Compound AgNORs were commonly present in the tumors but were essentially absent in controls. Antibody to PCNA did not stain normal choroid plexus cells (except for focal staining in one sample of normal choroid plexus adjacent to a carcinoma) but stained many papilloma and carcinoma cells. DNA ploidy analysis demonstrated aneuploidy in some papillomas and carcinomas but could not be used for the distinction of normal choroid plexus from papillomas. These results suggested that the AgNOR technique and PCNA immunohistochemistry could be used to distinguish normal choroid plexus from choroid plexus papilloma in small, diagnostically difficult biopsy specimens.