Diagnostic pitfalls leading to delayed diagnosis of human papillomavirus (HPV)–independent cervical malignancies in the era of HPV-based screening: clinicopathologic analysis of diagnostic pathways
Article information
Abstract
Background
Human papillomavirus (HPV)–independent cervical malignancies (HPV-IDCM) are a rare but clinically important subset that may escape detection by HPV-based screening. This study investigated diagnostic pitfalls contributing to delayed HPV-IDCM diagnosis and assessed the role of cervical cytology in the diagnostic pathway.
Methods
This multicenter retrospective case series was conducted as a policy research project of the Korean Society for Cytopathology. Patients with HPV-IDCM and repeated abnormal cytologic findings before definitive histologic diagnosis were identified. Clinical, cytologic, histologic, and HPV test results were reviewed to identify factors associated with diagnostic delay.
Results
Four patients were included, with a median age of 74 years (range, 62 to 79 years). Histologic diagnoses comprised three HPV-independent gastric-type adenocarcinomas and one HPV-independent squamous cell carcinoma. The median interval from the first abnormal cytologic finding to definitive diagnosis was 18.5 months (range, 11 to 30 months). Before diagnosis, all patients underwent a median of 3.5 cytologic examinations (range, 3 to 5). Associated factors included repeatedly negative HPV test results (3/4), nondiagnostic biopsy or conization (2/4), delayed tissue evaluation despite persistent cytologic abnormalities (1/4), deeply located endocervical lesions (3/4), and a concurrent HPV-associated squamous lesion (1/4). Re-review identified atypical or malignant cells in all cases, indicating that the delay did not result from failure of cytologic detection.
Conclusions
HPV-IDCM remain vulnerable to delayed diagnosis in the era of HPV-based screening. Persistent abnormal cytologic findings should not be dismissed solely because HPV test results are negative. Careful correlation of cytologic, clinical, and histologic findings remains essential for timely HPV-IDCM detection.
INTRODUCTION
The 2020 World Health Organization (WHO) classification of female genital tumors categorizes cervical neoplasms as human papillomavirus (HPV)–associated or HPV-independent [1-3]. Although HPV-independent cervical malignancies (HPV-IDCM) comprise a small proportion of cervical malignancies, they are increasingly recognized as a distinct group with unique clinicopathologic features and poor clinical outcomes. Approximately 5%–7% of cervical squamous cell carcinomas are HPV-independent and are associated with advanced stage, increased lymph node metastasis, and worse survival [4,5]. HPV-independent adenocarcinomas include gastric-type, clear cell, and mesonephric adenocarcinomas. Gastric-type adenocarcinoma is the most common subtype, accounting for approximately 10%–15% of cervical adenocarcinomas [6,7], and has significantly poorer survival than HPV-associated usual-type adenocarcinoma [8,9].
Advances in cervical cancer prevention have accelerated adoption of HPV-based screening. The 2021 WHO guideline strongly recommends primary HPV DNA testing because of its superior sensitivity for detecting cervical precancerous lesions and its potential to reduce cervical cancer incidence and mortality [10]. Similarly, the Korean cervical cancer screening guideline recognizes high-risk HPV testing as an alternative to cytology in women aged ≥25 years [11,12]. These recommendations reflect that most cervical cancers result from persistent high-risk HPV infection, particularly HPV types 16 and 18 [13].
Despite its success, HPV-based screening remains limited for HPV-IDCM. Because these tumors develop independently of HPV infection, HPV testing alone cannot detect them. Furthermore, some HPV-independent malignancies, particularly gastric-type endocervical adenocarcinoma, are clinically difficult to detect because they often arise deep within the endocervical canal and may be missed by colposcopy or limited biopsy [14,15]. Consequently, diagnosis often occurs at an advanced stage, contributing to poor prognosis. In these cases, abnormal cervical cytology may provide the earliest evidence of underlying malignancy.
In our previous nationwide study of HPV-IDCM, several patients had abnormal cytologic findings that preceded the final diagnosis by a prolonged interval [16]. Diagnosis was delayed despite abnormal cytology and subsequent evaluation because of persistently negative HPV test results, inconclusive biopsy findings, and subtle endocervical lesions. In this multicenter study, we describe HPV-IDCM with delayed diagnosis and analyze the clinical and cytologic factors underlying these diagnostic pitfalls. We also highlight the continuing value of cervical cytology and raise awareness of the diagnostic challenges posed by HPV-IDCM in the era of HPV-based screening.
