Limited antitumor activity of combined BET and MEK inhibition in neuroblastoma.

BACKGROUND: The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma. PROCEDURE: Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models. RESULTS: Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1. CONCLUSIONS: Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

BACKGROUND: High-risk neuroblastoma is a complex genetic disease that is lethal in more than 50% of patients despite intense multimodal therapy. Through genome-wide association studies (GWAS) and next-generation sequencing, we have identified common single nucleotide polymorphisms and rare, pathogenic or likely pathogenic germline loss-of-function variants in BARD1 enriched in neuroblastoma patients. The functional implications of these findings remain poorly understood. METHODS: We correlated BARD1 genotype with expression in normal tissues and neuroblastomas, along with the burden of DNA damage in tumors. To validate the functional consequences of germline pathogenic or likely pathogenic BARD1 variants, we used CRISPR-Cas9 to generate isogenic neuroblastoma (IMR-5) and control (RPE1) cellular models harboring heterozygous BARD1 loss-of-function variants (R112*, R150*, E287fs, and Q564*) and quantified genomic instability in these cells via next-generation sequencing and with functional assays measuring the efficiency of DNA repair. RESULTS: Both common and rare neuroblastoma-associated BARD1 germline variants were associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using isogenic heterozygous BARD1 loss-of-function variant cellular models, we functionally validated this association with inefficient DNA repair. BARD1 loss-of-function variant isogenic cells exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA double-strand break sites, and enhanced sensitivity to cisplatin and poly (ADP-ribose) polymerase (PARP) inhibition both in vitro and in vivo. CONCLUSIONS: Taken together, we demonstrate that germline BARD1 variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications.

BARD1 germline variants induce haploinsufficiency and DNA repair defects in neuroblastoma.

Importance: High-risk neuroblastoma is a complex genetic disease that is lethal in 50% of patients despite intense multimodal therapy. Our genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) within the BARD1 gene showing the most significant enrichment in neuroblastoma patients, and also discovered pathogenic (P) or likely pathogenic (LP) rare germline loss-of-function variants in this gene. The functional implications of these findings remain poorly understood. Objective: To define the functional relevance of BARD1 germline variation in children with neuroblastoma. Design: We correlated BARD1 genotype with BARD1 expression in normal and tumor cells and the cellular burden of DNA damage in tumors. To validate the functional consequences of rare germline P-LP BARD1 variants, we generated isogenic cellular models harboring heterozygous BARD1 loss-of-function (LOF) variants and conducted multiple complementary assays to measure the efficiency of DNA repair. Setting: (N/A). Participants: (N/A). Interventions/Exposures: (N/A). Main Outcomes and Measures: BARD1 expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associated BARD1 germline variants. Results: Both common and rare neuroblastoma associated BARD1 germline variants were significantly associated with lower levels of BARD1 mRNA and an increased burden of DNA damage. Using neuroblastoma cellular models engineered to harbor disease-associated heterozygous BARD1 LOF variants, we functionally validated this association with inefficient DNA repair. These BARD1 LOF variant isogenic models exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA doublestrand break sites, and enhanced sensitivity to cisplatin and poly-ADP ribose polymerase (PARP) inhibition. Conclusions and Relevance: Considering that at least 1 in 10 children diagnosed with cancer carry a predicted pathogenic mutation in a cancer predisposition gene, it is critically important to understand their functional relevance. Here, we demonstrate that germline BARD1 variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications, and these findings may also extend to other cancers harboring germline variants in genes essential for DNA damage repair. Key Points: Question: How do neuroblastoma patient BRCA1-associated RING domain 1 ( BARD1 ) germline variants impact DNA repair? Findings: Neuroblastoma-associated germline BARD1 variants disrupt DNA repair fidelity. Common risk variants correlate with decreased BARD1 expression and increased DNA double-strand breaks in neuroblastoma tumors and rare heterozygous loss-of-function variants induce BARD1 haploinsufficiency, resulting in defective DNA repair and genomic instability in neuroblastoma cellular models. Meaning: Germline variation in BARD1 contributes to neuroblastoma pathogenesis via dysregulation of critical cellular DNA repair functions, with implications for neuroblastoma treatment, risk stratification, and cancer predisposition.

Preclinical Development of [211At]meta- astatobenzylguanidine ([211At]MABG) as an Alpha Particle Radiopharmaceutical Therapy for Neuroblastoma.

