Biological purging of breast cancer cell lines using a replication-competent oncolytic virus in human stem cell autografts.

Autologous hematological stem cell transplantation (ASCT) is used for the treatment of many hematological and several solid cancers. ASCT, however, has proven disappointing as a therapeutic strategy for breast cancer. Our group and others have previously shown that breast cancer micrometastases found in patients' apheresis products (APs) predict shorter progression-free and overall survival. The implications of this finding are twofold: (i) contaminating tumor cells (CTCs) in AP reflect a higher systemic disease burden and/or (ii) reinfused CTCs contribute to relapse/progressive disease. To date, purging strategies have been disappointing. We have previously demonstrated the oncolytic properties of reovirus in in vitro, in vivo and ex vivo systems. In the present study, we tested the hypothesis that reovirus purges CTCs in a breast cancer cell line purging model. Reovirus-infected human breast cancer cell lines (HTB 133, HTB 132, SKBR3 and MCF7) exhibited cell death within days. Admixtures of AP with cells from breast tumor cell lines, which were then exposed to reovirus, showed complete purging of CTCs (assessed via flow cytometry/tumor cell outgrowth analysis) without deleterious effect on CD34+ cells. Our results provide preclinical support for the ex vivo use of reovirus as a purging modality for breast cancer during ASCT.

Bone marrow staging of patients with non-Hodgkin lymphoma by flow cytometry: correlation with morphology.

BACKGROUND: Immunophenotypic analysis is an established tool in the diagnosis and classification of many hematolymphoid disorders; however, the role of flow cytometry (FC) in detecting bone marrow involvement during the staging of non-Hodgkin lymphoma (NHL) has yet to be defined. METHODS: The authors retrospectively analyzed 157 staging and 70 restaging bone marrow biopsies on which morphologic and FC analyses were performed; these biopsies were taken from 195 consecutive patients. Bone marrow biopsies were blindly and independently reviewed and determined to be positive, negative, or suspicious for morphologic involvement by NHL, with disagreements settled by a third reviewer. A selected panel of monoclonal antibodies was used to determine whether bone marrow involvement was immunophenotypically positive (>5%), minimal (<5%), negative, or nondiagnostic. RESULTS: FC and morphology agreed in 78% of cases (178 of 227: 129 both negative, 49 both positive) and were discrepant in 22% (49 of 227). Seven percent (16 of 227) were morphologically positive but showed no evidence of disease on FC, whereas 12% (27 of 227) were positive by FC but had no morphologic involvement. Of the 162 morphologically negative or suspicious bone marrows, 27 were shown to be involved by FC, resulting in a false-negative detection rate of 17%. Most of these (22 of 27, 81%) had minimal detectable disease. Seven percent of Stage I and 26% of Stage II NHL cases with negative staging bone marrow morphologically were found to be involved by FC. CONCLUSIONS: Neither morphologic examination of bone marrow biopsy specimens nor FC alone is adequate to detect all cases of NHL with bone marrow involvement. FC is most sensitive for detecting minimal bone marrow lymphoma, whereas morphology will detect most cases in which involvement is >5%. Cases of early stage NHL with morphologically negative bone marrow could potentially be restaged as Stage IV on the basis of FC results. The clinical importance of minimal bone marrow involvement by NHL needs further evaluation.

Identification of new Fas mutations in a patient with autoimmune lymphoproliferative syndrome (ALPS) and eosinophilia.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare, newly recognized, chronic lymphoproliferative disorder in children and is characterized by lymphadenopathy, splenomegaly, pancytopenia, autoimmune phenomena and expansion of double-negative (DN) T lymphocytes (TCR alpha beta+, CD4-, CD8-). Defective lymphocyte apoptosis caused by mutations of the Fas (CD95) gene has been linked in the pathogenesis of ALPS, as binding of Fas-ligand to Fas can trigger apoptosis. Of the ALPS cases reported to date, point mutations, frameshifts and silent mutations in Fas all have been identified. We report two new point mutations in Fas in a child with ALPS and eosinophilia; studies on other family members established the pattern of inheritance for these mutations. Flow cytometric analysis of blood and tissues (spleen, lymph node, bone marrow) revealed abnormally expanded populations of DN T lymphocytes. Furthermore, activated lymphocytes and IFN gamma-activated eosinophils were resistant to Fas-mediated apoptosis. Eosinophil resistance to Fas-mediated apoptosis has not been previously described in ALPS. Sequencing of Fas revealed two separate mutations not previously reported. One mutation, a C to T change at base 836, was a silent mutation inherited from the mother, while the second mutation, a C to A change at base 916, caused a non-conservative amino acid substitution in the death domain of Fas, changing a threonine to a lysine. This mutation is associated with a predicted change in the structure of a part of the death domain from a beta-pleated sheet to an alpha-helix. We speculate that the mutation in the death domain prevents the interaction of Fas with intracellular mediators of apoptosis and is responsible for the autoimmune manifestations of ALPS and the abnormal lymphocytosis and eosinophilia in this patient.

