Lisa M. DiFrancesco

Reovirus Oncolysis of Human Breast Cancer

We have previously shown that human reovirus replication is restricted to cells with an activated Ras pathway, and that reovirus could be used as an effective oncolytic agent against human glioblastoma xenografts. This study examines in more detail the feasibility of reovirus as a therapeutic for breast cancer, a subset of cancer in which direct activating mutations in the ras proto-oncogene are rare, and yet where unregulated stimulation of Ras signaling pathways is important in the pathogenesis of the disease. We demonstrate herein the efficient lysis of breast tumor-derived cell lines by the virus, whereas normal breast cells resist infection in vitro. In vivo studies of reovirus breast cancer therapy reveal that viral administration could cause tumor regression in an MDA-MB-435S mammary fat pad model in severe combined immunodeficient mice. Reovirus could also effect regression of tumors remote from the injection site in an MDA-MB-468 bilateral tumor model, raising the possibility of systemic therapy of breast cancer by the oncolytic agent. Finally, the ability of reovirus to act against primary breast tumor samples not propagated as cell lines was evaluated; we found that reovirus could indeed replicate in ex vivo surgical specimens. Overall, reovirus shows promise as a potential breast cancer therapeutic.

Loss of Heterozygosity Associated with Uniparental Disomy in Breast Carcinoma

Loss of heterozygosity is commonly assumed to be due to deletion of the appropriate genomic region in one chromosome within a neoplastic cell but may be due to other mechanisms such as mitotic non-disjunction or somatic recombination leading to uniparental heterodisomy. We chose to study the genomic regions surrounding the p53 and RB1 tumor suppressor genes in breast carcinoma to evaluate the different mechanisms that could mediate loss of heterozygosity. A microsatellite analysis of polymorphic markers in 50 breast cancer samples showed loss of heterozygosity for at least 1 of the 10 markers analyzed in 50% of the tumors studied, and an overall 8.47% of the informative loci showed loss of heterozygosity. All of the cases with loss of heterozygosity were further analyzed for gene copy number of the tumor suppressor genes RB1 and p53 by fluorescence in situ hybridization of either tumor touch preparations or microdissected tumor nuclei with specific genomic probes. Surprisingly, all samples showed the presence of both copies of tumor suppressor genes, including 4/50 cases showing loss of heterozygosity of tumor suppressor gene-spanning markers. One of the 4 cases showed loss of heterozygosity of markers spanning a distance of 6 cM over the RB1 gene, with normal copy numbers of the gene. Three other cases showed loss of heterozygosity of markers within the tumor suppressor gene (RBI or p53) and at least one other spanning marker. No cases showed a simultaneous reduction to homozygosity of markers both near the tumor suppressor gene and distal loci. We suggest that the presence of both copies of the tumor suppressor gene in the cases with loss of heterozygosity of spanning markers and internal markers for that tumor suppressor gene could be explained by somatic recombination resulting in uniparental disomy, but not mitotic nondisjunction or deletion. As the mechanism for physical deletion of a chromosome may be different from those mediating somatic recombination, study of this phenomenon may identify different pathways of genomic instability that may be of diagnostic or treatment significance in breast or other cancers, particularly in those treatments based upon DNA-altering agents.

Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections.

Laser capture microdissection (LCM) has recently been identified as a quick, simple, and effective method by which microdissection of complex tissue specimens for molecular analysis can be routinely performed. Assessment of gene copy number by fluorescence in situ hybridization (FISH) is useful for the analysis of molecular genetic alterations in cancer. Unfortunately, the application of FISH to paraffin sections of tumor specimens is fraught with technical difficulty and potential artifacts. Our results demonstrate that LCM-microdissected nuclei are suitable for FISH gene copy analysis. Amplification of genes in cancer specimens can be detected as easily in LCM-prepared nuclei as in fresh nuclei from cancer tissue specimens. Furthermore, contamination of tumor specimens by normal cells can make interpretation of flow cytometric cell cycle analysis difficult. Our results show that LCM-microdissected nuclei can also be used for flow cytometric cell cycle and ploidy analysis.LCM/FISH offers the advantages of multicolor FISH in a morphologically defined cell population, without the technical problems of FISH performed on paraffin sections. This technique should further simplify the methodology required to perform copy number analysis of tumor suppressor or proto-oncogenes in archived cancer specimens. The use of LCM specimens will also improve the specificity and simplify the interpretation of flow cytometric cell cycle and ploidy analysis of breast cancer specimens.

Extra-Axial Chordoma

Chordomas are low-grade malignant tumors of bone that occur almost exclusively in the axial skeleton. Other neoplasms with a similar histologic picture but an extra-axial location have been described, including parachordoma, myxoid chondrosarcoma, and extra-axial chordoma. We herein present another case of the rare extra-axial chordoma. A 41-year-old woman developed an 8.3 cm mass in the pubic bone. The gross, microscopic, and immunohistochemical findings were identical to those of a classic chordoma. Parachordoma and myxoid chondrosarcoma were excluded from the differential diagnosis. Five previously reported cases of extra-axial chordoma were reviewed and found also to demonstrate clinical and pathologic features specific to chordoma, despite arising in an extra-axial location. Although rare, extra-axial chordoma does exist and should be recognized and managed in a similar fashion to its well-described counterpart. It must be differentiated from other histologic mimics, because the treatment and prognosis can differ significantly.

Superficial leiomyosarcoma: a clinicopathologic review and update.

Fauth CT, Bruecks AK, Temple W, Arlette JP, DiFrancesco LM. Superficial leiomyosarcoma: a clinicopathologic review and update.

Microstructural damage in arterial tissue exposed to repeated tensile strains.

