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Human papillomavirus negative cervical cancers and precancerous lesions: prevalence, pathological and molecular features, and clinical implications

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Abstract

Background: Cervical cancer is the fourth most common cancer in women globally. Although human papillomavirus (HPV) testing plays an increasing role in cervical cancer screening and treatment, HPV-negative cervical cancer remains a consistently reported entity globally. While numerous studies have focused on HPV-positive cervical cancers, detailed studies on HPV-negative counterparts remain limited.

Results: This literature review aims to discuss several aspects of HPV-negative cervical cancers including the prevalence of HPV-negative cervical carcinomas and precancerous lesions and their relation to sample types (paraffin-embedded tissue blocks or liquid samples). Additionally, we explore possible reasons for false HPV-negative cases. Furthermore, we review histomorphological, molecular and clinical features of HPV-negative cervical cancers.

Conclusions: The topic of HPV-negative cervical cancers is of importance given the drive towards HPV primary screening, initiation of self-collected HPV testing and widespread use of HPV vaccination.

Introduction

Cervical cancer is the fourth most common cancer in women globally, with 660 000 new cases and 350 000 deaths reported in 2022.1 2 Despite this calamity, there has been a significant decrease in the incidence of cervical cancer and an improvement in survival during recent decades due to improved screening practices and enhanced subsequent management of patients following diagnosis.3 Screening of cervical cancer has evolved steadily since the introduction of cervical cytology (Pap tests) in 1943 attributed to several milestones including (1) the first version of The Bethesda System for reporting cervical cytology developed in 1988 at a National Cancer Institute workshop in Bethesda, Maryland, USA, to standardise reporting Pap cytology results, (2) the introduction of liquid-based cytology (LBC) in the mid-1990s to standardise sample processing and improve the accuracy of Pap test cytology and (3) discovery of the association between human papillomavirus (HPV) and cervical cancer in the 1980s,4 which culminated in the use and improvement of HPV-based screening methods. The USA was the first country to approve and recommend high-risk HPV (hrHPV) testing for use in cervical cancer screening, initially for triage of atypical squamous cells of undetermined significance (ASC-US) detected on Pap test cytology in 2003 (reflex testing), and later for use as an adjunct to cytology in primary screening (cotesting). The Roche Cobas HPV test was approved by the US Food and Drug Administration for use in primary cervical cancer screening in 2014. Currently, guidelines from the US Preventive Service Task Force (USPSTF),5 the American Cancer Society (ACS),6 the American College of Obstetricians and Gynecologists (ACOG),7 8 and the US Centers for Disease Control and Prevention (CDC)9 all incorporate HPV testing, either Pap cytology/HPV cotesting or HPV primary screening, but with variations related to patient age and frequency of screening. No cervical cancer screening testing is recommended by any agency for individuals less than 21 years of age. For individuals aged 21–29 years, screening with Pap test cytology every 3 years is recommended by the USPSTF, ACOG and CDC, with HPV reflex testing for ASC-US in women >24 years. The ACS guidelines recommend HPV and/or Pap test testing to begin at age 25 years instead of 21 years. For individuals aged 30–65 years, HPV testing alone every 5 years is an accepted alternative to HPV and Pap cytology cotesting every 5 years or cytology alone every 3 years depending on resource availability. More recently, HPV genotyping has been used for defining risk thresholds and follow-up management, with positivity for HPV 16 or 18 warranting colposcopy with biopsy regardless of Pap cytology results.3

Given that HPV testing has played an increasingly important role in cervical cancer diagnosis and management, current recommendations and practices have reflected this. There has been a movement towards reduced Pap test cytology, and only screening with either HPV testing alone or cotesting. In fact, cotesting in several countries currently appears to become the most common screening method at large institutions.10 Furthermore, most studies pertaining to this topic have focused on HPV-positive cervical cancers. Additionally, new recommendations have been proposed that would accept self-collected vaginal HPV testing for cervical cancer screening.11 Given these trends, some arguments have been made that HPV-negative cervical malignancies will become negligible or discounted with a shift primarily towards HPV primary screening and more widespread HPV vaccination.12

Despite the trend principally towards HPV-based diagnostics, HPV-negative or HPV-independent cervical cancer cases continue to be reported consistently both within the USA and internationally.1 In these cases, cytology-based Pap tests with cytomorphological microscopic assessment remain potentially very useful for cervical cancer screening.13 Continued research and consideration of HPV-negative cervical cancer remain important as these HPV-negative cervical cancers demonstrate differences in patient population, presentation and clinical outcomes when compared with HPV-positive cervical cancer counterparts.1 This review highlights and discusses the distinguishing characteristics of HPV-negative cervical cancer and provides reasons to continue screening for and undertaking research regarding these distinct malignancies.