Metastatic dermatofibrosarcoma protuberans mimicking meningioma.

Dermatofibrosarcoma protuberans (DFSP) is a locally aggressive fibrohistiocytic tumor of intermediate malignancy with potential for local recurrences and with a small, but well recognized risk of distant metastases. We report a case of a 43-year-old Filipino male with an otherwise typical cutaneous DFSP followed 4 years later by a local recurrence of tumor with subsequent pulmonary metastasis at year 5, and 3 sequential central nervous system (CNS) metastases at years 5, 6, and 12 following the original diagnosis of DFSP. Recurrent and metastatic tumors showed areas of fibrosarcomatous change, high cellularity, high mitotic rate (10 - 15 per 10 high power fields), loss of CD34 expression, and aneuploidy. DFSP has not been previously reported to metastasize to the CNS. In addition, we report complex karyotypic aberrations within the metastasis, which have not been previously associated with DFSP. The dural-based location of the intracranial metastasis, together with the light- and electron-microscopic appearance, could cause diagnostic confusion with an anaplastic syncytial meningioma.

t(11;18)(q21;q21) is the most common translocation in MALT lymphomas.

BACKGROUND: Low grade malignant lymphomas arising from mucosa associated lymphoid tissue (MALT) represent a distinct clinicopathological entity. The cytogenetic findings and molecular genetics of MALT lymphomas remain minimally defined. Cytogenetic studies infrequently constitute part of the diagnostic work-up of MALT lymphomas, most commonly due to small biopsy size and their extranodal localization. Only 28 MALT cases with a clonal karyotype have been published to date. A number of chromosomal abnormalities have been observed with the majority of the cases featuring trisomy of chromosome 3 which is present in up to 78% of the cases. MATERIALS AND METHODS: A total of 116 cases of MALT lymphoma were diagnosed at BCCA between 1988 and 1997. Eleven cases of pathologically confirmed MALT lymphomas were subjected to cytogenetic analysis at the time of the initial evaluation. Eight of 11 cases yielded successful cultures and the presence of a clonal karyotype using standard cytogenetic methodology. In addition, a single case of orbital MALT lymphoma with a clonal karyotype has been obtained through our consultative practice from University of Nebraska Medical Center. These nine cases of MALT lymphoma with a clonal karyotype are the subject of this report. RESULTS AND CONCLUSION: In this study we report nine cytogenetically studied MALT lymphomas, three of which feature a novel t(11;18)(q21;q21) translocation which has also been observed in five other MALT cases described in the literature. This recurrent translocation is the most common translocation associated with MALT lymphomas being present in 33% (three of nine) of our cases and 18% (five of 28) of the previously published cases. The results suggest that a potentially important gene located at one of these breakpoints may be involved in the pathogenesis of MALT lymphomas.

Paired helical filament tau (PHFtau) in Niemann-Pick type C disease is similar to PHFtau in Alzheimer's disease.

Niemann-Pick Type C disease (NPC) is a cholesterol storage disease with defects in the intracellular trafficking of exogenous cholesterol derived from low density lipoproteins. In NPC cases with a chronic progressive course, neurofibrillary tangles (NFTs) that consist of paired helical filaments (PHFs) have been reported. To determine if NPC tangles contain abnormal tau proteins (known as PHFtau) similar to those found in Alzheimer's disease (AD) tangles, we examined the brains of five NPC cases by immunohistochemical and Western blot methods using a library of antibodies to defined epitopes of PHFtau. We show here that PHFtau in tangle-rich NPC brains is indistinguishable from PHFtau in AD brains. We speculate, that the generation of PHFtau in NPC may induce a cascade of pathological events that contribute to the widespread degeneration of neurons, and that these events may be similar in NPC and AD.