Douglas J. Demetrick

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.

The use of magnetic resonance imaging to noninvasively detect genetic signatures in oligodendroglioma.

Background: Some patients with low-grade glioma have extraordinarily long survival times; current, early treatment does not prolong their lives. For this reason, therapies that sometimes have neurologic side effects are often deferred intentionally. Methods: In a study of oligodendrogliomas, we used a quantitative method of MR analysis based on the S-transform to investigate whether codeletion of chromosomes 1p and 19q, a marker of good prognosis, could be predicted accurately by measuring image texture. Results: Differences in texture were seen between tumors with codeletion of chromosomes 1p and 19q and those with intact 1p and 19q alleles on contrast-enhanced T1-weighted and T2-weighted MR images. Quantitative MR texture on T2 images predicted codeletion of chromosomes 1p and 19q with high sensitivity and specificity. Conclusions: This new method of MR image interpretation may have the potential to augment the diagnostic assessment of patients with suspected low-grade glioma.

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.

Identification and characterization of demethylase JMJD1A as a gene upregulated in the human cellular response to hypoxia

Hypoxia is commonly found in human solid cancers and serves as a selective environment for the survival of aggressive cancer cells and as protection from anti-cancer therapies. In addition to a shift to anaerobic metabolism, the cellular response to hypoxia includes cessation of cell division and/or cell death. These mechanisms have still not been defined. Identification of the members of hypoxia-induced growth arrest pathways remain incomplete. We have undertaken an expression microarray analysis of the cellular response to hypoxia in diverse cell lines. An identified cohort of genes is reliably upregulated in various cells in response to hypoxia, as validated by reverse-transcriptase polymerase chain reaction (RT-PCR). One of the upregulated targets corresponds to an expressed sequence tag encoded by JMJD1A (a gene also known as JHDM2A), which has been identified as a histone demethylase that regulates the transcription of androgen receptor targets. We confirm, by RT-PCR, the upregulation of JMJD1A after hypoxia and desferroxamine treatment in multiple cell lines. We also show that JMJD1A is predominantly, but not exclusively, a nuclear protein. Immunofluorescent staining of HeLa cells shows a shift of cytoplasmic JMJD1A into the nucleus on hypoxia treatment. Immunohistochemical staining has revealed that JMJD1A is widely expressed in tissues, even in cells that are not known to express the androgen receptor, and is significantly increased in smooth muscle cells upon hypoxia treatment.

BRAF Mutations in Melanocytic Lesions and Papillary Thyroid Carcinoma Samples Identified Using Melting Curve Analysis of Polymerase Chain Reaction Products

CONTEXT Mutations of the proto-oncogene B-raf (BRAF) have been detected in melanocytic lesions and papillary carcinomas of the thyroid, and identification of these mutations could be useful in resolving some diagnostic problems. OBJECTIVE To develop a method to evaluate mutations of BRAF that could provide results much more rapidly than conventional polymerase chain reaction and DNA sequencing assays. DESIGN An assay using a LightCycler was developed to evaluate DNA sequences encoding amino acids within the activation loop of BRAF. RESULTS Using this real-time polymerase chain reaction method, we analyzed 55 paraffin-embedded melanoma or nevus samples. The V600E mutation was found in 0 (0%) of 13 samples diagnosed histologically as Spitz nevi, 9 (24.3%) of 37 invasive melanomas, and 5 (100%) of 5 other melanocytic nevi. Two additional mutations, V600K and VK600-1E, also were identified in cases of invasive melanoma. We analyzed 14 paraffin-embedded papillary thyroid cancer (PTC) samples, 6 of which showed the V600E mutation. We found that our test worked efficiently with fine-needle aspirate specimens, and it identified 6 V600E mutations in 10 fine-needle aspirate specimens diagnosed as PTC. We also identified 4 V600E mutations in 6 specimens of PTC metastatic to lymph node. Unlike the melanocytic lesions, the PTC specimens yielded only V600E mutations. Comparison of our real-time polymerase chain reaction results with conventional polymerase chain reaction and DNA sequencing demonstrated 100% concordance. Surprisingly, we did not identify the previously reported VK600-1E or K601E mutations in our PTC specimens. CONCLUSIONS Our results show that the real-time polymerase chain reaction method is a rapid and accurate method for identifying BRAF mutations, such as V600E, in both paraffin-embedded tissue and fine-needle aspirate specimens.

