Successful treatment of systemic and central nervous system lymphomatoid granulomatosis with rituximab.

Lymphomatoid granulomatosis (LYG) is a rare lymphoproliferative disorder with a mortality rate approaching 60% in the first year. The median survival is 14 months from the time of diagnosis. Although a variety of chemotherapeutic regimens have been utilized, there is no standard treatment. Studies have shown that in most cases the malignant cells are B-cells, which induce massive infiltration of reactive T-lymphocytes in the background. The disease is therefore considered as a T-cell rich B-cell lymphoproliferative disorder. We report a case of LYG with pulmonary, hepatic, central and peripheral nervous system involvement that was successfully treated with the anti-CD20 (B-cell) monoclonal antibody, Rituximab.

A novel multiparametric approach for analysis of cytoplasmic immunoglobulin light chains by flow cytometry.

We describe a novel flow cytometric approach using a two-step acquisition technique to determine the cytoplasmic immunoglobulin light chains (LC) expression. Samples were prepared by a lysed-whole-blood technique and incubated with CD38-PE (phycoerythrin) and CD45-FITC (fluorescein isothiocyanate). The cells were fixed and acquired on an FACSCalibur flow cytometer (first acquisition). The cells were then permeabilized, incubated with either kappa-FITC or lambda-FITC and reacquired (second acquisition). Analysis of the data was performed by gating on the differing intensities of CD38 and evaluating them for the presence of a shifting FITC-positive population from the first acquisition to the second acquisition. The FITC fluorescence intensity of the second acquisition was equal to the sum of surface CD45 expression obtained during the first acquisition and the cytoplasmic LC expression obtained during the second acquisition. Thus, the shifting (increase) of FITC fluorescence intensity during the second acquisition is specifically due to the cytoplasmic expression of either the kappa or lambda LC. We studied 15 multiple myeloma (MM) patients and 10 controls (samples from patients without plasma cell dyscrasias). None of the controls showed evidence of any clonal populations. Thirteen of 15 MM patients exhibited clonal plasma cells (CD38 bright), ranging from 0.01% to 34% of total events collected. In addition, we identified another minute clonal population of lymphocytes (CD38 dim, CD45 bright, low forward and side scatter) in 12 of 13 MM patients with clonal plasma cells. This population, ranging from 0.01% to 0.6% of total events collected, had the same LC restriction as the clonal plasma cells. Patients with a ratio of minor clonal population to clonal plasma cells less than 0.07 tended to remain in partial or complete remission than those with a ratio > or =0.07 (4/5 versus 1/4, P <.1, chi(2)). We conclude that this method is highly sensitive and permits us to identify the minute clonal population of lymphocytes in MM patients. Our preliminary observations with a small cohort of patients imply that this minute clonal population may have important prognostic significance. The prognostic significance should be confirmed by further studies.

Evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma: higher diagnostic accuracy with Melan-A and MART-1 compared with S-100 protein and HMB-45.

Accurate diagnosis of micrometastases in sentinel lymph nodes of cutaneous melanoma is critical for proper clinical management. S-100 protein and HMB-45 are the traditional immunomarkers widely used for this purpose. However, the interpretation of micrometastases by these markers is difficult with significant reduction in the diagnostic accuracy. S-100 protein demonstrates immunoreactivity for other nonmelanoma cells and obscures nuclear details, which are crucial for the interpretation of single cell metastases. We compared the new melanoma markers, Melan-A (clone A103) and MART-1 (clone M2-7C10), with S-100 protein and HMB-45, by examining 77 formalin-fixed paraffin-embedded sections of sentinel lymph nodes from 13 cases of primary cutaneous melanoma. CD68 (PG-M1) and hematoxylin-eosin-stained sections were also studied. Four pathologists interpreted the staining pattern after concealing the identity of each immunomarker. Az values (area under receiver operating characteristic curve) with receiver operating characteristic curve were higher with Melan-A (0.9742) and MART-1 (0.9779) compared with S-100 protein (0.8034) and HMB-45 (0.8651), demonstrating a higher diagnostic accuracy with Melan-A and MART-1 with superior detection of melanoma micrometastases. Melan-A and MART-1 showed sharp cytoplasmic immunoreactivity, almost exclusively restricted to the melanoma cells. Therefore, Melan-A and MART-1 are recommended for the evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma as a routine alternative to S-100 protein and HMB-45.