Prevalence of HPV-negative cervical carcinoma across different testing mechanisms

Formalin-fixed paraffin-embedded tissue assessment

The rates of HPV-negative cervical cancers vary depending on the sample collected and histological tumour types. In the USA, the most common type of cervical cancer is squamous cell carcinoma (SCC) (70%–85%), while the next most common type is adenocarcinoma (ADC) which comprises 15%–30% of total cases, followed by adenosquamous and other more rare subtypes comprising the remaining cases.14 A study conducted by the US CDC, in partnership with seven cancer registries in the USA, examining the presence of HPV DNA across different tissues in 2670 patients showed that most (90.6%) but not all invasive cervical cancers were HPV positive.15 A retrospective cross-sectional worldwide study including 38 countries using formalin-fixed paraffin-embedded (FFPE) blocks of invasive cervical cancer and PCR, immunoassay, and genotyping with a reverse hybridisation line showed that the HPV positive rate depended heavily on the types of cancer present. In cervical SCC cases, 87% were positive for HPV while only 62% of ADC cases were HPV positive.16 Similar rates of HPV-negative cervical cancer were identified in another large-scale study comparing the rate of HPV positivity in tissue blocks via genotyping across invasive cancers from multiple sites.17 In a similar study based in China, involving FFPE tissue blocks of cervical carcinoma cases, it was demonstrated that 2.4% of SCC and 25.5% of ADC cases were HPV negative.18 Additionally, in both SCC and ADC cases, there was a range of HPV viral loads, with highest viral loads being most common in SCC. Interestingly, 1.1% of SCC and 17% of ADC showed the lowest level of viral load (<1).18 Other studies based in China show a higher rate of HPV-negative cervical cancer, at 9.7%19, 9.8%20 and 10.4%.21 In other countries, a larger range of rates of HPV negativity in cases with SCC morphology have been reported: 5.6% in a study from Spain,22 11.4% in Turkey23 and 22.7% in a recent study from a Swedish cohort of 168 patients.24

Liquid-based sample assessment

LBC samples such as cervical scrapes or brushings can also be used for HPV DNA testing. Several studies of HC2 HPV testing on LBC samples collected either from the surgical specimen immediately prior to fixation or directly from the patient identified similar rates of HPV negativity in cases of invasive cervical carcinoma. In a study of 475 cases in China, HC2 was negative for HPV in 10.1% of patients and in Korea, Brazil and Slovenia, similar studies demonstrated rates of HC2 negativity for HPV at 6.6%, 14.14% and 12.6%, respectively.25–28

Cervical Pap tests prior to cervical cancer diagnosis

Another perspective on the prevalence of HPV-negative cervical cancers can be appreciated by retrospectively examining the history of HPV testing in patients diagnosed with cervical cancer. In a study of 70 patients with cervical carcinoma, the rate of HPV negative tests at <1 year, 1–3 years and 2–5 years was 9%, 23% and 25%.29 For comparison, Pap tests at the same intervals showed 3.4%, 33% and 44% negative tests. Within the 1-year period before diagnosis, the overall hrHPV-negative rate was 15.5% (74 of 477 patients) and the Pap-negative rate was also 15.5% (37 of 238 patients). Only 9 of 231 patients with both hrHPV testing and Pap testing (3.9%) had a double-negative result.29 30 In a study of 287 cases of cervical SCC conducted at Magee Women’s Hospital (MWH) of University of Pittsburgh Medical Center (UPMC) in the USA, 10% of HPV tests less than 1 year prior to a diagnosis of cervical cancer were negative.28 Another study of 256 648 cases across multiple clinical practices demonstrated that 11% of SCC and 37% of ADC patients had a negative HC2 HPV test less than 1 year prior to their histological diagnosis.31

