Reply to ‘comment on ‘circulating neutrophils in patients with hepatocellular carcinoma”

We thank Liu and Meng for their interest1 in our paper entitled ‘Neutrophils: driving progression and poor prognosis in patients with hepatocellular carcinoma?’.2 We would like to respond to

their comments. The authors point out that there were significant differences between the two cohorts that we have studied and suggest that our analytical approach was not consistent with

‘statistical rules’ and that these may have resulted in ‘incorrect conclusions’. The authors go on to suggest that unmatched conditions should have been eliminated in our combined cohort

analyses and reference two methods, namely Propensity Score Matching or Decision Tree and Random Forest. We have considered these suggestions carefully and on these statistical matters we

have to respectfully disagree. We entirely accept that there were differences between our UK and Hong Kong cohorts. Having demonstrated the key associations with neutrophils in our own large

UK cohort with hepatocellular carcinoma, we deliberately chose a validation cohort where other factors that could impact peripheral blood counts—such as race, aetiology, age, testing

laboratory—were different. While the combination analyses enhanced statistical significance, all of the reported associations were present in each independent cohort despite the differences

between them—which we see as a major strength of our study rather than a limitation. In the larger combined group, in which ‘cohort’ was included as a variable, a key added value was that it

provided sufficient numbers of cases to perform sub-analyses where numbers were smaller in individual cohorts. For example, the presence of cirrhosis may have been a confounder as it can

also affect cell number. By combining UK and Hong Kong cohorts, we were able to study associations in just over 400 patients and confirm that the reported cancer associations persisted in

the absence of cirrhosis. We could reiterate in more depth additional details of our study, but feel it is sufficient in addition to our explanation above, to state that we believe that our

approaches to the statistical analyses were biologically relevant and robust. Furthermore, suggesting some sort of matching would have improved the integrity of our study is simply not true.

As described in the papers referenced by Liu and Meng,1 matching methods are typically used when comparing a treatment arm of a clinical trial with a non-treatment arm—with the need to

ensure that reported outcomes are independent of differences—such as race, age, sex, severity of underlying disease—that may impact outcome. It is not appropriate to try and apply this kind

of matching to an observational study of a large heterogenous population in which many variables may influence outcome and the opportunity to assess their independent contributions is

related to the size of the cohort—and diminished by elimination of cases. In our combined analysis of factors influencing survival, the UK versus Hong Kong cohort was included as a variable

in the multivariate analyses and it was not significantly different—with a hazard ratio of 0.93 (0.82–1.05)—which indicates that cohort differences had little relevance. This was in contrast

to a number of other factors which had demonstrable importance. At a more practical level, any kind of matching has to be done in an inclusive and comprehensive fashion addressing all

potential confounders—not just one or two. Matching on race or aetiology alone would diminish our combined cohort to <100 patients, without even considering sex, age, cirrhosis, treatment

etc. The overall concern of Liu and Meng1 stems from an assumption that we propose neutrophils alone are central to disease progression and outcome for patients with HCC. Actually, this is

not our belief. Roles for lymphocytes in determining disease progression in the immunosuppressed tumour microenvironment (TME) are undoubtedly of paramount importance. In the Hong Kong

cohort rather than the UK cohort, peripheral lymphocyte was associated significantly with survival. Roles for platelets are also increasingly realized. It is the role of neutrophils that has

received far less attention and our study—in which circulating neutrophils were the only immune cell type independently associated with poorer prognosis in both of the cohorts despite their

differences—provides a compelling argument for turning our attention to them. In our study, we also demonstrated significant correlations between neutrophils and lymphocytes (negative) and

neutrophils and platelets (positive), and proposed that studying their interactions in more depth, as well as how levels in the circulation reflect the TME, would be very worthwhile. We

agree—as suggested by Liu and Meng—that studying relationships between cell counts in a longitudinal fashion may aid our understanding of their roles in disease progression and responses to

treatment. Not having done this as yet does not, however, invalidate the findings reported in our study of data generated from cell counts at the time of diagnosis. In short, we firmly stand

by our conclusions and discussion points. REFERENCES * Lui Y. and Meng X. Comment on ‘Neutrophils: driving progression and poor prognosis in hepatocellular carcinoma’. _Br. J. Cancer_ 119,

(2018) * Margetts, J., Ogle, L. F., Chan, S. L., Chan, A. W. H., Chan, K. C. A. & Jamieson, D. et al. Neutrophils: driving progression and poor prognosis in hepatocellular carcinoma?

_Br. J. Cancer_ 118, 248–257 (2017). Article  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * State Key Laboratory in Oncology of South China, Department of

Clinical Oncology, Chinese University of Hong Kong, Hong Kong, Hong Kong Stephen L. Chan * Institute of Digestive Disease, Chinese University of Hong Kong, Hong Kong, Hong Kong Stephen L.

Chan * Institute of Health & Society, Newcastle University, Sir James Spence Institute, Royal Victoria Infirmary, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP, UK Richard J. Q.

McNally * The Hepatopancreatobiliary Unit, Freeman Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, Freeman Road, Newcastle upon Tyne, NE7 7DN, UK Helen L. Reeves * Northern

Institute for Cancer Research, The Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK Helen L. Reeves Authors * Stephen L. Chan View author

publications You can also search for this author inPubMed Google Scholar * Richard J. Q. McNally View author publications You can also search for this author inPubMed Google Scholar * Helen

L. Reeves View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Helen L. Reeves. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no competing interests. FUNDING Cancer Research UK (CRUK)—C18342/A23390 [Reeves] Hong Kong Research Grants Council General Research Fund (number 462103) and Terry Rox

Run Hong Kong [Chan]. NOTE This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to

a Creative Commons Attribution-NonCommercial-Share Alike 4.0 Unported License. RIGHTS AND PERMISSIONS This article is distributed under the terms of the Creative Commons Attribution 4.0

International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the

original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chan,

S.L., McNally, R.J.Q. & Reeves, H.L. Reply to ‘Comment on ‘Circulating Neutrophils in patients with hepatocellular carcinoma”. _Br J Cancer_ 119, 781–782 (2018).

https://doi.org/10.1038/s41416-018-0250-3 Download citation * Received: 16 July 2018 * Revised: 01 August 2018 * Accepted: 03 August 2018 * Published: 12 September 2018 * Issue Date: 11

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