Classification of small B-cell lymphoid neoplasms using a paraffin section immunohistochemical panel.

Immunophenotypic analysis is critical in categorizing small B-cell neoplasms; however, many recommended antibody panels have required fresh or frozen tissue. Many paraffin-reactive antibodies are now available but have been studied mostly in isolation. Therefore, the utility of a panel of paraffin-reactive antibodies in differentiating small B-cell neoplasms was investigated. Paraffin-embedded sections of small lymphocytic lymphoma/B-chronic lymphocytic leukemia (SLL/B-CLL; 12), mantle cell (MCL; 15), follicular (FL; 11), and marginal zone B-cell (MZL; eight) lymphomas were stained with CD20/L26, CD3, CD43/DF-T1 or Leu22, CD5/4C7, CD23/BU38, cyclin D1/H295, and CD10/56C6 antibodies. For select antibodies, results were compared to flow cytometric data (FC). Formalin and B5 fixation were also compared. Seven of 11 SLL/B-CLL were CD43+ CD5+ CD23+ cyclin D1- CD10-; seven of 11 MCL were CD43+ CD5+ CD23- cyclin D1+ CD10-; nine of 10 FL were CD43- CD5- CD23- cyclin D1- CD10+; and five of six MZL were CD43+ CD5- CD23- cyclin D1- CD10-. CD5, CD23, and CD10 stains showed sensitivities of 81, 88, and 100%, respectively, compared to FC. With B5 fixation, cyclin D1 was more often negative and CD5 more often equivocal. A panel of paraffin-reactive antibodies aids in classification of small B-cell neoplasms, although a small number of cases have indeterminate phenotypes and MZL have no defining features. CD5 separates most SLL/B-CLL and MCL from FL and MZL. CD23 separates SLL/B-CLL from most MCL, but cyclin D1 is most important for identifying MCL. CD10 positivity distinguishes most FL from other small B-cell lymphoid neoplasms.

Hepatic epithelioid hemangioendothelioma: biological questions based on pattern of recurrence in an allograft and tumor immunophenotype.

Epithelioid hemangioendothelioma (EHE) is best considered a vascular neoplasm of intermediate malignancy. Although usually progressive, the clinical course is highly unpredictable. The present communication describes a case of extensive recurrent hepatic EHE, limited to the liver allograft and initially manifest as an insidious seeding of individual tumor cells in areas of perivenular inflammation associated with rejection. A detailed immunophenotypic characterization of this and a small series of EHE was carried out in an effort to highlight subtle disease recurrence and to gain possible insights into tumor biology associated with this intriguing disease. In a series of five cases of hepatic EHE, CD34 (QB-END/10) was found to be more sensitive than Factor VIII (F-VIII) for recognition of the disease, similar to previous reports. The former diffusely and distinctly stained both epithelioid and dendritic tumor cells, whereas staining for the latter was focal, indistinct, and showed a high background. Although the tumor cells were negative for some markers of dendritic or macrophage maturation, such as CD1a, S100 protein, Mac 387, CD68, and LN3, there was marked infiltration of hepatic EHE by factor XIIIa + (F-XIIIa), Mac 387+, CD68+, and LN3+ macrophages and dendrocytes, most of which were interpreted as reactive. The "reactive" macrophage and dendrocyte populations were present throughout the fibrotic stroma and intermingled with the epithelioid clusters of EHE. Interestingly, a small subset of tumor cells coexpressed CD34 or F-VIII and F-XIIIa, the last of which is normally restricted to cells of the monocyte/macrophage lineage and cytokine activated microvascular endothelium in vitro. The known association of F-XIIIa+ dendrocytes with granulation tissue, repair and fibrogenesis, and the modulation of F-XIIIa and F-VIII expression by inflammatory cytokines led us to speculate that EHE lesions may derive from primitive "reticuloenothelial" cells that can differentiate along endothelial and dendritic pathways. The EHE lesions may represent a neoplastic analogue of wound healing. Thus, the variability in F-VIII staining, the strong expression of CD34, the infiltration of EHE lesions with F-XIIIa+ dendrocytes, and the coexpression of CD34 and F-XIIIa on a subset of tumor cells may have an important biological basis.

Congenital muscular dystrophy with primary laminin alpha2 (merosin) deficiency presenting as inflammatory myopathy.

