Initial experience in a community hospital with sentinel lymph node mapping and biopsy for evaluation of axillary lymph node status in palpable invasive breast cancer.

BACKGROUND AND OBJECTIVES: To determine the sentinel node detection rate and the accuracy with which the sentinel node histology reflects that of the axilla in a series of patients with palpable invasive breast cancer. METHODS: Forty-four patients with clinically node-negative palpable invasive T1 or T2 breast tumors underwent sentinel node biopsy using isosulfan blue dye, followed immediately by either local excision of the primary lesion with standard axillary lymph node dissection or modified radical mastectomy. All surgeries were performed at Northwest Hospital, Seattle, Washington, between January 1996 and October 1997. RESULTS: The sentinel node was successfully identified in 73% of the patients (32/44). The frequency of sentinel node detection was greater for tumors in the outer quadrants than the inner quadrants (z-test, P < 0.001). Of the 32 patients in whom a sentinel node was identified, 10 (31%) had histologically positive sentinel nodes: 5 (16%) by frozen section, 2 additional patients (6%) after permanent hematoxalin-eosin (H&E) stained sections, and the remaining 3 (9%) after immunohistochemical stains for cytokeratins when the FS and permanent H&E-stained sections were benign. Twenty patients had benign axilla. The sentinel node was falsely negative in 2 patients, yielding an accuracy of 93.8%, sensitivity of 83.3%, and negative predictive value of 91%. CONCLUSIONS: Lymphatic mapping is technically feasible for patients with small (T1 or T2) palpable invasive breast tumors. The sentinel node can be reliably identified in the majority of these patients, and its histology reflects that of the axilla with a high degree of accuracy. Immunohistochemical stains and permanent H&E-stained sections of the sentinel node increased the test's ability to correctly identify axillary metastases. Improving this sensitivity remains a primary goal, however, if benign sentinel node histology is to be used as a criterion to preclude axillary dissection.

Cell adhesion molecule Nr-CAM is over-expressed in human brain tumors.

Using the technique of differential display-polymerase chain reaction (DD-PCR), we isolated a cDNA fragment that is over-expressed in glioblastoma multiforme tissue as compared to normal brain tissue. Sequence analysis indicated that this sequence is identical to the previously isolated human neuron-glia-related cell adhesion molecule hNr-CAM. Gene-specific RT-PCR analysis indicated that hNr-CAM is over-expressed in high-grade astrocytomas, gliomas and glioblastoma tumor tissues as compared to normal brain tissue. High levels of hNr-CAM expression also were observed in cell lines derived from astrocytomas, gliomas and glioblastoma multiforme tumors. Low levels of hNr-CAM expression were observed in neuroblastoma, meningiomas, melanoma, normal breast and prostate tumor tissues. Northern blot analysis showed an alternatively spliced mRNA of 1.4 kb in several tumors as compared to the 7.5 kb transcript found in normal brain tissue. Genomic Southern blot analysis of DNA from 3 brain tumor cell lines showed that over-expression of hNr-CAM in brain tumors was not due to gene amplification. In situ hybridization analysis indicated that 11 of the 20 human brain tumor samples studied showed hNr-CAM over-expression. Our results suggest that hNr-CAM is over-expressed in malignant brain tumors and can serve as a novel marker for brain tumor detection and perhaps therapy.

Application of the differential hybridization of Atlas Human expression arrays technique in the identification of differentially expressed genes in human glioblastoma multiforme tumor tissue.

BACKGROUND AND OBJECTIVES: Several molecular biology techniques are utilized to study changes in gene expression during the genesis of human tumors. Our objective was to identify genes that showed altered expression between normal brain tissue (NBT) and glioblastoma multiforme tumor tissue (GMTT). METHODS: The technique of differential hybridization of two Atlas Human cDNA expression array was used. In this technique, dCTP32-labeled complimentary DNA from NBT and GMTT was hybridized to two identical human cDNA expression array membranes containing 588 known genes. RESULTS: Autoradiographic analysis showed that of the 588 genes analyzed, 52 are overexpressed in GMTT and 57 in NBT. A gene-specific semiquantitative reverse transcription polymerase chain reaction (RT-PCR) method was used to confirm the expression pattern of seven known genes. RT-PCR results demonstrate that the expression pattern of a majority of genes agreed with the expression pattern observed on expression array. The known tumor suppressor genes retinoblastoma (RB) and p53 showed loss of expression in GMTT compared with NBT. CONCLUSIONS: We conclude that the differential hybridization technique of Atlas Human cDNA expression array can be a useful method in identifying genes that are differentially expressed either in NBT or GMTT.

