HPV-negative Tumors in a Swedish Cohort of Cervical Cancer

Abstract

Despite the common perception that the human papilloma virus (HPV) is a requirement for the development of cervical cancer (CC), a considerable number of CCs test HPV negative. Presently, many countries are shifting to HPV primary CC screening, and it is of importance to increase the knowledge about the group of CCs that test HPV negative. The aim of this study was to reinvestigate a proportion of cervical tumors with a primary negative or invalid test result. Reinvestigation with repeated genotyping (targeting L1) was followed by analysis with an alternative target method (targeting E6/E7) on existing or additional tumor material. Consistently negative tumors were histologically evaluated, and cases with low or lacking tumor cell content, consistent invalid test results, or with suspicion of other than cervical origin were excluded. HPV-negative cases were thereafter subjected to immunohistochemistry (Cytokeratin 5, pan cytokeratin, protein 63, P16, and P53). The HPV-negative proportion could after reinvestigation be reduced by one-half (14%-7%). Additional positive samples were often detected in late polymerase chain reaction cycles, with an alternative (E6/E7) or the same (L1) target, or with a method using shorter amplicon lengths. Confirmed HPV negativity was significantly associated with worse prognosis, high patient age, longer storage time, and adenocarcinoma histology. Some of the HPV-negative cases showed strong/diffuse p16 immunoreactivity, indicating some remaining false-negative cases. False HPV negativity in this cohort was mainly linked to methodological limitations in the analysis of stored CC material. The small proportion of presumably true HPV-negative adenocarcinomas is not a reason for hesitation in revision to CC screening with primary HPV testing.

FIG. 1

Flowchart of the reinvestigation chain. Samples previously tested negative or invalid (AnyplexTM II HPV28, targeting L1) were reanalyzed with the same method in step 1 of the reinvestigation chain. In step 2, an alternative target method (in-house real-time PCR, targeting E6/E7) was used. Persistently human papilloma virus (HPV)-negative samples with alternative tumor tissue block were in step 3 analyzed with the same algorithm (L1*2+E6/E7) as above. The remaining HPV-negative cases were evaluated by a pathologist in step 4. Consistent invalid test result, lack of tumor in sample, and suspicion of other than cervical origin led to exclusion.

FIG. 2

The reinvestigation process of 37 tumors previously tested human papilloma virus (HPV) negative or invalid. (A) All samples were retested with the same method (step 1), and consistently negative samples were tested with an alternative target method (step 2). Cases with alternative tumor tissue available were analyzed with both methods, L1*2+E6/E7 (step 3) and repeated negative results led to evaluation by a pathologist (step 4). Reasons for exclusion were consistent invalid test result, lack of tumor material, or suspicion of other than cervical origin. (B) Sixteen additional samples tested positive in steps 1 to 3 of the reinvestigation chain, and, after step 4, 14 samples still had a HPV-negative test result. An overview of sample storage time (diagnosis decade), histology of the tumor, amplification cycle for positive result in reinvestigation, result previous to reinvestigation, and results from additional HPV analysis using MGP is shown in the color chart.