Ten laminin alpha2-deficient patients were identified by both immunofluorescence and immunoblotting (30% of congenital muscular dystrophy patients tested). Three of the laminin alpha2-deficient patients were carrying a diagnosis of infantile polymyositis prior to immunostaining studies. The clinical features in the 10 merosin-deficient patients were homogeneous, with severe floppiness at birth, delay in achievement of motor milestones, and magnetic resonance imaging findings of white matter changes with normal intelligence. The 10-kb laminin alpha2-coding sequence was screened for causative mutations by reverse transcriptase-polymerase chain reaction/single-stranded conformational polymorphism analysis in muscle biopsy specimens from 5 patients, followed by automatic sequencing of aberrant conformers. Clear loss-of-function deletion mutations were identified in both alleles of 1 patient. Muscle histopathology in this patient showed a striking inflammatory infiltrate of T cells and B cells. Reexamination of biopsy specimens from other laminin alpha2-deficient patients showed minor signs of inflammation in each. Based on these findings and the histological and clinical picture suggesting failure of muscle regeneration, a pathogenesis model for this major subset of congenital muscular dystrophy is proposed. Our data show that muscle histopathology showing a neonatal inflammatory process should be considered consistent with congenital muscular dystrophy.

Human polyoma virus infection of renal allografts: histopathologic diagnosis, clinical significance, and literature review.

Human polyoma virus infection was diagnosed by a needle biopsy of the allograft in two kidney transplant recipients. Viral infection was initially suggested by the occurrence of markedly enlarged tubular epithelial cells with nuclear atypia and chromatin basophilia. Confirmatory evidence was obtained by immunohistochemistry in both cases, and electron microscopy in one instance. Case 1 presented as a refractory interstitial nephritis and underwent allograft nephrectomy. Case 2 showed viral infection concurrent with acute cellular rejection. The rejection initially responded to treatment, but recurred twice on subsequent followup. A review of the literature indicates that asymptomatic infection, ureteric stricture and hemorrhagic cystitis are other possible manifestations of polyoma virus in the human urogenital tract.

Extramedullary hematopoiesis in the allograft liver.

The clinicopathologic correlates of extramedullary hematopoiesis were studied in 77 allograft biopsies from 27 patients who underwent liver transplantation for end-stage liver disease. The patient cohort consisted of 19 men and 8 women, ranging in age from 23 to 75 yr (median 41). The causes of end-stage liver disease included viral hepatitis (n = 20), ethanol abuse (n = 6), and congenital hepatic fibrosis (n = 1). Most patients (23 of 27) had significant septic complications in the postoperative period. The hematocrit was typically low (25 to 31%), and a history of allograft hepatectomy with retransplantation was available in 10 of 27 (37%) patients. Extramedullary hematopoiesis was first diagnosed 5 to 461 days (median 275) post-transplant and persisted 7 days to 36 mo (median 1 mo) thereafter. Pathologic findings concurrent with extramedullary hematopoiesis were acute cellular rejection/central venulitis (n = 7), ischemic preservation injury (n = 10), chronic rejection (n = 5), and chronic hepatitis/cirrhosis (n = 5). The pathogenesis of extramedullary hematopoiesis in these cases is not clear, but a low hematocrit may have been a stimulant for the observed hematopoiesis. In addition, the frequent coexistence of infectious, immunologic, or ischemic injury within the allograft suggests that reparative responses can stimulate intrahepatic stem cells to undergo hematopoietic differentiation. The cytokines likely involved in this process are discussed.

Serum antibody production to botulinum A toxin.

PURPOSE: Conflicting data have been reported regarding development of serum antibodies to botulinum A toxin. The purpose of this study is to determine conclusively whether antibody production to this toxin occurs in humans, and, if so, to determine its relationship, if any, to length of treatment, total cumulative dose, and clinical response to treatment. METHODS: Sixty-five sera samples from 42 adults treated with botulinum A toxin for essential blepharospasm, hemifacial spasm, or spasmodic torticollis were analyzed via a sphere-linked immunodiagnostic assay for antibody production. Results were plotted against length of treatment, number of injections, cumulative dose, and treatment effect produced. RESULTS: Twenty-four (57%) of the 42 patients produced antibodies in all three diagnostic groups. No significant differences were found between antibody producers and nonproducers with respect to age (P = 0.216), length of treatment (P = 0.586), number of injections (P = 0.619), or total cumulative dose (P = 0.286). Within the antibody-producing group, there was no significant correlation between amount of antibody and length of treatment (P = 0.081), number of injections (P = 0.134), or cumulative dose (P = 0.250). The presence of demonstrable antibodies in serum did not affect the clinical responsiveness to injection. CONCLUSION: Antibody production is present in a majority of patients treated with botulinum A toxin. The sphere-linked immunodiagnostic assay is a reliable and reproducible method for detecting and quantifying these antibodies. When antibody production occurs, it is likely due to variations in individual immune responsiveness and appears to have no direct effect on the patient's clinical response to treatment.