PURPOSE: [131I]meta-iodobenzylguanidine ([131I]MIBG) is a targeted radiotherapeutic administered systemically to deliver beta particle radiation in neuroblastoma. However, relapses in the bone marrow are common. [211At]meta-astatobenzylguanidine ([211At] MABG) is an alpha particle emitter with higher biological effectiveness and short path length which effectively sterilizes microscopic residual disease. Here we investigated the safety and antitumor activity [211At]MABG in preclinical models of neuroblastoma. EXPERIMENTAL DESIGN: We defined the maximum tolerated dose (MTD), biodistribution, and toxicity of [211At]MABG in immunodeficient mice in comparison with [131I]MIBG. We compared the antitumor efficacy of [211At]MABG with [131I]MIBG in three murine xenograft models. Finally, we explored the efficacy of [211At]MABG after tail vein xenografting designed to model disseminated neuroblastoma. RESULTS: The MTD of [211At]MABG was 66.7 MBq/kg (1.8 mCi/kg) in CB17SC scid-/- mice and 51.8 MBq/kg (1.4 mCi/kg) in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Biodistribution of [211At]MABG was similar to [131I]MIBG. Long-term toxicity studies on mice administered with doses up to 41.5 MBq/kg (1.12 mCi/kg) showed the radiotherapeutic to be well tolerated. Both 66.7 MBq/kg (1.8 mCi/kg) single dose and fractionated dosing 16.6 MBq/kg/fraction (0.45 mCi/kg) × 4 over 11 days induced marked tumor regression in two of the three models studied. Survival was significantly prolonged for mice treated with 12.9 MBq/kg/fraction (0.35 mCi/kg) × 4 doses over 11 days [211At]MABG in the disseminated disease (IMR-05NET/GFP/LUC) model (P = 0.003) suggesting eradication of microscopic disease. CONCLUSIONS: [211At]MABG has significant survival advantage in disseminated models of neuroblastoma. An alpha particle emitting radiopharmaceutical may be effective against microscopic disseminated disease, warranting clinical development.

Pre-clinical investigation of astatine-211-parthanatine for high-risk neuroblastoma.

Astatine-211-parthanatine ([211At]PTT) is an alpha-emitting radiopharmaceutical therapeutic that targets poly(adenosine-diphosphate-ribose) polymerase 1 (PARP1) in cancer cells. High-risk neuroblastomas exhibit among the highest PARP1 expression across solid tumors. In this study, we evaluated the efficacy of [211At]PTT using 11 patient-derived xenograft (PDX) mouse models of high-risk neuroblastoma, and assessed hematological and marrow toxicity in a CB57/BL6 healthy mouse model. We observed broad efficacy in PDX models treated with [211At]PTT at the maximum tolerated dose (MTD 36 MBq/kg/fraction x4) administered as a fractionated regimen. For the MTD, complete tumor response was observed in 81.8% (18 of 22) of tumors and the median event free survival was 72 days with 30% (6/20) of mice showing no measurable tumor >95 days. Reversible hematological and marrow toxicity was observed 72 hours post-treatment at the MTD, however full recovery was evident by 4 weeks post-therapy. These data support clinical development of [211At]PTT for high-risk neuroblastoma.

PARP Targeted Alpha-Particle Therapy Enhances Response to PD-1 Immune-Checkpoint Blockade in a Syngeneic Mouse Model of Glioblastoma.