OBJECTIVES Vertebral artery (VA) damage has been anecdotally linked to high-speed, low-amplitude spinal manipulative treatments (SMTs) of the neck. Apart from a single study quantifying the maximum stresses and strains imposed on the VA during cervical SMT, there are no data on the mechanics of the VA for this treatment modality, and there is no information on the possible long-term effects of repeat exposure to cervical SMT. The purpose of this study was to quantify microstructural damage in arterial tissue exposed to repeat strain loading of a magnitude similar to the maximum strains measured in the VA during high-speed, low-amplitude cervical SMT. METHODS Twenty-four test specimens from cadaveric rabbit ascending aorta were divided into 2 control groups (n = 12) and 2 experimental groups (n = 6 each). Specimens were exposed to 1000 strain cycles of 0.06 and 0.30 of their in situ length. A pathologist, blinded to the experimental groups, assessed microstructural changes in the arteries using quantitative histology. Pearson chi(2) analysis (alpha = .05) was used to assess differences in tissue microstructure between groups. RESULTS Control and 0.06 strain tissues were statistically the same (P = .406), whereas the 0.30 strain group showed microstructural damage beyond that seen in the control group (P = .024). CONCLUSIONS We conclude that cadaveric rabbit arterial tissue similar in size and mechanical properties of that of the human VA can withstand repeat strains of magnitudes and rates similar to those measured in the cadaveric VA during cervical SMT without incurring microstructural damage beyond control levels.

Mutation or polymorphism of the CD20 gene is not associated with the response to R-CHOP in diffuse large B cell lymphoma patients.

BACKGROUND Rituximab, a chimeric antibody targeted to the human CD20 molecule, is a major component of the R-CHOP protocol used to treat patients with diffuse large B cell lymphoma (DLBCL). It is also a very expensive drug. Though the response rate of R-CHOP is higher than that of CHOP alone, patients may still experience a poor response. One possible mechanism that may mediate the poor response to R-CHOP is poor binding of the drug to the CD20 molecule, perhaps due to mutations or polymorphisms of the CD20 gene that affect its structure. To test this hypothesis and perhaps define a new predictor of R-CHOP response, our pilot study evaluated the CD20 gene sequence from patients exhibiting a poor outcome to R-CHOP. METHODS Eleven patients with DLBCL who exhibited a recurrence of their disease and/or died within 24 months of R-CHOP treatment were categorized as showing poor progression-free survival (poor outcomes). Paraffin-embedded tissue specimens from the original tumors were evaluated for mutations or polymorphisms of the CD20 gene. Furthermore, sections from these patients and as well as from matched control patients who were categorized as good outcome patients (no progression of their disease within 36 months) were stained with anti-CD20 antibody and compared for CD20 protein expression. RESULTS DNA sequence analyses revealed that no mutations were observed in DNA from the coding region of the CD20 gene in any of the initial tumors from 11 patients who showed poor outcomes with R-CHOP therapy. One case showed a synonymous single nucleotide polymorphism in exon 2 (C216T; rs2070770; Ile>>Ile). No significant differences in CD20 expression was observed between good and poor outcome patients with R-CHOP. CONCLUSIONS Our results do not support association of CD20 mutations in specimens of the initial tumor or structural polymorphisms of the CD20 gene with patients who exhibited poor outcomes to R-CHOP.

Costs Associated with Diffuse Large B-Cell Lymphoma Patient Treatment in a Canadian Integrated Cancer Care Center

OBJECTIVE The objective of this study was to provide a detailed comparative microcosting analysis for two cancer treatment pathways to contribute evidence for resource allocation and operational decision-making in a Canadian cancer care context. METHODS We estimated direct medical costs (in 2004 CAN

Chondroblastoma: An Update

) of the entire pathway of care for diffuse large B-cell lymphoma (DLBCL) patients in a large Canadian cancer treatment center. Patient samples were defined as those who received R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; n = 85) or CHOP (i.e., without rituximab; n = 86) as first-line treatment. All subsequent treatments including palliative care for these patient samples were assessed. Hospitalization costs and unit costs of medical resources were collected from integrated medical organizations. Individual patient resource consumption was assessed via medical chart review. RESULTS For first-line treatment, drug cost was the largest contributor to total cost, followed by hospitalization cost. Rituximab was the largest contributor to mean cost differences between R-CHOP and CHOP treatments. For treatments subsequent to first-line treatment, no significant cost differences were found. Hospitalization and transplantation costs were the two largest constituents of total costs subsequent to first-line treatment, followed by drug cost. Patients with advanced stage disease cost significantly more than patients with limited stage disease. CONCLUSION This is the first detailed microcosting study that has employed consistent local data to estimate total medical costs for DLBCL patients in Canada. This information is useful for resource allocation planning and operational decisions, because it provides more substantive, relevant evidence as compared to aggregated, literature or extrapolated information.

Adhesive-Enhanced Sternal Closure: Feasibility and Safety of Late Sternal Reentry

Chondroblastoma is a rare primary bone tumor of young people that typically arises in the ends of the long bones. Radiologic investigations show a small, circumscribed, lytic lesion. The tumor is characterized histologically by the proliferation of chondroblasts along with areas of mature cartilage, giant cells, and occasionally, secondary aneurysmal bone cyst formation. Chondroblastoma, however, may also present with atypical features, such as prominent hemosiderin deposition, numerous giant cells, or the presence of a large aneurysmal bone cyst component. Malignant entities such as clear cell chondrosarcoma and chondroblastic osteosarcoma must also be considered. Recently, immunohistochemical stains such as DOG1 and SOX9 have been described in chondroblastoma, and K36M mutations in either the H3F3A or H3F3B genes have also been identified. While generally regarded as a benign entity, chondroblastoma manifests an intermediate type of behavior, given its ability to recur locally, and rarely, metastasize.