Immunohistochemical evidence for C3bi involvement in Graves ophthalmopathy.

PURPOSE: To determine by immunohistochemical methods if components of the complement system are present in Graves ophthalmopathy extraocular and periocular tissues compared with non-Graves ophthalmopathy ocular tissues, and, if so, whether a qualitative difference exists. METHODS: Orbital muscle, periorbital muscle, and adipose tissue from 10 Graves ophthalmopathy patients were studied with in situ assays using monoclonal antibodies for C3bi and C5b-9 (the terminal attack complex) complement components. Extraocular muscle, periocular muscle, and adipose tissue from 12 patients treated for unrelated orbital disorders were used as controls. RESULTS: All nine Graves extraocular and periocular muscle tissues exhibited C3bi positive staining in an intense, localized oval- to spindle-shaped reaction that appeared to represent cells on a diffuse staining background of the endomysial and perimysial connective tissues with no staining of the muscle fibers themselves. Some reactivity was seen in 6 of the 12 control muscles, but this was much less intense than that of Graves ocular muscle tissue. Only two Graves muscle samples stained minimally with the monoclonal antibody for the C5b-9 terminal attack complex while none of the control muscle samples demonstrated reactivity. Orbital fat from Graves and control patients did not demonstrate any reactivity for C3bi or C5b-9. CONCLUSION: C3bi and not C5b-9 (the terminal attack complex) is present in Graves ophthalmopathy extraocular and periocular tissues in a qualitatively greater way than in control non-Graves ophthalmopathy ocular tissue. Consequently, C3bi may contribute to the pathophysiology of Graves ophthalmopathy.

Re-examination of peripheral blood T cell subsets in dysthyroid orbitopathy.

Patients with dysthyroid orbitopathy (DO) were grouped according to a multifactorial assessment of disease severity and the rate of disease progression. Using this system and flow cytometric measurements of T cell subsets in the peripheral blood, a significant increase in the percentage of CD4+ lymphocytes correlated with disease severity in DO patients with progressive disease. These observations are consistent with the hypothesis that the CD4+ peripheral blood T helper cells play a significant role in the progression of DO.

Correlation of serum immunoglobulin E elevations with clinical stages of dysthyroid orbitopathy.

Total immunoglobulin E (IgE) was measured by an enzyme-linked immunoassay in serum samples from patients with dysthyroid orbitopathy and from a group of healthy volunteers. All the serum donors had no symptoms of allergy or infection and were not given any immunoregulative treatments for at least 6 months before the sampling. One hundred thirty-seven dysthyroid orbitopathy patients were rated clinically as belonging to one of the following groups: (1) stable dysthyroid orbitopathy; (2) active dysthyroid orbitopathy; (3) chronic or recurrent dysthyroid orbitopathy; or (4) dysthyroid orbitopathy characterized by limited myopathy. The serum IgE levels of all these groups were compared with 26 healthy, nonatopic volunteers. The mean IgE levels of groups 3 and 4 were significantly higher than the mean IgE level of the control group as well as that of the group with stable dysthyroid orbitopathy. Furthermore, serial readings on several patients were consistent with the hypothesis that serum IgE is elevated in connection with certain stages of rapid dysthyroid orbitopathy progression and also with two unusual clinical forms of dysthyroid orbitopathy.

Immunohistochemical evidence for IgA1 involvement in Graves ophthalmopathy.

Orbital muscle, adipose tissues, and periorbital muscle from 11 patients with Graves ophthalmopathy were studied with in situ assays using monoclonal antibodies for IgA1, IgA2, IgM, and IgG. Tissue biopsies were taken from varied extraocular muscles and orbital sites. All cases were from patients with severe disease or disease of long duration. Control specimens of extraocular muscle tissues were obtained from nine patients treated for unrelated orbital disorders. Only connective tissue associated with the extraorbital muscles and periorbital muscles showed any reactivity. Of the muscle tissue obtained from patients with Graves disease all exhibited IgA1 positive staining of the endomysium and perimysium, without staining of the muscle fibers themselves. Parallel sections of orbital muscles reacted with anti-IgA2 or anti-IgM antibody failed to demonstrate staining. Control extraocular muscle tissue did not stain with anti-IgM and one control muscle of seven reacted minimally with anti-IgA2. Some reactivity with anti-IgA1 was seen in four of the seven control muscles but this was qualitatively much less than that of muscle tissue from patients with Graves disease. Monoclonal anti-IgG did not stain tissue from the six Graves specimens and three control specimens tested.

