Evidence for virus-mediated oncogenesis in bladder cancers arising in solid organ transplant recipients
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Curated by eLife
eLife assessment
The fundamental work by Starret et al advances the understanding of the etiological roles of viruses and other environmental factors in bladder cancers after solid organ transplantation. The evidence is compelling using cutting edge sequencing approaches of patient samples. This work will be immediately interesting to multiple fields (of viral oncogenesis, BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV), solid organ transplantation and whole genome and transcriptome sequencing) and may eventually influence care after organ transplantation.
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Abstract
A small percentage of bladder cancers in the general population have been found to harbor DNA viruses. In contrast, up to 25% of tumors of solid organ transplant recipients, who are at an increased risk of developing bladder cancer and have an overall poorer outcomes, harbor BK polyomavirus (BKPyV). To better understand the biology of the tumors and the mechanisms of carcinogenesis from potential oncoviruses, we performed whole genome and transcriptome sequencing on bladder cancer specimens from 43 transplant patients. Nearly half of the tumors from this patient population contained viral sequences. The most common were from BKPyV (N=9, 21%), JC polyomavirus (N=7, 16%), carcinogenic human papillomaviruses (N=3, 7%), and torque teno viruses (N=5, 12%). Immunohistochemistry revealed variable Large T antigen expression in BKPyV-positive tumors ranging from 100% positive staining of tumor tissue to less than 1%. In most cases of BKPyV-positive tumors, the viral genome appeared to be clonally integrated into the host chromosome consistent with microhomology-mediated end joining and coincided with focal amplifications of the tumor genome similar to other virus-mediated cancers. Significant changes in host gene expression consistent with the functions of BKPyV Large T antigen were also observed in these tumors. Lastly, we identified four mutation signatures in our cases, with those attributable to APOBEC3 and SBS5 being the most abundant. Mutation signatures associated with an antiviral drug, ganciclovir, and aristolochic acid, a nephrotoxic compound found in some herbal medicines, were also observed. The results suggest multiple pathways to carcinogenesis in solid organ transplant recipients with a large fraction being virus-associated.
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Author Response
Reviewer #1 (Public Review):
Starrett, Gabriel et al. investigated 43 bladder cancers (primary tumors), 5 metastases and 14 normal tissues from 43 solid organ transplant recipients of 5 Transplant Cancer Match Study participating registries (US) for the presence of viral genetic signatures, their host genome integration and possible contribution in carcinogenesis. They isolated DNA and RNA from FFPE tissues to perform state of the art whole genome and transcriptome sequencing. They find that 20 of the primary tumors, 3 of the metastases and 7 of the normal tissues harbor viral signatures with BKPyV and JCPyV being the most prevalent viruses detected. The bulk of the experiments focuses on the 9 BKPyV-positive primary tumors. They report that several of the BKPyV-positive tumors show host genome integration of BKPyV …
Author Response
Reviewer #1 (Public Review):
Starrett, Gabriel et al. investigated 43 bladder cancers (primary tumors), 5 metastases and 14 normal tissues from 43 solid organ transplant recipients of 5 Transplant Cancer Match Study participating registries (US) for the presence of viral genetic signatures, their host genome integration and possible contribution in carcinogenesis. They isolated DNA and RNA from FFPE tissues to perform state of the art whole genome and transcriptome sequencing. They find that 20 of the primary tumors, 3 of the metastases and 7 of the normal tissues harbor viral signatures with BKPyV and JCPyV being the most prevalent viruses detected. The bulk of the experiments focuses on the 9 BKPyV-positive primary tumors. They report that several of the BKPyV-positive tumors show host genome integration of BKPyV with associated focal amplifications of adjacent host chromosome regions, with chromosome 1 being the most prevalent. Furthermore, BKPyV-positive tumors show a distinct transcriptomic signature with gene expression changes related to DNA damage responses, cell cycle progression, angiogenesis, chromatin organization, mitotic spindle assembly, chromosome condensation/separation and neuronal differentiation. The authors only touch the features of other virus-positive tumors, e.g. those with JCPyV and HPV signals, without offering further detail or thought. The overall mutation signature analysis reveals no clear correlation between presence of viral sequences and tumor mutation burden suggesting that many different, virus-unrelated, factors possibly contribute to bladder cancer genesis and progression. Most striking are cases potentially linked to aristolochic acid, ABOBUCK3 and SBS5. Thus, while the approach is state-of-the-art, the causality of viral signatures and oncogenesis and vice versa remains unsolved.
Strengths:
- The study assesses 43 primary tumors, 5 metastases and 14 normal tissues from 43 solid organ transplants of different kinds (24x kidney, 4x liver, 14x heart and/or lung, 1x pancreas) rather than just focusing on a particular organ.
- The study makes use of whole genome sequencing and transcriptomics and the assayed material is extracted from FFPE tissue, which shows a high level of practical, technical and computational skills and expertise.