We have previously demonstrated potent antitumor effects of PARP targeted alpha-therapy with astatine-211-MM4 ([211At]MM4) in neuroblastoma preclinical models, although differential sensitivity suggests it is unlikely to be curative as a single-agent in all tumor types. Alpha-particle induced DNA damage can elicit an immune response that results in T-cell activation against tumor cells; however, tumor cells can evade immune surveillance through expression of programmed death ligand 1 (PD-L1). Therefore, we investigated the effects of α particle therapy in combination with immune-checkpoint blockade using astatine-211-MM4 and anti-programmed death receptor 1 (anti-PD-1) immunotherapy in a syngeneic mouse model of glioblastoma. We characterized the sensitivity of four human glioblastoma cell lines to [211At]MM4 in vitro. To evaluate [211At]MM4 treatment effects on hematological tissues, complete blood counts were performed after a single dose at 12, 24, or 36 MBq/kg. In vivo efficacy was evaluated in a syngeneic mouse model of glioblastoma using GL26 glioblastoma cells in CB57BL/6J mice treated with either 36 MBq/kg [211At]MM4, anti-PD-1 antibody, or a combination of the two. Following a single dose of [211At]MM4, lymphocytes are significantly decreased compared to control at both 72 h and 1 week following treatment followed by recovery of counts by 2 weeks. However, neutrophils showed an increase with all dose levels of [211At]MM4 exhibiting higher levels than control. The average best tumor responses for combination, anti-PD-1, and [211At]MM4 were 100%, 83.6%, and 58.2% decrease in tumor volume, respectively. Average progression free intervals for combination, anti-PD-1, [211At]MM4, and control groups was 65, 36.4, 23.2, and 3 days, respectively. The percentages of disease-free mice at the end of the study for combination and anti-PD-1 were 100% and 60%, while [211At]MM4 and control groups were both 0%. In summary, combination therapy was more effective than either single agent in all response categories analyzed, highlighting the potential for PARP targeted alpha-therapy to enhance PD-1 immune-checkpoint blockade.

Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo.

Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To address this issue, we created a series of CARs that contain the T cell receptor-zeta (TCR-zeta) signal transduction domain with the CD28 and/or CD137 (4-1BB) intracellular domains in tandem. After short-term expansion, primary human T cells were subjected to lentiviral gene transfer, resulting in large numbers of cells with >85% CAR expression. In an immunodeficient mouse xenograft model of primary human pre-B-cell acute lymphoblastic leukemia, human T cells expressing anti-CD19 CARs containing CD137 exhibited the greatest antileukemic efficacy and prolonged (>6 months) survival in vivo, and were significantly more effective than cells expressing CARs containing TCR-zeta alone or CD28-zeta signaling receptors. We uncovered a previously unrecognized, antigen-independent effect of CARs expressing the CD137 cytoplasmic domain that likely contributes to the enhanced antileukemic efficacy and survival in tumor bearing mice. Furthermore, our studies revealed significant discrepancies between in vitro and in vivo surrogate measures of CAR efficacy. Together these results suggest that incorporation of the CD137 signaling domain in CARs should improve the persistence of CARs in the hematologic malignancies and hence maximize their antitumor activity.

PARP-1-Targeted Auger Emitters Display High-LET Cytotoxic Properties In Vitro but Show Limited Therapeutic Utility in Solid Tumor Models of Human Neuroblastoma.

The currently available therapeutic radiopharmaceutical for high-risk neuroblastoma, 131I-metaiodobenzylguanidine, is ineffective at targeting micrometastases because of the low-linear-energy-transfer (LET) properties of high-energy ß-particles. In contrast, Auger radiation has high-LET properties with nanometer ranges in tissue, efficiently causing DNA damage when emitted near DNA. The aim of this study was to evaluate the cytotoxicity of targeted Auger therapy in preclinical models of high-risk neuroblastoma. Methods: We used a radiolabled poly(adenosine diphosphate ribose) polymerase (PARP) inhibitor called 125I-KX1 to deliver Auger radiation to PARP-1, a chromatin-binding enzyme overexpressed in neuroblastoma. The in vitro cytotoxicity of 125I-KX1 was assessed in 19 neuroblastoma cell lines, followed by in-depth pharmacologic analysis in a sensitive and resistant pair of cell lines. Immunofluorescence microscopy was used to characterize 125I-KX1-induced DNA damage. Finally, in vitro and in vivo microdosimetry was modeled from experimentally derived pharmacologic variables. Results:125I-KX1 was highly cytotoxic in vitro across a panel of neuroblastoma cell lines, directly causing double-strand DNA breaks. On the basis of subcellular dosimetry, 125I-KX1 was approximately twice as effective as 131I-KX1, whereas cytoplasmic 125I-metaiodobenzylguanidine demonstrated low biological effectiveness. Despite the ability to deliver a focused radiation dose to the cell nuclei, 125I-KX1 remained less effective than its α-emitting analog 211At-MM4 and required significantly higher activity for equivalent in vivo efficacy based on tumor microdosimetry. Conclusion: Chromatin-targeted Auger therapy is lethal to high-risk neuroblastoma cells and has the potential to be used in micrometastatic disease. This study provides the first evidence for cellular lethality from a PARP-1-targeted Auger emitter, calling for further investigation into targeted Auger therapy.