Cloning, sequence, and developmental expression analysis of C4-2, a potential brain tumor-suppressor gene.

BACKGROUND: Previously, we reported the isolation of C4-2 as a potential tumor suppressor gene in human brain tumors. To understand the function of this gene, we investigated its molecular characterization and expression during development. METHODS: Human fetal brain library screening and 5'RACE-PCR method was used to isolate the full-length cDNA. The coding region of C4-2 was used for in situ hybridization to study its expression during development. RESULTS: We report here the complete sequence of this gene. Sequence analysis indicated that C4-2 has a 94% sequence identity to a family of cAMP-regulated phosphoproteins (ARPP-16/19) in the coding region. C4-2 has a 3.1 Kb long 3'UTR with variable identity to ARPP-16 and ARPP-19. Northern blot analysis indicated that C4-2 is expressed at high levels in normal brain compared to other tissues. Zoo blot analysis demonstrated that the coding region of C4-2 is highly conserved among different animals. In situ hybridization using C4-2 coding region demonstrated that it follows a unique expression pattern during mouse brain development. High level of C4-2 expression was also observed in the spinal cord and somites of the developing embryo. CONCLUSION: Expression analysis during brain development strongly suggests that this family of proteins may play an important role not only in normal functioning of the brain, but also during brain development.

Isolation and characterization of a novel gene from human glioblastoma multiforme tumor tissue.

Using the technique of DD-PCR (differential display-polymerase chain reaction) we isolated a novel gene (D2-2) that is overexpressed in glioblastoma multiforme tissue (GMT) as compared to normal brain tissue (NBT). D2-2 is also highly expressed in recurrent glioma, colon tumor metastatic to brain, breast tumors, prostate tumors and a prostate tumor cell line (LNCaP). Northern blot analysis showed that D2-2 is highly expressed in several tumor cell lines (MOLT lymphoblastic leukemia, SW480 colorectal adrenocarcinoma, A549 lung carcinoma, HL-60 promyelocytic leukemia, S3 HeLa cells, K-562 chronic myelogeneous leukemia and G361 melanoma) as compared to NBT. Additionally, D2-2 is very highly expressed in cell lines derived from glioblastomas, grade IV astrocytomas, normal human fetal astrocytes (NHFA) and glioma. D2-2 is moderately expressed in neuroblastoma, neuroectodermal and medulloblastoma tumor cell lines. D2-2 expression is localized to the frontal lobe, occipital lobe and the cerebellum in the normal brain. Normal tissues such as thyroid, stomach, adrenal cortex, small intestine and pancreas show high expression of D2-2. We also show that D2-2 is expressed 28-fold higher in fetal brain (20 weeks) than in adult brain. Sequence analysis of a 2.0-kb fragment for D2-2 shows no homology to known sequences in the data base.

Characterization of C4-2 as a tumor-suppressor gene in human brain tumors.

BACKGROUND: Brain tumors claimed the lives of 13,300 people in 1995. Our objective was to isolate and characterize unique tumor-suppressor genes from human brain tumors derived from patients in the United States. METHODS: Differential display-polymerase chain reaction was used to isolate tumor suppressor genes. RESULTS: Clone C4-2 was isolated and is expressed in normal adult human brain, but not in brain tissue from glioblastoma multiforme tumors. C4-2 has 66% homology to the previously isolated ARPP-16 (cAMP-regulated phosphoprotein of Mr = 16,000) based on limited sequencing. C4-2 is expressed at high levels in normal brain and is not expressed or expressed at low levels in several brain tumor cell lines. Expression of C4-2 was also either not expressed or expressed at low levels in meningioma, B-cell lymphoma, recurrent glioma, LNCAP (prostate tumor cell line), breast tumor, or prostate tumor tissue. CONCLUSION: We conclude that C4-2 may function as a potential tumor-suppressor gene.