Weaknesses:
- There have been multiple inconsistencies in sample number and figure references throughout the publication. Is it 19 or 20 cases that have viral sequences detected? A comprehensive checker board table showing all cases, the available tissue samples and respective analyses would be in order.
We would like to thank the reviewer for their detailed assessment of the manuscript. A checkerboard table of all samples tissues and analysis has been added as supplemental table 1 (Supplementary file 1a).
- The overall low coverage of the whole genome sequencing, which the authors mention, and the relatively big variation in coverage in both datasets (WGS, transcriptomics) are major limitations of the study. Possibly, this was done to increase specificity, but sorting out and discarding reads may also be problematic. Please comment.
Besides performing quality and adapter trimming as described in the methods, we did not discard any reads. Experimental design and analysis were conducted to be as inclusive as possible considering the rarity of these specimens.
Reviewer #2 (Public Review):
Starrett et al performed whole genome and transcriptome sequencing of bladder cancers from 43 organ transplant recipients. They found that most of these tumors contained DNA from one of four viruses (BKPyV, JCPyV, HPV, and TTV). Viral genomes are most often integrated into the genomes of these tumor cells and the authors provide evidence that the integration utilized the POL theta-mediated end joining pathway. In most cases, viral RNA was detected in tumors with viral DNA. This suggests that the viruses are actively altering the cellular environment. Frequently, this resulted in similarities for overall gene expression patterns in the tumors that were grouped by the type of virus present in the tumor. Moreover, the changes in expression linked with viral gene expression were found in genes relevant to tumorigenesis. Immunohistochemical detection of viral proteins in these tumors also demonstrated active viral gene expression. However, the presence of viral proteins was heterogenous within the tumor, with between 1 and 100% of the tumor staining positive for BKPyV large T antigen. An analysis of mutational signatures in these tumors indicate that the viruses are also shaping the tumor genome by inducing mutations. Evidence that specific viruses are contributing to tumorigenesis in organ transplant patients has fundamental implications for preventing tumorigenesis in these patients.
The conclusions of this paper are generally well supported by the data provided. Indeed, there is little doubt that viral infections are more likely in these tumors. However, there are aspects of the paper that could be improved and or clarified. Most importantly, despite the strong evidence that the viruses are altering the tumor cell environment, it is unclear if these changes are necessary for tumorigenesis or less excitingly the result of an even more immune suppressive environment within the tumor. The heterogeneity of the LT expression suggests that the presence of the viral DNA and RNA may not be enough to assess whether it is actively contributing to the tumor. Is an increased frequency of viral protein staining linked with any evidence of an active contribution to tumorigenesis (fewer tumor-suppressor/oncogene mutations). that they reduced mutations in tumor suppressors. This might be easiest to assess with the tumors that have oncogenic HPV DNA. If those tumors lacked p53 and RB mutations, it would support a causative role for the virus.
We thank the reviewer for their thoughtful review. Indeed, in Figure 6 we show that no BKPyV-positive or HPV-positive tumor harbored mutations in RB1. Additionally, only one BKPyV-positive tumor and none of the HPV-positive tumors had a mutation in TP53. We have added further emphasis to this point on page 14, “None of the HPV-positive tumors with WGS harbored mutations in TP53 or RB1. Similarly, none of the polyomavirus-positive tumors harbored mutations in RB1 and only TBC08 had a frameshift mutation in TP53.”
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eLife assessment
The fundamental work by Starret et al advances the understanding of the etiological roles of viruses and other environmental factors in bladder cancers after solid organ transplantation. The evidence is compelling using cutting edge sequencing approaches of patient samples. This work will be immediately interesting to multiple fields (of viral oncogenesis, BK polyomavirus (BKPyV) and JC polyomavirus (JCPyV), solid organ transplantation and whole genome and transcriptome sequencing) and may eventually influence care after organ transplantation.