In the USA, three large-scale studies performed using data from MWH of UPMC, Quest Diagnostics and Kaiser Permanente Northern California examining the rate of HPV-negative tests less than 12 months and more than 12 months prior to the diagnosis of SCC or ADC showed HPV screening results that were more likely to be negative greater than 12 months prior to a diagnosis of cervical cancer32–34 (table 1). The highest rate of HPV-negative screening results was 45% reported in a study by Quest Diagnostics.33 Two large-scale studies performed at the Obstetrics and Gynecology Hospital of Fudan University and West China Second University Hospital, examining the rate of HPV-negative screening tests less than 6 months prior to diagnosis of cervical cancer, showed that cervical ADC demonstrated the highest HPV-negative rate (16.7%–29.7%), while cervical SCCs showed an HPV-negative rate of 6.3%–6.4%35 36 (table 2). When the time interval prior to diagnosis was extended, the HPV-negative rate of cervical cancers was even higher. For example, HPV-negative rates in women who developed incidental cervical cancer 2.5–8 years after the start of this study trial were 42%.37 These results suggest that more consideration should be given when determining the recommended time interval between HPV screening, owing to the aforementioned complexity of hrHPV negative testing results.

Table 1

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Comparison of the prevalence of HPV-negative tests less than 12 months or greater than 12 months prior to diagnosis of cancer or precancer

Table 2

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Comparison of the prevalence for HPV-negative tests 6 months or less prior to cervical histological diagnosis

Prevalence of HPV test negative precancerous lesions of the cervix

Negative hrHPV screening tests prior to the diagnosis of precancerous cervical lesions, including cervical intraepithelial neoplasia 2/3 (CIN2/3) and cervical adenocarcinoma in situ (AIS), have been consistently reported. In the large cohort studies that resulted from MWH, KPNC and Quest diagnostics, the HPV-negative rate of screening tests less than 12 months prior to a diagnosis of CIN3/AIS ranged from 2.4% to 4.1%, while the HPV-negative rate ranged from 19.9% to 29.4% in screening studies greater than 12 months prior to the diagnosis of CIN3/AIS32–34 (table 1). In two large-scale studies from China, the HPV-negative rate in screening tests less than 6 months from a diagnosis of CIN3 and AIS was similar, but slightly higher ranging between 4.5% and 12.3%35 36 (table 2). In another study based in China, with a total of 5699 patients with histologically diagnosed CIN2/3, 8.3% had a previous negative HPV HC2 test within 6 months, and on average within 1.4 months.38 Similarly, in another study of 1387 CIN2/3 cases, 8.4% of patients had negative hrHPV tests on average 0.4 months prior to diagnosis.39 Another report of 2274 cases of CIN 2/3 demonstrated 9.9% hrHPV-negative screening test 6 months or less prior to diagnosis.21 Another study that examined 2827 cases with a diagnosis of CIN 2/3 on biopsy found that 3% of HPV tests performed immediately prior to biopsy and 17% of tests performed within 1–3 years prior to biopsy were negative.40

The prevalence of HPV-negative precancerous lesions has varied across studies. In a study from Japan, of 256 women found to have LSIL on cytology but had hrHPV-negative screening results who then underwent colposcopy, 3.9% of those were found to have CIN3 on the final histological diagnosis and 9.4% were found to have CIN2.41 A direct study of the HPV types in histologically verified cervical dysplasia showed 22.7% of HSIL and 43.8% of LSIL cases were HPV-negative.24 In a multi-institution study from Italy of 722 cases with FFPE blocks, 4.7% of CIN3 cases were found to be HPV-negative while 13.2% of CIN2 cases were HPV-negative.42 Overall, the ability to detect precursor lesions of the cervix allows for the development of appropriate screening guidelines, and consideration of HPV-negative precursor lesions remains an important topic.

True and false HPV-negative cervical cancers and precancerous lesions

HPV-negative cervical carcinoma cases may be due to both true HPV negativity and false-negative test results. True HPV-independent cervical SCC are recognised by the WHO fifth edition as a separate entity with an estimated prevalence rate of 5%–7%.2 16 22 43 Several studies have investigated the true negativity rate.43–45 PCR-based techniques from cancer tissue blocks used to reanalyse HPV-negative cases by HC2 testing from previous cervical LBC samples demonstrate that a significant portion of HPV-negative cases remain negative on reanalysis; roughly around 50% of SCC cases.43 45 These studies demonstrate that while a proportion of HPV-negative SCC cases of the cervix can be attributed to false negatives in HPV testing, a significant number are likely truly negative. However, as there has been a range of reported prevalences for HPV-negative precursor and invasive cervical lesions, false negative results and their causes warrant further consideration.