Procainamide-induced agranulocytosis differs serologically and clinically from procainamide-induced lupus.

Agranulocytosis is a well recognized but uncommon complication of procainamide (PA) therapy, whereas a lupus-like syndrome occurs in approximately 20% of patients treated chronically with PA. In order to gain insight into the immunopathogenic relationships among these conditions, we compared the humoral immune abnormalities in these patient groups as well as in asymptomatic PA-treated patients. A relatively uniform profile of IgM but not IgG autoantibody reactivity with a set of chromatin-related antigens was observed in eight elderly men who developed agranulocytosis after treatment with PA. In contrast PA-induced lupus patients had predominant reactivity with [(H2A-H2B)-DNA] in both IgM and IgG classes. Five of eight patients with agranulocytosis had elevated levels of neutrophil-reactive IgG which appeared to be due to immune complexes based on Fc gamma receptor blocking studies. However, 12 of 15 patients with PA-induced lupus, none of whom had neutropenia, had similar levels of neutrophil-reactive IgG, suggesting that this reactivity was not causally related to agranulocytosis. Agranulocytosis developed after less than 3 months treatment with PA in six of eight patients. This time course was similar to that seen in 77 PA-induced agranulocytosis patients reported in the literature plus 127 patients reported to the U.S. Food and Drug Administration in whom 90% developed agranulocytosis within 3 months of starting PA. In contrast, the mode duration of treatment with PA before lupus-like symptoms develop is 10-12 months. These findings, together with the different profiles of autoantibodies and clinical presentations, suggest that agranulocytosis arises from a different mechanism than that underlying PA-induced lupus.

Multiple plasmacytomas in skin. Harbinger of aggressive B-immunocytic malignancy.

A 55-year-old alcoholic man with chronic atypical mycobacterial lung disease developed multiple plasmacytomas in the skin over a 6-month period. There was no evidence of either multiple myeloma or plasma cell leukemia at the time of diagnosis; however, evidence of multiple myeloma became apparent about 7 months later, and the man died, despite therapy for this condition, about 22 months after the initial diagnosis of his plasma cell disorder. Despite the apparent rarity of this clinical presentation, the available evidence suggests it is a harbinger of a relatively aggressive B-immunocytic malignancy.

Effect of iron status on reticulocyte mean channel fluorescence.

Flow cytometric reticulocyte enumeration measures the fluorescence intensity of the reticulocyte population, the reticulocyte mean channel fluorescence. Reticulocyte mean channel fluorescence, used as an indicator of reticulocyte maturation, is directly proportional to the amount of intracellular RNA. Other factors, such as iron stores, may affect reticulocyte mean channel fluorescence. Iron status in normal controls, patients with anemia of chronic disease, and pregnant women was evaluated by hemoglobin, hematocrit, red blood cell indices, iron, total iron-binding capacity, and ferritin. Reticulocyte mean channel fluorescence was significantly elevated (P less than 0.0001) to 85.6 +/- 4.6 (mean +/- 1 standard deviation) in iron-deficient anemic patients and to 81.1 +/- 8.4 in iron-depleted patients compared to healthy individuals (69.7 +/- 2.6). The reticulocyte mean channel fluorescence in anemia of chronic disease was 71.3 +/- 5.8 and was not significantly different from that of normal controls. Reticulocyte mean channel fluorescence showed significant correlations with total iron-binding capacity (P less than 0.0001, r = 0.62) and ferritin (P less than 0.0001, r = 0.40). A possible explanation for these findings, describing differences in cytoplasmic levels of transferrin receptor mRNA, is discussed.

Coexisting Hodgkin's disease and mycosis fungoides. Immunohistochemical proof of its existence.