Inactivation of wild-type p53 protein function by reactive oxygen and nitrogen species in malignant glioma cells.

Malignant gliomas are the most common primary brain tumors in adults, and the most malignant form, glioblastoma multiforme (GBM), is usually rapidly fatal. Most GBMs do not have p53 mutations, although the p53 tumor suppressor pathway appears to be inactivated. GBMs grow in a hypoxic and inflammatory microenvironment, and increased levels of the free radicals nitric oxide (NO) and superoxide () occur in these malignancies in vivo. Peroxynitrite (ONOO(-)) is a highly reactive molecule produced by excess NO and that can posttranslationally modify and inactivate proteins, especially zinc finger transcription factors such as p53. We demonstrated previously that GBMs have evidence of tyrosine nitration, the "footprint" of peroxynitrite-mediated protein modification in vivo, and that peroxynitrite could inhibit the specific DNA binding ability of wild-type p53 protein in glioma cells in vitro. Here we show that both authentic peroxynitrite and SIN-1 (3-morpholinosydnonimine hydrochloride), a molecule that decomposes into NO and to form peroxynitrite, can inhibit wild-type p53 function in malignant glioma cells. Concentrations of peroxynitrite associated with a tumor inflammatory environment caused dysregulation of wild-type p53 transcriptional activity and downstream p21(WAF1) expression.

Human cytomegalovirus infection and expression in human malignant glioma.

Malignant gliomas are the most common primary brain tumors in adults, have no known etiology, and are generally rapidly fatal despite current therapies. Human cytomegalovirus (HCMV) is beta-herpesvirus trophic for glial cells that persistently infects 50-90% of the adult human population. HCMV can be reactivated under conditions of inflammation and immunosuppression, and HCMV gene products can dysregulate multiple cellular pathways involved in oncogenesis. Here we show that a high percentage of malignant gliomas are infected by HCMV and multiple HCMV gene products are expressed in these tumors. These data are the first to show an association between HCMV and malignant gliomas and suggest that HCMV may play an active role in glioma pathogenesis.

Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAFV600E Conformation.

The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAFV600E) accounts for about 90% of these activating mutations. While BRAFV600E-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK, or splicing variants. RAF kinases signal as homo- and heterodimers, and another complication associated with small molecule BRAFV600E inhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called "transactivation" or "paradoxical activation." Here, we used BRAFV600E and vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells but promotes an inactive dimeric BRAFV600E conformation unable to undergo transactivation. The crystal structure of a BRAFV600E/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over an active dimeric BRAFV600E conformation. These studies have implications for targeting BRAFV600E/RAF heterodimers and other kinase dimers for therapy.

Targeting Notch enhances the efficacy of ERK inhibitors in BRAF-V600E melanoma.

The discovery of activating BRAF mutations in approximately 50% of melanomas has led to the development of MAPK pathway inhibitors, which have transformed melanoma therapy. However, not all BRAF-V600E melanomas respond to MAPK inhibition. Therefore, it is important to understand why tumors with the same oncogenic driver have variable responses to MAPK inhibitors. Here, we show that concurrent loss of PTEN and activation of the Notch pathway is associated with poor response to the ERK inhibitor SCH772984, and that co-inhibition of Notch and ERK decreased viability in BRAF-V600E melanomas. Additionally, patients with low PTEN and Notch activation had significantly shorter progression free survival when treated with BRAF inhibitors. Our studies provide a rationale to further develop combination strategies with Notch antagonists to maximize the efficacy of MAPK inhibition in melanoma. Our findings should prompt the evaluation of combinations co-targeting MAPK/ERK and Notch as a strategy to improve current therapies and warrant further evaluation of co-occurrence of aberrant PTEN and Notch activation as predictive markers of response to therapy.

Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma.

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

Characterization CSMD1 in a large set of primary lung, head and neck, breast and skin cancer tissues.