Decreased CD10 expression in grade III and in interfollicular infiltrates of follicular lymphomas.

CD10 expression in various grades and interfollicular infiltrates of follicular lymphoma (FL) has not been well documented. Immunohistochemical staining for CD10 (clone 56C6) was performed on paraffin-embedded tissue from 26 cases of classic FL. Negative or weak expression of CD10 was more frequent in grade III (5/6 [83%]) than in grade I FLs (3/15 [20%]). CD10+ interfollicular infiltrates were present in 16 cases. Six (38%) of 16 cases showed that CD10 expression was strong or moderate in follicular areas but weak or negative in interfollicular infiltrates. Our results suggest that CD10 expression is frequently weak to negative in grade III and in interfollicular infiltrates of FLs. Therefore, lack of CD10 expression on small specimens, such as from needle core biopsy or fine-needle aspiration, does not preclude the possibility of a diagnosis of FL. Furthermore, lack of CD10 expression in diffuse large B-cell lymphoma does not exclude the possibility that the neoplastic lymphocytes are of follicle center cell origin.

Polyoma virus infection after renal transplantation. Use of immunostaining as a guide to diagnosis.

BACKGROUND: Polyoma virus infection is characterized by lymphocytic interstitial infiltrate in the kidney, and it mimics acute rejection. The purpose of this study is to estimate renal allograft outcome with this infection and characterize the lymphocytic infiltrates in polyoma virus-infected renal allografts. METHODS: Patients who had polyoma virus inclusions in renal allograft biopsies were identified. Other viral inclusions were excluded by immunohistochemistry. The lymphocytic infiltrates of six cases of polyoma virus infection were compared with six cases of definite acute rejection by immunostaining for T and B cells. RESULTS: There were 10 cases of polyoma virus infections in renal transplant recipients. Immunosuppressants consisted of mycophenolate mofetil with tacrolimus in eight cases and mycophenolate mofetil with cyclosporine in two. The median time of diagnosis of polyoma virus infection after transplantation was 9.5 months, and the time to graft failure after the diagnosis was 4 months. Reduced allograft survival was seen in patients who had polyoma virus infection. Immunostaining for T and B cells revealed marked increase in the B cells (CD20) in renal allografts with polyoma virus infection of 21% (range, 5-40%) compared with 6% (range, 0-10%) in those with acute rejection (P=0.039). Reduced cytotoxic T cells (TIA-1: median, 7%; range, 2-15%) were seen in polyoma virus-infected allografts compared with 24% (range, 15-30%) in those patients who had acute rejection (P=0.0159). CONCLUSION: Irreversible graft failure is more prevalent with polyoma virus infection. Enhanced immunosuppressants with mycophenolate mofetil with tacrolimus may play a role in the development of this infection. An increase in CD20 and a decrease in cytotoxic T cells in allografts is characteristic of polyoma virus infection.

Routine air drying of all smears prepared during fine needle aspiration and intraoperative cytology studies. An opportunity to practice a unified protocol offering the flexibility of choosing a variety of staining methods.

OBJECTIVE: To evaluate the possibility of routine use of air-dried smears (ADS) instead of wet-fixed smears (WFS). STUDY DESIGN: Intraoperative cytology (IC) smears from 293 specimens and fine needle aspiration cytology (FNAC) smears from 118 cases were studied. Cytomorphology of ADS processed with our protocol for hematoxylin-eosin (HE) and Papanicolaou (PAP) staining after saline rehydration and postfixation in 95% ethanol with 5% acetic acid were compared with respectively stained WFS. Additional ADS were stored up to 72 hours at room temperature prior to HE, PAP and Diff-Quik (DQ) staining to evaluate the effects of postponing rehydration and postfixation. Special stains for fungi were also studied in four cases. RESULTS: ADS were easy to prepare without air-drying artifact in the final HE- and PAP-stained smears. ADS were more cellular than WFS. Erythrocyte interference was frequent in WFS. HE and PAP staining of ADS stored up to 72 hours showed cytomorphology comparable to that of the similarly stained fresh smears. However, DQ staining was better if ADS were processed before 24 hours. ADS stained with special stain for fungi showed good morphology, similar to that in WFS. CONCLUSION: All ADS showed results comparable to or better than WFS. ADS could be stored up to 72 hours before staining with HE and PAP. ADS offers the flexibility of selecting a variety of staining methods and is a practical alternative to WFS.

