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Comments on: Limitations of HPV DNA Testing in Screening of Cervical Adenocarcinomas
10 June 2019 (online)
We read with interest the article on human papillomavirus (HPV) DNA testing for cervical cancer in Brazil, authored by Zeferino et al (Brazilian Association for the Lower Genital Tract Pathology and Colposcopy (ABPTGIC, in the Portuguese acronym), as well as the subsequent letter to the editor, by Dr de Alcantara Segura, and the authors' reply.  We agree that detection of adenocarcinomas represents a significant challenge for cervical cancer screening programs. In the United States, squamous cancer rates have seen a decline since the introduction of the Pap smear and molecular testing, but adenocarcinoma rates have remained unchanged ([Fig. 1]). Moreover, Australia, which arguably has a best in class vaccination and screening program, is reporting a steady increase in adenocarcinoma rates. As Dr. de Alcantara Segura points out, cytology is an insensitive method for screening adenocarcinomas. This has also been shown to be the case in the US, where Katki et al reported that 63% of adenocarcinomas showed normal cytology results. In addition, screening for adenocarcinoma precursors by HPV is clearly less sensitive than squamous cancer precursors, as evidenced by the lack of progress in reducing the adenocarcinoma incidence since the introduction of molecular testing in the 2000s ([Fig. 1]). A review of the recently published literature has identified a number of potential causes for the apparent lack of HPV detection within cervical adenocarcinoma, including the following: (i) Historical use of the Hybrid Capture 2 (HC2) assay and an associated higher rate of HPV negative cervical adenocarcinoma cases reported ; (ii) low viral load of HPV and detection only with nested polymerase chain reaction (PCR) methods; (iii) L1 vs E6/E7 detection (HPV integration and L1 deletion) ; (iv) non-HR HPV genotypes associated with some adenocarcinomas; and (v) rare histological subtypes that either have low viral loads or are HPV-negative.  There are some cervical adenocarcinoma tissues that appear to be non-HPV derived. Clear cell adenocarcinoma can arise from prenatal diethylstilbestrol exposure that are not believed to be associated with high-risk HPV infections. Likewise, gastric-type adenocarcinomas are considered to be either HPV-negative, or at least, have very low levels of HPV present.
In particular, target region selection has a significant impact on an HPV DNA test's ability to detect cancers. The majority of assays target the L1 region of the virus, and the gene is prone to deletion during virus integration as disease progresses.   Viral integration can also occur early in the disease process and is detected in routine liquid-based cytology specimens of precancer patients.  While it has long been reported that E6/E7 target regions are more sensitive than L1, some have argued that this is due to technical difficulties with isolating DNA from fixed tissue (formalin-fixed, paraffin embedded [FFPE]) samples. The use of L1-target region assays, and the older detection methods in the cross-sectional study by de Sanjose et al may have contributed to the quoted poor detection rate (62%) of adenocarcinomas. More recent studies using more robust methods and shorter target sequences estimate the rate of HPV-negative cancers at approximately 10% using L1-based PCR assays.  While an improvement, it still represents an unacceptably high false-negative rate. Assays that target the E6/E7 genes rarely undergo deletion because of the requirement for E6/E7 gene products in oncogenesis. Thus, assays that target this region of the virus should be more sensitive in detecting cancers. Recent publications confirm that this is in fact the case. Molijn et al reported a 7% increase in adenocarcinoma detection using type-specific E6 PCR versus a very sensitive L1-based SPF10-PCR assay. Lagheden et al also reported the results of a comprehensive analysis of HPV type detection using tissue blocks from the Swedish cancer registry collected from 2002 to 2011. The authors were successful in obtaining 69% of all cell blocks (from 22 different biobanks), and samples were extracted using standardized procedures with hematoxylin & eosin (H&E) confirmatory staining of the first and last sections of each block for re-review. A total of 37 different HPV types were detected using L1 Modified General Primers (MGP) and a Luminex bead readout. Of the 2,932 retrieved blocks, 2,850 (97%) gave valid PCR results (beta globin internal cellularity control detected). A total of 527 (18%) samples were found to be HPV-negative but approximately 8% of these were negative for invasive cancer on reviewing the histology, and were thus excluded from the analysis. The DNAs from the 483 remaining L1-negative cases were re-tested using E6/E7 type specific primers, and 89 additional cancer cases were detected. This corresponds to an 18% (89/483) improvement or a 3.2% (89/2806) gain in overall HPV-negative cancer detection using just the HPV16/18 target regions (65% of the cancer cases in the study cohort). If one assumes an equal detection rate for all 14 high-risk types, this would result in an approximately 4.9% (136/2806) improvement in overall cancer detection. Thus, based on this large cancer cohort (representing approximately 1.57 million screened women), an estimated 28% (136/483) increase in HPV-negative cancer detection might be obtained using E6/E7 versus L1 primer sets.
While it is not possible to detect all cancer using either cytology and or HPV testing, there is little doubt that HPV testing is the most sensitive tool. In addition to the Ronco et al study cited by Zeferino and colleagues, Schiffman et al performed a comprehensive analysis in a cohort of over 1.2 million women with screening histories preceding cancers (n = 623) and precancers (n = 5369). They found that HPV testing identified more women subsequently diagnosed with cancer (P < 0.001) and precancer (P < 0.001), except for within 12 months when cytology showed improved cancer detection (P = 0.10). However, this represented, at most, 5 cases per million women per year, and the authors questioned the utility of continued co-testing versus primary HPV screening – “Excessive screening in an attempt to prevent every case could have minimal cancer prevention benefits while increasing the harms of screening”.
The decision on which screening paradigm to adopt is often driven by both the science as well as regional or country-specific parameters, including infrastructure, societal risk tolerance, and funding/reimbursement. However, it is important that clinicians and decision makers carefully weigh the safety and effectiveness of different approaches so as to maximize the impact of any screening program. Adenocarcinomas continue to represent a significant screening challenge, but it is hoped that with advancements in screening tools and the impact of vaccination, this performance gap will soon begin to close.
Finally, the issue with HPV- negative cervical adenocarcinoma poses both a challenge for cervical cancer screening programs and an important scientific and clinical question about the etiology of cervical glandular lesions. In our opinion, this situation necessitates a “call to arms” within the scientific and clinical community, in which a systematic investigation of cervical adenocarcinoma cases using deep nucleic acid sequencing analysis is warranted to answer the question about HPV association with all cervical adenocarcinomas. It is likely that these studies will lead to next generation cervical cancer screening technologies and improved adenocarcinoma detection.
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