False negativity may also be due to testing mechanism differences. A study of five cases with a negative HPV-ISH (in situ hybridisation) surgical specimen showed that only 2/5 of these cases were negative for hrHPV on the biopsy specimen.46 In comparison, a study of 670 cases from multiple countries using a microarray and ISH for both high-risk and low-risk HPV found that 2% of cervical SCC cases were HPV independent.11 In studies employing Aptima hrHPV mRNA testing, 538 of 701 hrHPV mRNA-positive cases (76.8%) and 15 of 36 hrHPV mRNA negative cases (41.7%) were diagnosed with CIN2+/HSIL on histological follow-up.47

In cytology specimens, no significant difference in HPV-positivity rate was found between HC2 (88.1%), Cervista (90.7%) and Cobas (93.9%) testing methods in a study with 2092 HSIL cytology cases when factoring in age stratification.48

False negative results may also be ascribed to the deletion of target HPV DNA fragments, low viral load, HPV types not detected by commonly used testing platforms or inadequate sampling.49 Moreover, HPV detection may present challenges in populations where the HPV genotype prevalence differs. For example, in the Chinese population, CIN2/3 most commonly showed HPV16, HPV52 and HPV58 infection while SCC most commonly showed HPV16, HPV58, HPV52, HPV18 and HPV33 infection.35 There is some belief that HPV false negativity can depend on the sensitivity of testing for specific genotypes. Lower HPV-negative rates were found in studies in an immunocompromised HIV-positive population, although sensitivity among women with and without HIV was comparable.50 Additionally, coinfection with multiple hrHPV infections is common, accounting for 24.7% of cases in a study of 21 070 cases of patients with cervical squamous lesions. Interestingly, certain combinations of multiple hrHPV infections demonstrated a decrease in the detection rates of CIN3+lesions.51 Further, multiple HPV infections were most commonly noted in cases with histopathological diagnoses of CIN1 and negative cases.21 These data pose interesting questions of whether other viral comorbidities and intergenotypic competition of different HPV types may affect viral load, which would in turn have an effect on the sensitivity of HPV testing. These studies suggest the need for continued research and improvement of HPV testing to most accurately predict the false negative rate of HPV tests in cervical carcinomas.

Clinical characteristics and prognosis of HPV-negative cervical cancer

Multiple studies have demonstrated that compared with HPV-positive cervical cancers, patients with HPV-negative cervical cancers are significantly older (median age 49 vs 72 years) and diagnosed at a higher stage.11 18 21 23 24 41 52 Interestingly, the age of diagnosis for patients with HPV-negative cervical ADC was similar to those with HPV-negative SCC.18 In general, HPV-negative cervical SCC has the same clinical presentation as its HPV-associated counterpart, but haemorrhage and abdominal pain may be more frequent at diagnosis, in line with presentation at a later stage.22 43 Several studies have demonstrated worse clinical outcomes for patients with HPV-negative cervical cancers.1 11 23 43 53–56 One study demonstrated patients with HPV-negative cervical cancer had 59.8-month disease-free survival (DFS) compared with 132.2-month DFS in HPV-positive tumours, and overall survival (OS) of 77.0 months compared with 153.8 months OS for HPV-positive neoplasms. However, this was due to advanced FIGO stage and lymph node metastasis on multivariate analysis.22 Such studies suggest a continued need to research the clinical impact of HPV-negative cervical cancers, particularly in larger cohorts over longer periods of time.

Histological and molecular features of HPV-negative cervical cancers and precancerous lesions

The pathogenesis of HPV-negative carcinomas is still not well understood. Studies examining the histological patterns of HPV-negative cervical SCC demonstrate varied morphologies including keratinising, non-keratinising and warty subtypes.11 In a study by Nicolás et al, 1 of 12 cases of HPV-negative SCC was keratinising, one was sarcomatoid and the others were all non-keratinising.22 Other studies have also demonstrated an increased proportion of non-keratinising SCC in HPV-independent carcinomas.43

The p53 status of HPV-negative carcinomas also varies greatly between studies. A study by Stolnicu et al demonstrated that 2/10 HPV-negative SCC cases showed null p53 expression, while the others were wild type.11 In the study by Nicolás et al, 9/12 cases showed abnormal p53 expression.22 Other studies demonstrated intermediate frequencies of p53 mutation at 35.7%23 and 4/743 of examined HPV-negative SCC samples. Similarly, the p16 immunostain showed varied rates of positivity in HPV-negative SCC cases, ranging between 7/1222 and 5/743 cases.