Immunophenotypic profile of myeloid cells in granulocytic sarcoma by immunohistochemistry. Correlation with blast differentiation in bone marrow.

The present study was designed to evaluate the lineage differentiation (particularly monocytic differentiation) of immature myeloid cells in granulocytic sarcoma (GS) by immunohistochemistry and correlate the results with lineage differentiation of blasts in the bone marrow and to determine the degree of maturation of the infiltrating myeloid cells in GS by immunohistochemistry using CD34 and HLA-DR. Immunohistochemical stains were performed on paraffin-embedded tissue from 17 GS lesions with lineage-associated markers: myeloperoxidase, CD68 (KP1), CD68 (PG-Ml), glycophorin A, factor VIII, and CD56; and with markers for blasts and immature myeloid cells: CD34 and HLA-DR. Our results show that positive staining with PG-M1, but not KP1, suggests monocytic differentiation of myeloid cells in GS and correlates with the monocytic differentiation of blasts in the bone marrow. Expression of CD56 is frequent in GS, especially when the marrow blasts have monocytic differentiation, and should not be interpreted as a primary natural-killer cell process. The immature myeloid cells in GS are frequently HLA-DR positive. However, CD34 positivity of the immature myeloid cells is relatively uncommon, except in cases with underlying myelodysplastic syndrome or chronic myelogenous leukemia.

Human parvovirus B19 infection presenting as persistent anemia in renal transplant recipients.

BACKGROUND: Immunosuppression cannot be achieved without immunosuppressive effects. Human Parvovirus infection is known to occur after organ transplantation. We present our experience with Parvovirus infection in two cases. METHODS AND RESULTS: Two kidney transplant recipients developed symptomatic anemia requiring blood transfusions. Common causes of anemia, such as gastrointestinal bleeding, iron/vitamin deficiencies, hemolysis, and drug toxicities, were ruled out. A peripheral smear revealed low reticulocyte count. Bone marrow examination showed hypoplastic bone marrow with intranuclear inclusions suggestive of human Parvovirus. This was confirmed by immunohistochemical analysis. Treatment with i.v. immunoglobulin G resulted in a dramatic sustained response. Transplant kidney function remained stable. CONCLUSION: Human Parvovirus infections should be considered in immunosuppressed individuals with anemia with poor bone marrow response. Bone marrow examination can reveal viral inclusions and can be confirmed by immunohistochemical analysis. Intravenous immunoglobulin G results in resolution of anemia.

Reduced CD4 and HLA-DR expression in neonatal monocytes.

Three-color flow cytometry was used to assess the immunophenotypic characteristics of normal cord blood monocytes after labeling with a variety of antibodies against myeloid/monocyte-specific markers. Monocytes both in cord blood and in peripheral blood from normal adults were defined in a backgating procedure as cells with the light-scattering characteristics of monocytes that also expressed CD14. The percentage of monocytes, defined in this fashion, that also displayed CD4 receptors was significantly lower in cord blood (mean +/- SD = 29.3 +/- 13.9%) than in peripheral blood from normal adult controls (mean +/- SD = 68.9 +/- 13%) (P < 0.005). Similarly, HLA-DR expression was found on only 86 +/- 6.6% of monocytes in cord blood but on 99 +/- 1% of monocytes in adults (P < 0.005). The percentage of monocytes displaying CD16 receptors in cord blood did not show any significant difference in comparison with adult monocytes. When coexpression of CD14, CD16, and CD4 was assessed, cord blood showed a predominant population of monocytes bearing the phenotype CD14+/CD16-/CD4-. Similarly, approximately 10% of CD14+ monocytes in cord blood expressed neither CD4 nor HLA-DR. Cytochemically, monocytes from cord blood revealed intense granular staining for PAS and marginal or absent staining for nonspecific esterase (NSE). These results raise the possibility that reduced expression of CD4 and HLA-DR receptors on cord blood monocytes may contribute to their impaired immune response. Additionally, the high percentage of CD14+/CD16-/CD4- cells in cord blood suggests that these cells may represent a phenotypically immature population of monocytes. Likewise, the unusual cytochemical staining patterns suggest that these cells are biochemically immature as well.