Hodgkin's disease and mycosis fungoides have been rarely reported in the same patient. This coexistence has been debated in the medical literature. We studied such a patient and report, to our knowledge, the first immunophenotypic evidence for such a coexistence. Reed-Sternberg cells and their variants stained with anti-Leu-M1, Hefi-1, anti-Tac, anti-HLA-DR, and OKT9, but were negative for T cell markers 3A1, Leu-1, Leu-2a, and Leu-3a, a phenotype typical of Hodgkin's disease; infiltrating small lymphocytes were predominantly T cells and were phenotypically normal. In the skin lesions, cells with the phenotype of Hodgkin's disease were not present; the infiltrate was composed of helper T lymphocytes that were 3A1-negative, a phenotype characteristic of the malignant cells of mycosis fungoides. Unexpectedly, a dermatopathic lymph node from the same patient showed the presence of the Leu-M1 antigen on the majority of normal-appearing interdigitating reticulum cells; this was not the case with control dermatopathic lymph nodes from patients without a malignancy. The significance, implications, and possible interrelationships of the findings are discussed.

Lymphokine-induced phagocytosis in angiocentric immunoproliferative lesions (AIL) and malignant lymphoma arising in AIL.

A factor that augmented the phagocytosis of IgG-coated ox red blood cells by the human monocyte/macrophage line U937 was identified in cell culture supernatants from two of two patients with angiocentric peripheral T cell lymphomas, three of three patients with angiocentric immunoproliferative lesions that were not frankly malignant, and one of two patients with T lymphoblastic malignancies. The factor was not present in supernatants derived from 14 nonangiocentric peripheral T cell lymphomas of other histologic types nor in ten cases of B cell lymphoma and two cases of Hodgkin's disease. A similar factor was present in the supernatants of concanavalin A (Con A)-stimulated normal peripheral blood mononuclear cells and in the supernatants of IL-2-dependent T cell lines derived from normal peripheral blood. The factor had an apparent mol wt of greater than 50,000 daltons, was heat labile (100 degrees C for two minutes), and stable at pH 2.0. Its stimulation of phagocytosis was independent of any increase in number of Fc receptors. Thus, this factor is probably not gamma-interferon. This factor may play a pathogenetic role in the hemophagocytic syndromes associated with certain T cell malignancies and immunodeficient states.

Sarcoma arising in a residual testicular teratoma after cytoreductive chemotherapy.

A case of testicular teratoma metastasized to the retroperitoneum and after cytoreductive chemotherapy was noted to contain areas of frank sarcoma. Sarcomatous areas included embryonal rhabdomyosarcoma with a pattern of sarcoma botyroides, alveolar rhabdomyosarcoma, and fibrosarcoma. These areas differed markedly from areas of immature teratoma, which composed the remainder of the retroperitoneal lesion and which also characterized the primary tumor. These sarcomatous areas were characterized by numerous mitoses, marked cellular pleomorphism and diagnostic histologic, ultrastructural, and immunocytochemical features. Residual germ cell tumors following cytoreductive chemotherapy are traditionally categorized as teratoma only or teratoma with embryonal carcinoma or choriocarcinoma for therapeutic and prognostic reasons. This case does not conform to this simple categorization and raises serious questions concerning subsequent therapeutic decisions.

Autopsy pathology in the acquired immune deficiency syndrome.

The acquired immune deficiency syndrome (AIDS) is a devastating new illness which appears to be sexually and parenterally transmissible. AIDS was first described in the male homosexual community; however, the disease has more recently been described among intravenous drug abusers, Haitians, hemophiliacs, and others. The etiologic agent is unknown. AIDS may represent an infection by a previously undescribed organism, a mutant of a known microorganism, or a multifactorial combination of environmental, immunologic, and genetic factors. As a consequence of the disease's seemingly irreversible ablation of the cell-mediated immune system, AIDS victims succumb to a variety of infections and/or unusual neoplasms. In its fully developed form, mortality approaches 100%. At autopsy the gross and microscopic pathology of the syndrome can be divided into three general categories: 1) morphologic manifestations of profound lymphoid depletion; 2) infections, usually with mixed opportunistic pathogens; and 3) unusual neoplasms, most frequently Kaposi's sarcoma or high-grade lymphomas.

Production of monoclonal antibodies against a cell surface concanavalin A binding glycoprotein.

Concanavalin A-binding (Con-A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate--polyacrylamide gels than did the Triton-soluble surface components, which were not retarded by the Con-A-Sepharose column. [125I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate--polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten alpha-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [125I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [125I]-labeled CHO surface component of approximately 265,000 daltons.