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Reviewer #1 (Public Review):
Starrett, Gabriel et al. investigated 43 bladder cancers (primary tumors), 5 metastases and 14 normal tissues from 43 solid organ transplant recipients of 5 Transplant Cancer Match Study participating registries (US) for the presence of viral genetic signatures, their host genome integration and possible contribution in carcinogenesis. They isolated DNA and RNA from FFPE tissues to perform state of the art whole genome and transcriptome sequencing. They find that 20 of the primary tumors, 3 of the metastases and 7 of the normal tissues harbor viral signatures with BKPyV and JCPyV being the most prevalent viruses detected. The bulk of the experiments focuses on the 9 BKPyV-positive primary tumors. They report that several of the BKPyV-positive tumors show host genome integration of BKPyV with associated focal …
Reviewer #1 (Public Review):
Starrett, Gabriel et al. investigated 43 bladder cancers (primary tumors), 5 metastases and 14 normal tissues from 43 solid organ transplant recipients of 5 Transplant Cancer Match Study participating registries (US) for the presence of viral genetic signatures, their host genome integration and possible contribution in carcinogenesis. They isolated DNA and RNA from FFPE tissues to perform state of the art whole genome and transcriptome sequencing. They find that 20 of the primary tumors, 3 of the metastases and 7 of the normal tissues harbor viral signatures with BKPyV and JCPyV being the most prevalent viruses detected. The bulk of the experiments focuses on the 9 BKPyV-positive primary tumors. They report that several of the BKPyV-positive tumors show host genome integration of BKPyV with associated focal amplifications of adjacent host chromosome regions, with chromosome 1 being the most prevalent. Furthermore, BKPyV-positive tumors show a distinct transcriptomic signature with gene expression changes related to DNA damage responses, cell cycle progression, angiogenesis, chromatin organization, mitotic spindle assembly, chromosome condensation/separation and neuronal differentiation. The authors only touch the features of other virus-positive tumors, e.g. those with JCPyV and HPV signals, without offering further detail or thought. The overall mutation signature analysis reveals no clear correlation between presence of viral sequences and tumor mutation burden suggesting that many different, virus-unrelated, factors possibly contribute to bladder cancer genesis and progression. Most striking are cases potentially linked to aristolochic acid, ABOBUCK3 and SBS5. Thus, while the approach is state-of-the-art, the causality of viral signatures and oncogenesis and vice versa remains unsolved.
Strengths:
The study assesses 43 primary tumors, 5 metastases and 14 normal tissues from 43 solid organ transplants of different kinds (24x kidney, 4x liver, 14x heart and/or lung, 1x pancreas) rather than just focusing on a particular organ.
The study makes use of whole genome sequencing and transcriptomics and the assayed material is extracted from FFPE tissue, which shows a high level of practical, technical and computational skills and expertise.
Weaknesses:
There have been multiple inconsistencies in sample number and figure references throughout the publication. Is it 19 or 20 cases that have viral sequences detected? A comprehensive checker board table showing all cases, the available tissue samples and respective analyses would be in order.
The overall low coverage of the whole genome sequencing, which the authors mention, and the relatively big variation in coverage in both datasets (WGS, transcriptomics) are major limitations of the study. Possibly, this was done to increase specificity, but sorting out and discarding reads may also be problematic. Please comment.
-
Reviewer #2 (Public Review):
Starrett et al performed whole genome and transcriptome sequencing of bladder cancers from 43 organ transplant recipients. They found that most of these tumors contained DNA from one of four viruses (BKPyV, JCPyV, HPV, and TTV). Viral genomes are most often integrated into the genomes of these tumor cells and the authors provide evidence that the integration utilized the POL theta-mediated end joining pathway. In most cases, viral RNA was detected in tumors with viral DNA. This suggests that the viruses are actively altering the cellular environment. Frequently, this resulted in similarities for overall gene expression patterns in the tumors that were grouped by the type of virus present in the tumor. Moreover, the changes in expression linked with viral gene expression were found in genes relevant to …
Reviewer #2 (Public Review):
Starrett et al performed whole genome and transcriptome sequencing of bladder cancers from 43 organ transplant recipients. They found that most of these tumors contained DNA from one of four viruses (BKPyV, JCPyV, HPV, and TTV). Viral genomes are most often integrated into the genomes of these tumor cells and the authors provide evidence that the integration utilized the POL theta-mediated end joining pathway. In most cases, viral RNA was detected in tumors with viral DNA. This suggests that the viruses are actively altering the cellular environment. Frequently, this resulted in similarities for overall gene expression patterns in the tumors that were grouped by the type of virus present in the tumor. Moreover, the changes in expression linked with viral gene expression were found in genes relevant to tumorigenesis. Immunohistochemical detection of viral proteins in these tumors also demonstrated active viral gene expression. However, the presence of viral proteins was heterogenous within the tumor, with between 1 and 100% of the tumor staining positive for BKPyV large T antigen. An analysis of mutational signatures in these tumors indicate that the viruses are also shaping the tumor genome by inducing mutations. Evidence that specific viruses are contributing to tumorigenesis in organ transplant patients has fundamental implications for preventing tumorigenesis in these patients.
The conclusions of this paper are generally well supported by the data provided. Indeed, there is little doubt that viral infections are more likely in these tumors. However, there are aspects of the paper that could be improved and or clarified. Most importantly, despite the strong evidence that the viruses are altering the tumor cell environment, it is unclear if these changes are necessary for tumorigenesis or less excitingly the result of an even more immune suppressive environment within the tumor. The heterogeneity of the LT expression suggests that the presence of the viral DNA and RNA may not be enough to assess whether it is actively contributing to the tumor. Is an increased frequency of viral protein staining linked with any evidence of an active contribution to tumorigenesis (fewer tumor-suppressor/oncogene mutations). that they reduced mutations in tumor suppressors. This might be easiest to assess with the tumors that have oncogenic HPV DNA. If those tumors lacked p53 and RB mutations, it would support a causative role for the virus.
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