Classical epidemiology is poorly equipped to determine multifactorial causality for common commensal tumor viruses, such as EBV.

<p>Multifactorial causal reasoning is shown for a simple electrical circuit (inset) with two switches, Switch 1 and Switch 2, either of which can “cause” the light bulb to turn on. An analogous pathway is shown for the genesis of Burkitt lymphoma, in which EBV is responsible for a portion of tumors, but also only in the biological context of other factors, such as cMYC translocations. Since EBV is nearly ubiquitous, teasing out its contribution to a rare cancer like Burkitt lymphoma is supremely difficult using standard epidemiologic methods, but is readily evident using molecular biologic information that has been available for decades. EBV is clonal in these tumors based on terminal repeat copies and Epstein–Barr encoding region (EBER) in situ hybridization typically reveals the presence of EBV genome in all tumor cells but not surrounding nontumor cells. These facts are biologically implausible for a non-causal passenger infection [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006078#ppat.1006078.ref006" target="_blank">6</a>].</p

Kaplan-Meier curves of multiple MCC mouse xenograft models on different treatments.

<p><b>A</b>) Estimated survival means and 95% confidence intervals are reported along compressed survival summaries per cell line and treatment arm, where open circles correspond survival of individual mice. <b>B</b>) Mice with MKL-1 xenografts exhibit significantly prolonged survival (****P < 0.0001) on any of the three YM155 treatment groups (3-weeks at 2mg/kg = red; continuous treatment at 2mg/kg = purple; continuous treatment at 4mg/kg = orange) relative to saline treatment (green). Increasing the duration of YM155 treatment from 3-weeks to continuous treatment at the 2mg/kg dose significantly prolongs survival (****P < 0.0001). Increasing the dose of YM155 from 2mg/kg to 4mg/kg on continuous treatment significantly prolongs survival (****P < 0.0001). <b>C</b>) Mice with MS-1 xenografts do not exhibit prolonged survival with YM155 continuous treatment (either at 2mg/kg or 4mg/kg) relative to saline treatment (NS = not significant). One mouse on saline treatment spontaneously regressed for over 5-weeks and was euthanized early (as indicated by <b>x</b>). <b>D</b>) Mice with WaGa xenografts exhibit significantly prolonged survival (**P = 0.0034) with continuous YM155 treatment at 4mg/kg relative to saline treatment. <b>E</b>) Mice with MKL-2 xenografts exhibit significantly prolonged survival (****P < 0.0001) with continuous YM155 treatment at 4mg/kg relative to saline treatment. Two mice did not reach the final 20mm tumor dimension by day 105 and were euthanized early (as indicated by <b>##</b>). </p

Mouse weights by treatment regimen.

<p>Average mouse weights with standard deviations are reported according to treatment regimen, where weights were normalized to day zero of treatment (100%): <b>A</b>) mouse weights on saline, continuous-treatment (green line); <b>B</b>) mouse weights on 2mg/kg YM155, continuous-treatment (purple line); and <b>C</b>) mouse weights on 4mg/kg YM155, continuous-treatment (orange line). Mouse weights were adjusted to remove the weight of tumors prior to normalization. Weights from mice with significant liver metastases were not included as metastatic-tumor weights could not be determined during the course of treatment. </p

Various chemotherapeutics combined with YM155 induce MCC cell death in an additive manner, <i>in</i><i>vitro</i>.

<p>CellTiter-GLO assays were performed using multiple MCC cell lines as well as the control primary human fibroblast, BJ. Corresponding dose-response curves are shown for the following chemotherapeutic agents and drug combinations: <b>A</b>) YM155; <b>B</b>) Bortezomib; <b>C</b>) Bortezomib + 3nM YM155; <b>D</b>) Docetaxel; <b>E</b>) Docetaxel + 3nM YM155; <b>F</b>) Etoposide; <b>G</b>) Etoposide + 3nM YM155 <b>H</b>) Topotecan; and <b>I</b>) Topotecan + 3nM YM155. </p

MCC mouse xenograft treatment groups and experimental outline.

<p><b>A</b>) NSG mice were subcutaneously injected in the right flank with 2x10⁷ MCV-positive, MCC cells (MKL-1, MS-1, WaGa, or MKL-2). <b>B</b>) NSG mice were monitored for palpable tumors (~2mm x 2mm) to determine start of treatment. <b>C</b>) Mice with palpable tumors were randomly assigned to either saline treatment, YM155 treatment for 3-weeks at 2mg/kg, YM155 continuous treatment at 2mg/kg, or YM155 continuous treatment at 4mg/kg. Each week of treatment consisted of a single intraperitoneal injection per day for 5 days, followed by 2 days of rest. </p

Immunohistochemistry of MCV-LT in a MKL-1 xenograft primary tumor and a liver metastasis.

<p>Shown are paired hemotoxylin & eosin (H&E) stained slides and adjacent sections stained with CM2B4, the MCV-LT antibody (LT-IHC), in mice with MKL-1 xenografts: <b>A</b>) MKL-1 xenograft primary tumor, H&E; <b>B</b>) MKL-1 xenograft primary tumor, LT-IHC; <b>C</b>) MKL-1 xenograft liver metastasis, H&E; and <b>D</b>) MKL-1 xenograft liver metastasis, LT-IHC. MKL-1 cells contains nuclear staining of LT, consistent with an intact nuclear localization signal (NLS). Original magnification = 200X; insets = 600X. </p

Time-to-Palpability.

<p>The length of time lapsed after initial cell line injection to detection of palpable tumors (~2mm x 2mm) is indicated for each of the four MCC cell lines tested (MKL-1, WaGa, MKL-2, and MS-1). </p

MCV genome replication.

<p>Southern blot (right panel) for MCV for MCV-HF (lane 1), MCV-Rep⁻ (lane 2) and MCV-hVam6p⁻ (lane 3) viruses four days after transfection of 1 µg circular genomic DNA into 293 cells. Panel on left shows the ethidium bromide-stained gel prior to transfer indicating equal DNA loading. Bands for the full-length 5.4 kb MCV genome are present as <i>Dpn</i>I-resistant bands in MCV-HF and MCV-hVam6p⁻ viruses (lanes 1 and 3) but not in the replication deficient MCV-Rep⁻ virus (lane 2). The replication efficiency was measured by the ratio between the <i>DpnI</i>-resistant 5.4 kb band and the <i>DpnI</i>-sensitive band. The MCV-hVam6p⁻ virus generates ∼2-fold more full length genome compared to wild-type MCV-HF virus. Replicated viral DNAs also show the presence of extensive subgenomic fragments.</p

Quantitative PCR for MCV virion production for MCV-HF, MCV-Rep⁻ and MCV-hVam6p⁻ viruses.

<p>One microgram MCV clone DNAs were transfected into 293 cells stably transduced to express MCV sT or LT proteins (not shown). DNA was extracted and treated with benzonase and RNase to discriminate packaged viral DNA. The nuclease-resistant MCV genome was precipitated and measured by quantitative PCR after proteinase K treatment. Cellular sT expression increases virion production for both MCV-HF and MCV-hVam6p⁻ viruses. Comparison of MCV-HF and MCV-hVam6p⁻ shows that loss of the hVam6p binding site also increases virus production. Coexpression of sT and mutation of the hVam6p binding site in the MCV genome are additive in virion production compared to MCV-HF without sT coexpression.</p

Optimization of MCV production in various cell lines and effect of co-expression of viral proteins.

<p>*<i>Note: Relative expression was determined in individual experiments by PCR(§) , MCV protein expression (¶), or both.</i></p