The Cub and Sushi Multiple Domains-1 (CSMD1) is a tumor suppressor gene on 8p23.2, where allelic loss is both frequent and associated with poor prognosis in head and neck squamous cell carcinoma (HNSCC). To understand the extent of CSMD1 aberrations in vivo, we characterized 184 primary tumors from the head and neck, lung, breast and skin for gene copy number and analyzed expression in our HNSCCs and lung squamous cell carcinomas (SCCs). We detected loss of CSMD1 in a large proportion of HNSCCs (50%), lung (46%) and breast cancers (55%), and to a lesser extent in cutaneous SCCs (29%) and basal cell carcinomas (BCCs, 17%) using array-based comparative genomic hybridization (aCGH). Studying the region more closely with quantitative real-time PCR (qPCR), the loss of CSMD1 increased to 80% in HNSCCs and 93% in lung SCCs. CSMD1 expression was decreased in tumors compared to adjacent benign tissue (65%, 13/20) and was likely due to gene loss in 45% of cases (9/20). We also identified truncated transcripts lacking exons due to DNA copy number loss (30%, 5/17) or aberrant splicing (24%, 4/17). We show loss of CSMD1 in primary HNSCC tissues, and document for the first time that CSMD1 is lost in breast, lung and cutaneous SCCs. We also show that deletions of CSMD1 and aberrant splicing contribute to altered CSMD1 function in vivo.

Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions.

Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.

High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma.

PURPOSE: Recent epidemiological data indicate that a history of increased exposure to sexually transmitted diseases is associated with an increased risk of prostate cancer. Human cytomegalovirus (HCMV) is a member of the herpesvirus family, is sexually transmitted in adults and can persistently infect prostatic epithelium in non-immunocompromised hosts. Based on increased awareness of the oncogenic potential of this virus, we decided to reexplore the issue of whether HCMV might be involved in prostate cancer pathogenesis. MATERIALS AND METHODS: Paraffin embedded biopsy specimens from 22 randomly selected patients with prostatic intraepithelial neoplasia (PIN) lesions and prostatic carcinoma were analyzed by immunohistochemistry, in situ hybridization, polymerase chain reaction and DNA sequencing to detect HCMV nucleic acids and determine whether HCMV gene products were specifically associated with neoplastic cells. RESULTS: We detected HCMV proteins and/or nucleic acids in all 22 of the 22 preneoplastic and neoplastic prostate lesions evaluated. HCMV proteins were specifically and often highly expressed in basal cell hyperplasia and PIN lesions, and to a lesser degree in carcinoma cells. RESULTS: To our knowledge these data demonstrate for the first time the specific localization of HCMV nucleic acids and proteins in a high percent of PIN and prostate carcinoma lesions, and raise the possibility that HCMV might contribute to the natural history of prostatic cancer.

Specific localisation of human cytomegalovirus nucleic acids and proteins in human colorectal cancer.

BACKGROUND: Colorectal cancer is the second most frequent cause of death from cancer in the USA, and most tumours arise sporadically with no clear cause or genetic predisposition. Human cytomegalovirus is a beta-herpesvirus that is endemic in the human population and can cause life-threatening disease in immunosuppressed adults. In vitro, human cytomegalovirus can transform cells and dysregulate many cellular pathways relevant to colon adenocarcinoma pathogenesis, especially those affecting the cell cycle, mutagenesis, apoptosis, angiogenesis, and cyclo-oxygenase-2 (COX-2) expression. We aimed to assess whether gene products of human cytomegalovirus could be detected in colorectal cancers. METHODS: We obtained formalin-fixed, paraffin-embedded pathological specimens of colorectal polyps, adenocarcinomas, and adjacent normal mucosa from 29 patients. To detect human cytomegalovirus proteins and nucleic acids, we used immunohistochemistry with two different monoclonal antibodies, in-situ hybridisation, and PCR with DNA sequencing. FINDINGS: Human cytomegalovirus proteins IE1-72 and pp65 were detected in a tumour cell-specific pattern in 14 (82%) of 17 and seven (78%) of nine colorectal polyps, respectively, and 12 (80%) of 15 and 11 (92%) of 12 adenocarcinomas, respectively, but not in adjacent non-neoplastic colon biopsy samples from the same patients (none of seven and none of two, respectively). Human cytomegalovirus infection of colon-cancer cells (Caco-2) in vitro resulted in specific induction of Bcl-2 and cyclo-oxygenase-2 proteins, both of which are thought to contribute to progression of colon cancer. INTERPRETATION: Human cytomegalovirus nucleic acids and proteins can be found that specifically localise to neoplastic cells in human colorectal polyps and adenocarcinomas, and virus infection can induce important oncogenic pathways in colon-cancer cells.