Immunohistochemical evidence for IgE involvement in Graves' orbitopathy.

Orbital muscle and adipose tissues from seven Graves' orbitopathy patients were studied with in situ assays for IgE. The cases varied in disease severity and site biopsied. Two monoclonal and one polyclonal anti-IgE reagents produced similar results. Identically prepared monoclonal anti-IgM and anti-IgG antibodies and tissues obtained from five patients treated for unrelated orbital disorders were used as controls. Graves' tissues exhibited extravasated leukocytes and leukocyte-rich vessels. These leukocytes were mostly lymphoid. Some basophils and mast cells were identified and polymorphonuclear cells were abundant within vessels of adipose tissue. IgE-positive material was found in association with the majority of leukocytes and with muscle fibers. Parallel sections reacted with anti-IgM antibody were negative, whereas anti-IgG produced diffuse staining with no specific structures highlighted. Control, non-Graves' tissues had no evidence of immune cell activity and were either negative or displayed reactions with anti-IgE reagents that were in most cases different from those of Graves' tissue. Serum IgE was measured in six of the seven patients and was elevated in the two patients with fulminating disease.

Flow cytofluorometric analysis of choriogonadotropin-like material on the surface of human and mouse malignant cells.

Quantitation by flow cytofluorometry of the distribution of human choriogonadotropin (hCG)-like material on the surface of various human and mouse tumor cells grown in tissue culture and as solid tumors has been done using fluorescein-tagged rabbit antisera (IgG fraction) to intact hCG and, in one experiment, by use of two monoclonal antibodies specific for hCG. Fibroblasts were used as a negative (nontumorigenic) cell control, and a rabbit antiserum to human hemoglobin was used as reagent control. All malignant cells tested stained more intensely with the anti-hCG serum than with the antihuman hemoglobin serum. Positive reaction with the monoclonal antibodies specific for hCG provided strong evidence that the material stained was identical to hCG. Heterogeneity of the expression of the hCG-like material was notable both within each cell line and between different cell lines. This heterogeneity was not associated with cell-cycle phase. 3T3 fibroblast-like cells in vitro were originally negative for hCG but acquired reactivity with anti-hCG serum after ten passages.

Effects of immunization against human choriogonadotropin on the growth of transplanted Lewis lung carcinoma and spontaneous mammary adenocarcinoma in mice.

We studied the effects of preimmunization with a synthetic carboxy-terminal peptide of the beta-subunit of human choriogonadotropin (hCG) conjugated to diphtheria toxoid on the growth of two tumor models, the transplantable Lewis lung carcinoma in C57BL/6J mice and the spontaneous mammary carcinoma in C3H/OuJ mice. Immunization with the conjugate prior to Lewis lung tumor implantation significantly (P less than 0.05) retarded the growth of tumors as measured by tumor weight 18 days following transplantation. The weights of Lewis lung tumors in animals preimmunized with the hCG immunogen were inversely correlated (r = 0.61) with the levels of circulating antibodies against human chorionic gonadotropin, whereas no statistical correlation was found between tumor weights and the levels of antibodies reactive to diphtheria toxoid. The number of conjugate-treated C3H/OuJ mice that developed mammary tumors was significantly (P less than 0.05) reduced compared to their vehicle-treated cohorts. Pretreatment with the synthetic muramyl dipeptide analog utilized as an adjuvant with both immunogens did not show any effect on the tumor growth in either tumor system.

Humoral response of normal and athymic (nude) mice to human choriogonadotropin immunogens.

The production of antibodies against human choriogonadotropin (hCG) was studied in normal and athymic (nu/nu) mice of two strains (C57/BL and Balb/c), injected with native (whole) hCG or an immunogen consisting of a synthetic hCG beta COOH-terminal peptide, residues 109-145, conjugated to diphtheria toxoid and mixed with a synthetic muramyl dipeptide analog (nor-MDP) as adjuvant. Both the short-term effect of native hCG dissolved in saline and injected IM (primary response), and the long-term effect of the native hCG and of the hCG immunogen dissolved in saline, emulsified in squalene-Arlacel A, and injected SC as a depot injection (secondary or memory response), were considered. The results obtained indicate that native hCG may be classified as a T-cell independent antigen in the sense that it can elicit low levels of IgM antibodies on a short term basis in athymic mice that have either no or very low T-cell levels. In long-term studies using hCG and the hCG immunogen no antibodies could be detected in athymic mice 14 days after a booster inoculation given 28 days after primary immunization, a regimen that produced high levels of antibodies in normal mice. Because of their inability to sustain humoral responses to native hCG as well as to other hCG immunogens, athymic mice seem well suited for in vivo studies of some of the biological effects of hCG.