Obliterative central bronchitis due to mineral dust in patients with pneumoconiosis.

OBJECTIVE: We studied at autopsy a distinctive obliterative bronchitis in three persons with pneumoconiosis and hilar node fibrosis. METHODS: Lungs were evaluated macroscopically, microscopically, and with energy-dispersive spectroscopy. RESULTS: Chest roentgenogram demonstrated right middle lobe syndrome in one patient; bronchostenosis was seen at bronchoscopy in another. The stenotic sites were in perihilar bronchi and showed an upper lobe predominance. Fibrosis with silicotic nodules involved the bronchus, peribronchial tissue, and adjacent lymph nodes. Simple coalworkers' pneumoconiosis was observed in two patients; the third had complicated, mixed dust fibrosis. CONCLUSION: Obliterative bronchitis represents an unusual fibrotic response to free crystalline silica. The process may occur simultaneously in the adjacent lymph node and the bronchial wall; however, it need not be associated with complicated pneumoconiosis. Clinically, obliterative bronchitis may masquerade as bronchogenic carcinoma.

Destruction and loss of bronchial cartilage in cystic fibrosis.

We studied by means of serial sections of intact isolated bronchi, the distribution and morphology of bronchial cartilage in lobar and segmental airways of 6 patients with cystic fibrosis (CF). Findings were compared to those of 4 young adults without CF who served as controls. Compared to the controls, cartilage in CF airways extended for a shorter absolute distance along the bronchial tree and disappeared at a more proximal branching level. Loss of cartilage appeared to correlate with the severity of bronchiectasis. In proximal airways chronic inflammation, destruction and fibrous replacement of cartilage preceded its disappearance. Immunohistochemical staining indicated that cells of monocyte/macrophage lineage (CD68, MAC387 positive) were most closely associated with chondrolysis. Dystrophic calcification and ossification were more commonly seen in CF bronchi and dystrophic calcification was present even in the lobar branches. Destruction of bronchial cartilage is the result of sustained bronchial infection and chronic inflammation and is an additional contributory factor to bronchiectasis and airway instability in patients with CF.

Pharmacokinetic study of radioactive antineoplaston A10 following oral administration in rats.

Radiolabelled (3H-labelled) Antineoplaston A10 was administered in a single dose of 220 mg to 230 mg/kg to female Sprague Dawley rats. Blood and urine samples for determination of radioactivity were collected one hour prior to, and then at different time intervals after, the administration of the drug. Rats were sacrificed 6 h or 36 h later for the study of radioactivity in the various organs. The concentration of radioactivity in blood reached a maximum after 2 to 3 h after the administration of Antineoplaston A10, whereas the highest concentration of radioactivity in urine was observed in the 3.5-h to 4-h samples. It was observed by quantitative HPLC analysis that in rats sacrificed 6 h after Antineoplaston A10 administration, between 61% to 69% of the drug was absorbed, whereas between 37% to 28% was found in the stomach and between 2% to 3% was present in the intestinal contents and faeces. After 36 h, none could be detected in the stomach, intestinal contents or faeces. Organ distribution studies indicated greater accumulation of radioactivity in ileum, bladder, duodenum, kidneys and jejunum, and relatively low accumulation in the heart, lung, liver and brain. The concentration of radioactivity after 36 h was very low. By quantitative measurement, between 40% to 42% of the drug was excreted in the urine in 6 h and 75% of the radioactive material was in the form of Antineoplaston A10. The identification of the major radioactive material as Antineoplaston A10 was confirmed by TLC and analysis of the products of acid hydrolysis and by determination of melting range.

Chemoprevention by antineoplaston A10 of benzo(a)pyrene-induced pulmonary neoplasia.

The effect of Antineoplaston A10 (AA10), an amino acid derivative isolated from human urine, has been studied on pulmonary adenoma formation resulting from intragastric administration of benzo(a)pyrene(BP) to A/HeJ mice. Two doses of BP, 3 mg each, administered two weeks apart, induced an average of 6.86 tumours within 157 days in the control animals (Tumorigenic Index 437). One per cent of AA10 (w/w) given in mouse food for one week prior to, and then continued after the administration of BP, produced a 70% reduction in the total number of tumours in the test groups.