Copy number analysis of c-erb-B2 (HER-2/neu) and Topoisomerase IIα genes in breast carcinoma by quantitative real-time polymerase chain reaction using hybridization probes and fluorescence in situ hybridization

CONTEXT The Topoisomerase IIalpha (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents. TOP2A is frequently coamplified with c-erb-B2 and consequently might be a prognostic and/or predictive factor for breast cancer patients when anthracycline-based chemotherapy is a consideration. A total of 20% to 35% of breast carcinomas show amplification of the erb-B2 gene, some of which also have coamplification of the TOP2A gene. Investigation of the prognostic or predictive significance of these gene amplifications requires a reliable and sensitive method for the measurement of gene copy number in clinical tumor samples. OBJECTIVE To assess 2 different assay methods that might allow accurate, reproducible, quantitative, and high-throughput estimation of gene copy number in fresh, frozen, or paraffin-embedded breast cancer specimens. DESIGN We developed an assay and analyzed the gene copy numbers of the erb-B2 and TOP2A genes in 8 breast cancer cell lines, 6 fresh frozen samples, and 38 paraffin-embedded breast tumor specimens by a novel real-time polymerase chain reaction (PCR) assay using hybridization probes. The results were compared with standard fluorescence in situ hybridization. RESULTS We discovered a 100% concordance between assessment of gene copy number of erb-B2 and TOP2A between quantitative PCR and fluorescence in situ hybridization (FISH). Quantitative PCR also had the additional feature of uncovering an erb-B2 gene polymorphism. Finally, we observed that TOP2A amplification only occurred in conjunction with erb-B2 amplification in our paraffin-embedded cases of invasive breast carcinoma and that this event was present in 5 (42%) of 12 erb-B2 amplified cases. CONCLUSIONS We conclude that the potentially automatic, real-time PCR analysis using hybridization probes is an efficient method to perform copy number analysis, with results that appear identical to the FISH technique and with the benefit of identifying HER-2 polymorphisms.

Human papillomavirus type 16 associated with oral squamous carcinoma in a cardiac transplant recipient

Human papillomavirus type 16 (HPV 16) has been associated with a variety of squamous carcinomas, particularly those involving the anogenital tract. The authors report the development of an oropharyngeal carcinoma in a 43‐year‐old man approximately 20 months after cardiac transplantation while he was on a maintenance regimen of cyclosporine A and prednisone. The carcinoma was resistant to treatment, and he died of complications related to metastatic disease 3 years posttransplantation. Molecular biologic studies using nonisotopic‐labeled viral DNA probes were done. In situ hybridization demonstrated the presence of HPV 16 DNA in the tumor cells. DNA dot blot analysis confirmed the presence of multiple copies of HPV 16 DNA within the tumor cells and their absence from adjacent normal‐appearing tissue. Southern blot analysis suggested that the HPV 16 DNA was integrated into the tumor cell genome. With increasing recognition of the carcinogenicity of HPV type 16 infection, a role for this virus in the development of squamous cell malignancies in immunosuppressed organ transplant recipients is likely to be noted with increasing frequency.

Epstein–barr virus-associated primary b-cell lymphoproliferative disorder of the cerebellum in an immune competent man

Background. The role of the Epstein‐Barr virus (EBV) in lymphoproliferative lesions has been widely accepted. Most of these lesions occur in patients who have deficiencies in their immune status. Lymphomatoid granulomatosis (LG) is a lymphoproliferative disorder originally characterized as an angiocentric, necrotizing, pleomorphic infiltrate of mononuclear cells. The etiology of LG is unknown. It was originally hypothesized that LG may represent an unusual lymphoid response to an infective organism, possibly EBV.

Expression of MS4A and TMEM176 Genes in Human B Lymphocytes

The MS4A gene family in humans includes CD20 and at least 15 other genes. CD20 exists as homo-oligomers in the plasma membrane, however different MS4A proteins expressed in the same cell may hetero-oligomerize. Given the importance of CD20 in B-cell function and as a therapeutic target, we sought to explore the potential for CD20 hetero-oligomerization with other MS4A proteins. We investigated expression in primary human B-cells of the four MS4A genes previously shown to be expressed in human B-cell lines (MS4A4A, MS4A6A, MS4A7, MS4A8B), as well as two genes comprising the closely related TMEM176 gene family, with a view to identifying candidates for future investigation at the protein level. TMEM176A and TMEM176B transcripts were either not detected, or were detected at relatively low levels in a minority of donor B-cell samples. MS4A4A and MS4A8B transcripts were not detected in any normal B-cell sample. MS4A6A and MS4A7 transcripts were detected at low levels in most samples, however the corresponding proteins were not at the plasma membrane when expressed as GFP conjugates in BJAB cells. We also examined expression of these genes in chronic lymphocytic leukemia (CLL), and found that it was similar to normal B-cells with two exceptions. First, whereas MS4A4A expression was undetected in normal B-cells, it was expressed in 1/14 CLL samples. Second, compared to expression levels in normal B-cells, MS4A6A transcripts were elevated in 4/14 CLL samples. In summary, none of the MS4A/TMEM176 genes tested was expressed at high levels in normal or in most CLL B-cells. MS4A6A and MS4A7 were expressed at low levels in most B-cell samples, however the corresponding proteins may not be positioned at the plasma membrane. Altogether, these data suggest that CD20 normally does not form hetero-oligomers with other MS4A proteins and that there are unlikely to be other MS4A proteins in CLL that might provide useful alternate therapeutic targets.