Molecular studies performed on HPV-negative carcinomas, while limited, have demonstrated a pathogenic germline polymorphism Q472H in the KDR gene (7/9 patients) and chromosome 3q gains (4/9 patients).57

HPV-negative cervical ADC represents a significant proportion of cervical ADC. These malignancies generally present at more advanced stages. These cancers also have a higher rate of HPV negativity than in SCC19–22 30; where 15%–40% of cervical ADC cases are HPV-negative.18 21 58 59 Cervical ADC can be subdivided into several morphological subtypes including usual, villoglandular, mucinous, serous, gastric, clear cell, mesonephric and endometrioid types. HPV infection varies significantly with these subtypes. Gastric type is the most common HPV-independent carcinoma (with an almost 100% HPV-negative rate), followed by endometrioid type (81.1%). The usual type has the lowest rate of HPV negativity (9.6%). Clear cell, serous and not otherwise specified types have rates of HPV negativity that range between gastric and usual types.60 Five cases of HPV testing negative for cervical carcinoma were demonstrated in figures 1–5. Immunohistochemical studies in HPV-negative ADC demonstrated mixed p53 and p16 staining patterns, similar to those of HPV-negative SCC.22 43 Molecular studies have revealed mutations in HPV-negative ADC including TP53, STK11, KRAS, ARID1A, BRCA2, CDKN2A in the gastric type, KRAS, NRSA, ARID1A/B, SMARCA4, BCOR/BCORL1, FGFR2 in the mesonephric type, and PIK3CA, PTEN, CTNNB1, FBXW7, KRAS, AKT1, MSI-H in the endometrioid type.58 59

Figure 1

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Case of a 60-year-old woman with a false negative Aptima HR-HPV test. (A) HPV and Pap cotesting showed a cluster of squamous cells with marked atypia but HPV negative by Aptima HPV assay. (B) RNAscope in situ hybridisation (ISH) was positive in the cell block. (C) Biopsy specimen with squamous cell carcinoma. (D) RNAscope ISH is positive for high-risk HPV in the biopsy specimen. *The Aptima HPV test detects E6/E7 messenger RNA (mRNA) of 14 high-risk HPV types, including HPV-16, HPV-18, HPV-31 to HPV-33, HPV-35 to HPV-39, HPV-45 to HPV-51, HPV-52 to HPV-56, HPV-58 to HPV-59, HPV-66 and HPV-68, from Pap liquid specimens. RNAscope ISH detects E6/E7 mRNA of 18 high-risk HPV types, including 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82 in FFPE tissue specimens. The negative Aptima HPV test and positive ISH assay indicated this cervical cancer was positive for one of the four HPV types, HPV26, 53, 73, 83, which was not covered by the Aptima HPV test. This case demonstrated a false negative HPV test for the Aptima HPV assay. FFPE, formalin-fixed paraffin-embedded; HR-HPV, high-risk human papillomavirus.

Figure 2

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Case of a 62-year-old woman with Pap and HPV cotesting showing HSIL and HPV negative. (A) H&E of the biopsy showing poorly differentiated squamous cell carcinoma. (B) P63 staining was positive in tumour cells. (C) P16 was negative in tumour cells. (D) HPV RNAscope in situ hybridisation was negative for high-risk HPV in tumour cells. HPV, human papillomavirus.

Figure 3

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Case of a 35-year-old woman with gastric-type endocervical adenocarcinoma. Pap and HPV cotesting screen were both negative. The patient had continued increased vaginal secretion, and a cervical mass was found by imaging. Cervical biopsy and LEEP/Cone biopsy were negative. Finally, hysterectomy was performed. (A) Low power H&E showing infiltration of mucinous glands into the cervical stroma deeply. (B) Higher power H&E showing the lack of marked cytological atypia in the mucinous glands. (C) Ki67 proliferative index is mildly to moderately increased. (D) RNAscope in situ hybridisation is negative for high-risk HPV. HPV, human papillomavirus.