Prevention of oncogenic viral infections in mice with CGP 11637, a synthetic muramyl dipeptide analog.

The efficacy of N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (nor-MDP) in controlling viral oncogenesis in mice was investigated. The tumors studied were blood cell malignancies induced by Friend leukemia virus in SJL/J mice, spontaneous mammary neoplasms in RIII/Imr and C3H/OuJ mice, and spontaneous lymphocytic leukemia in AKR/J mice. A transplantable tumor, Lewis lung carcinoma, in C57BL/6J mice was used as a nonvirally induced control model. The nor-MDP was dissolved in saline and made into an emulsion with an equal volume of squalene-Arlacel A and injected subcutaneously at 1- to 2-month intervals. Test and control mice were challenged with exogenous virus or tumor transplant 2 weeks after a second injection of nor-MDP. Administration was started at around 2 months of age in mice that develop spontaneous neoplasms. Electron microscopy studies were done on neoplastic tissues of selected test and control mice. This administration of nor-MDP prevented erythroleukemia in SJL/J mice caused by low doses of Friend leukemia virus (although erythroleukemia survivors were not protected from a late-developing lymphoma) and also delayed (possibly prevented) the development of a spontaneous mammary neoplasm in RIII/Imr mice. No antitumor effects were observed on the spontaneous neoplasms of C3H/OuJ and AKR/J mice or on the Lewis lung carcinoma implanted into C57BL/6J mice. Electron microscopic examinations of the various neoplastic tissues indicated that nor-MDP administration eliminated or reduced extracellular viruses. The results suggested that under the experimental conditions used nor-MDP appears to effect the viruses and not the malignant cells per se.

Effect of propane sultone pretreatment on Friend virus leukemogenesis in mice.

Propane sultone (PS) injected i.p. 24 or more hours before Friend leukemia virus increased the incidence of lymphoma in SJL/J mice and at a higher dose increased the incidence of erythroleukemia in B10SJF1 mice. PS at the same time also decreased hematopoietic stem cell clonogenicity.

Effect of cyclophosphamide on Friend virus leukemogenesis in virus-sensitive and virus-resistant mice.

The influence of cyclophosphamide (CY) on Friend virus leukemogenesis was studied in SJL/J, C57BL/10J, and C57BL/10J X SJL/J F1 (hereafter called B10SJF1) mice. All three differ in their susceptibility to the viral oncogenic effect. Immunosuppressive doses of CY, which by themselves produced no cancer, were followed 2 days later by injection of Friend leukemia virus. The virus doses were the same as used previously. Although in other experiments preinjection of various chemical carcinogens augmented leukemogenesis by Friend leukemia virus in SJL/J mice, in the present study, pretreatment by CY had no such effect. In contrast, CY increased Friend erythroleukemia incidence from 15 to 100% in B10SJF1 mice and from 0 to 85% in C57BL/10J mice. The disease in C57BL/10J mice had a 190-day incubation period, which is approximately 5 times that in the SJL/J and B10SJF1 mice. During this latent period, the C57BL/10J mice harbored infectious Friend leukemia virus in their plasma.

Potentiation of Friend viral leukemogenesis by 9,10-dimethyl-1,2-benzanthracene in two strains of mice.

The polycyclic aromatic hydrocarbon, 9,10-dimethyl-1,2-benzanthracene (DMBA) produced malignancy involving the spleen in SJL/J and B10SJF1 mice when injected ip at 500 micrograms per mouse either alone or in combination with threshold doses of Friend leukemia virus (FLV). The mice that received both chemical and virus died significantly sooner than mice that received either chemical or virus alone, and a synergism between DMBA and FLV was demonstrated in both the virus-resistant B10SJF1 hybrids and virus-sensitive SJL/J mice.

Effects of benzo(a)pyrene on Friend viral leukemogenesis in B10SJF1 mice.

A single intraperitoneal injection of benzo(a)pyrene (BP) given 1, 2, or 3 days before an ip injection of Friend leukemia virus (FLV) significantly increased the leukemogenic effect of the virus in B10SJF1 mice. These hybrids are the offspring of C57BL/10 females and SJL/J males and are highly resistant to FLV leukemogenesis when the virus is injected alone.