The co-presence of high-risk human papillomaviruses and Epstein-Barr virus is linked with tumor grade and stage in Qatari women with breast cancer
Authors: Reem Al-Sarraf
Ayesha Jabeen
Ali A Sultan
Semir Vranic
Hamda Al-Thawadi
Ishita Gupta
Ala-Eddin Al Moustafa
Hanan Farghaly
Journal: Human Vaccines & Immunotherapeutics (2021)
Discipline: IMMUNOLOGY
Keywords: EBV; HPV; Qatari population; breast cancer; tumor grade
Affiliations: College of Medicine, QU Health, Qatar University, Doha, Qatar, Biomedical Research Centre, QU Health, Qatar University, Doha, Qatar, Department of Pathology, Hamad General Hospital, Doha, Qatar, Department of Microbiology and Immunology, Weill Cornell Medicine – Qatar, Doha, Qatar

Abstract:  High-risk human papillomaviruses (HPV) can be present and cooperate with Epstein-Barr virus (EBV) to promote the onset and/or progression of various cancers including cervical, breast, head and neck as well as colorectal. In this investigation, we explored the co-prevalence of high-risk HPV and EBV in 74 breast cancer tissues from Qatari women using polymerase chain reaction. We found that high-risk HPV and EBV are present in 48/74 (65%) and 36/74 (49%) of the cases, respectively. While we noted that the presence of HPV presence is associated with triple-negative breast cancer (TNBC) (p = .008), however, the presence of EBV did not correlate with any breast cancer subgroup. Moreover, our data revealed that high-risk HPV and EBV are co-present in 35/74 (47%) of the samples and their co-presence is significantly associated with tumor grade (p = .04) and tumor stage (p = .04). These data indicate that HPV and EBV are commonly co-present in breast cancer and their association could be linked with a more aggressive tumor phenotype. Thus, further investigations are essential to understand the underlying mechanisms of HPV and EBV cooperation in breast carcinogenesis. 

Retracted: No

PubPeer comments:
 There are multiple potential flaws in Fig.1 and Fig.2: (1) Gels have non-matching numbers of lanes in HPV vs. GAPDH (2) GAPDH in Fig.1 and Fig.2 appear more similar then expected (3) Some bands appear to be spliced (indicated by arrows) Could the authors please clarify these concerns ![file] ![file] , 
 #1 Dear Mr Heterochasta We thank you for the concern. We apologize for any issue. 1. 1. We ran 37 samples, and while cropping, we miscalculated. We are sincerely sorry about this 2. Gapdh was run only once for all samples and hence the similarity. 3. Since these are representative images, we took bands from the gel image to show best image. Hope this clarifies the issue. We are extremely sorry for any error. , 
 Dear authors, Thank your for your response. (1) Cutting and pasting gel bands is usually not seen as good scientific practice. (2) With  similarity you mean that they are the same? , 
 #3 Thank you for your comments. We appreciate your concern. 1. We totally agree with you, but, it was a representative image only and not for any quantitative purpose. The image was just to reflect the presence of the subtypes in whole cohort. 2. Since GAPDH was run only once for all the samples, the image is same for both HPV and EBV. Hope this clarifies your doubt. Thank you again , 
 #4 I thank the authors for engaging in this discussion. If I understand correctly, the reason the authors ran GAPDH was to ensure that a sample NOT showing a signal for HPV was truly negative (i.e. the lack of signal was not due to issues with sample degradation, DNA quality etc.). Therefore, it is essential that all the HPV samples are correctly matched to their corresponding GAPDH controls. Whether the data are a representative image only and not for any quantitative purpose is irrelevant. At least for the images highlighted by Heterochasta, please can the authors provide original images with the DNA ladder included? This will clarify if the original image also shows what appears to be a splice but could also be something to do with image processing. , 
 Dear Miss Antarctica We thank you for joining this discussion and showing your concern. Please note that GAPDH was run for all the samples. The images highlighted have been attached below for your reference. Kindly note that we ran 3 gels in total for all the 74 samples. For HPVs- 45, 51 and 52, the negative control was not loaded in the same gel from where the data has been derived for the manuscript and hence, that area was spliced from the either of the two gels run for the respective HPV subtypes. However as requested by you the gel images with the controls and the ladder have been added. HPV 45![file] ![file] HPV 51 ![file] ![file] ![file] HPV52 ![file] ![file] Hope this clarifies the issue. Since these are only a few number of samples represented in the figure you cannot comprehend from this a negative impact on our data integrity. We believe, if we had to manipulate data, we would not have attached the figures. Please focus on the statistics rather than the figures as this is study is to determine the prevalence of the HPV subtypes rather than quantifying or analyzing PCR images , 
 Dear authors, The data you published has clinical implications, so it is even more important that the paper is robust. The PCR data have been used for quantification in the paper, based on the following text: **Forty-eight of the 74 samples in our cohort are positive for high-risk HPVs (64.8%) (Table 2); the most commonly present high-risk HPVs are HPV52 (51%) followed by HPV56 (44%), HPV45 (22%), HPV58 (15%), HPV18 (11%), HPV35 (3%) and HPV39 (1%) (Figure 3). HPV types 16, 31, 33 and 51 were not detected in our examined samples (Figure 3). On the other hand, we found that 36 74 of the samples are positive for EBV (49%) (Table 2); of these 36 cases, 33 74 (45%) and 28 74 (38%) are positive for EBNA1 and LMP1 of EBV, respectively.** Please see link for Taylor and Francis policy regarding image manipulation (Science, technology and medicine section): https: authorservices.taylorandfrancis.com editorial-policies images-and-figures Specifically, this paragraph: **Regarding images of electrophoretic gels and immunoblots, where parts of the same gel or blot are spliced together, this should be indicated on the figures with a dividing line, making it clear where the image has been joined. Areas from different gels should not be spliced together. Where loading controls are present, these should always be included in the image; if spliced together, any modifications to the loading control and area of interest must be identical.** 1. Given that you acknowledge splicing in your gels, it might be a good idea to update your paper by publishing a correction to fit the journal s image manipulation policy. This will improve the reader s confidence in the dataset. 2. In addition, a large number of samples show smearing. Based on the % values reported, it looks like a smeared sample was classified as negative. For example, HPV45 positive % was 22% (I calculated 21.6% by counting sharp bands and ignoring smears). I am wondering if the authors quantified DNA in their samples prior to electrophoresis? Smearing could be an issue if sample concentration is too high. By discounting smeared samples, I am wondering if the authors are underestimating the number of positive samples. Again, thank you for clarifying and providing original images. , 
 #7 Thank you for your comments. 1. We will surely do the same. 2. We calculated only samples with bands and smears were classified as negative. The smear is present in samples as they were FFPE samples . Moreover, smears were not within the same base pair size and hence, were calculated as negative. DNA was quantified prior to electrophoresis and all the samples were diluted to a concentration of 25ng ul. The percentage calculated is correct and is the same as reported. Hope all is clarified. Thank you , 
 #8 The authors make a good point about the samples being prepared from cross-linked tissues and I agree that this could contribute to the smearing observed in many analyzed samples (even though a de-crosslinking step is described in the paper). The failure to successfully reverse crosslinking is a technical problem and could make it difficult to classify a sample as positive or negative. Do the GAPDH gels show smearing too? Please can the authors check or provide the originals? The data in the manuscript show clear GAPDH bands but it is hard to evaluate smearing without looking at the full gels. ,