Figure 4

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Case of a 55-year-old woman with clear cell carcinoma of the cervix. (A) Screening Pap and HPV cotesting showed marked atypical glandular cells and was HPV negative. (B) H&E of the biopsy showing clear cell carcinoma morphology with hobnail cells, eosinophilic cytoplasm and high nuclear grade. (C) HNF1β immunohistochemical stain was positive in tumour cells. (D) RNAscope in situ hybridisation is negative for high-risk HPV in the biopsy specimen. HPV, human papillomavirus.

Figure 5

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Case of a 58-year-old woman with mesonephric adenocarcinoma. Pap and HPV cotesting showed atypical glandular cells and was HPV negative. (A) H&E of the biopsy shows the tubular pattern of mesonephric adenocarcinoma with focal benign endocervical glands (left down). (B) Immunohistochemical stain for Müllerian marker PAX8 was positive. (C) Immunohistochemical stain for TTF-1 was positive. (D) RNAscope in situ hybridisation is negative for high-risk HPV. HPV, human papillomavirus.

More rare types of HPV-negative cervical cancer include mesonephric ADC. One case series demonstrated HPV negativity in all eight such cases, but five cases showed positive p16 staining.61

HPV-negative precursor lesions also show large variation in features. In some studies, precursor lesions showed extensive basaloid morphology with eosinophilia or a dense inflammatory infiltrate.11 57 Immunostaining with p16 showed block positive staining in one of six cases, and heterogeneous staining in one case while the others were negative. The adjacent invasive components were keratinising and p16-negative.57 HPV-negative AIS lesions demonstrated decreased apoptotic bodies and mitotic activity compared with HPV-positive cases. These HPV-negative AIS lesions also showed positive immunohistochemical staining with CK7 and MUC6, but a varied rate of positive staining with CK20, CDX2, PAX 8 and CEA (2/6 cases).62 Oestrogen receptor and progesterone receptor stains were usually negative. A p53 mutant pattern of staining was found in two of nine cases.62

Conclusions

In this article, we have reviewed the prevalence of HPV-negative cervical cancers and precursor lesions with consideration of both true HPV-negative cases and false negative testing. Differences regarding clinical characteristics and histomorphology were also highlighted between HPV-negative and positive lesions. Increased cervical cancer screening and HPV vaccination have led to a continual increase in the overall ratio of HPV-negative to HPV-positive cases. Concurrently, consideration of HPV-negative lesions remains important when managing individual patients, as well as in assessing and updating cervical cancer screening guidelines. While some published estimates deem the 5-year risk of CIN3+/HSIL to be less than 0.15% if HPV status is negative and up to 0.54% in HPV-negative ASC-US63 or 0.034%, 0.041% and 0.016% for one, two or three consecutive Pap and HPV cotests, respectively,64 other studies demonstrate a decreased ability for a negative HPV test to predict cervical carcinoma in the 1–5 years interval. Overall, continued research about HPV-negative cervical cancer prevalence is imperative, especially since new cervical cancer screening recommendations and techniques such as self-collection for HPV testing get launched. Furthermore, research into the pathogenesis underlying HPV-negative cervical carcinomas and how HPV may interact differently with the cervical epithelium compared with the squamous epithelium in other tissue types may reveal novel mechanisms that help explain discrepancy rates in the HPV negativity rate across SCC of different organs. Additional discussion around limitations in this specific field is presented by Lee et al.1 These limitations include only few biomarkers and classification of HPV-negative carcinomas and the need for a robust model system for understanding cervical cancer. Overall, HPV-negative cervical cancers remain an important entity that will benefit from further investigation.

Contributors: WD: conceptualisation, investigation and writing–original draft. XZ: conceptualisation, investigation and writing–review and editing. LP: conceptualisation, investigation and writing–review and editing. CZ: conceptualisation, investigation, writing–review and editing, and supervision. CZ is the guarantor.
Funding: The authors declared no any finding support for this review article.
Competing interests: CZ has served as the editorial member of GOCM. There are no competing interests for other authors.
Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review: Not commissioned; externally peer reviewed.

Ethics statements

Patient consent for publication:

Acknowledgements

The authors would like to thank Dr. Marshall Austin (University of Pittsburgh Medical Center) for his careful reading of the manuscript and insightful suggestions.

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