MATERIALS AND METHODS
Case selection
This multicenter study was conducted as part of a policy research project organized by the Korean Society for Cytopathology. Patients with HPV-IDCM were identified from participating institutions [16]. These patients represented a subset of the HPV-IDCM cohort from our previous nationwide study and were selected for delayed diagnosis. Whereas the previous study examined the overall frequency of HPV-IDCM and the diagnostic yield of cytology, the present study focuses on individual diagnostic pathways and cytologic features associated with delayed diagnosis and therefore does not overlap in objectives or analyses.
Patients were eligible for inclusion if they (1) had histologically confirmed HPV-IDCM, (2) had one or more abnormal cervical cytologic findings before definitive histologic diagnosis, and (3) had an interval of ≥6 months between the first abnormal cytologic finding and definitive histologic diagnosis. This threshold was consistent with the definition applied in our previous nationwide study [16]. In addition, according to the Korean cervical cancer screening guideline, abnormal cytologic findings warrant follow-up within 6 months or earlier diagnostic evaluation depending on the severity of the cytologic abnormality [17]. Therefore, an interval of ≥6 months was considered a conservative threshold for defining delayed diagnosis after an abnormal cytologic finding. Patients with available clinical records and cytology slides underwent detailed clinicopathologic review. Only cases with cervical cytology, histologic evaluation, and clinical follow-up available within the same institution were included to ensure reliable assessment of the diagnostic interval. Cross-institutional records were unavailable for verification.
HPV-IDCM were defined according to the 2020 WHO classification of female genital tumors based on histologic features, HPV test results, and immunohistochemical findings, including p16 expression when available [1,6,7]. Cases with incomplete clinical information or unavailable cytology slides were excluded.
Data collection and review
Clinical data, including age, cervical cytology, HPV test results, final histologic diagnosis, treatment history, and clinical outcome, were obtained from medical records. The interval between the first abnormal cytologic finding and definitive histologic diagnosis was defined as the diagnostic delay period.
Available cytology slides, histologic specimens, and immunohistochemical results were independently reviewed by three pathologists (H.E.P., H.-R.J., and H.-J.K.). All archived cytology slides (12 slides from four patients) were independently reexamined to determine whether atypical or malignant cells were identifiable and to assign a Bethesda category [18]. Discrepancies were resolved by consensus, and the final interpretation was compared with the original screening result. Histologic diagnoses were reviewed according to the 2020 WHO Classification of Female Genital Tumors [1]. Following consensus review, the principal factors associated with diagnostic delay were identified by correlating the clinical course with cytologic, histologic, and HPV test findings. Given the descriptive nature of this case series and the limited sample size, no comparative or inferential statistical analyses were performed.
RESULTS
Clinicopathologic characteristics
Among 208 patients with HPV-IDCM identified in the nationwide cohort [16], four (1.9%) met the criteria for delayed diagnosis and comprised the study population. The median age at diagnosis was 74 years (range, 62 to 79 years). Histologic diagnoses included three HPV-independent gastric-type adenocarcinomas and one HPV-independent squamous cell carcinoma. The median interval from the initial abnormal cytologic finding to definitive histologic diagnosis was 18.5 months (range, 11 to 30 months). Clinicopathologic characteristics are summarized in Table 1.
Clinicopathologic characteristics of patients with delayed diagnosis of HPV-IDCM
Cytologic findings and diagnostic course
All patients had abnormal cytologic findings before definitive diagnosis. The median number of cytologic examinations before diagnosis was 3.5 (range, 3 to 5). Cytologic abnormalities included atypical glandular or endocervical cells in three patients, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) in one patient, and high-grade squamous intraepithelial lesion (HSIL) in one patient. Persistent abnormal cytologic findings were documented in all patients.
On re-review of the 12 available cytology slides from the four patients, the consensus interpretation agreed with the original diagnosis in 11 slides. One slide from case 1 was reclassified from ASC-H to atypical glandular cells. At least one slide from each patient contained atypical or malignant cells, confirming that abnormal cytologic findings were detectable throughout the diagnostic course. These findings indicate that diagnostic delay resulted from events after cytologic evaluation rather than failure of cytologic detection.
Factors associated with diagnostic delay
Recurring factors associated with delayed diagnosis are summarized in Table 2, and the diagnostic courses of all four patients are shown in Fig. 1. Repeated HPV-negative results occurred in three patients. Two patients underwent biopsy or conization that remained nondiagnostic despite persistent cytologic abnormalities. Three patients had deeply located endocervical glandular lesions that were difficult to detect by routine evaluation. In one patient, tissue diagnosis was delayed despite persistent abnormal cytologic findings, including repeated HSIL results. In another, a concurrent HPV-associated squamous lesion may have masked an underlying HPV-independent glandular neoplasm. Collectively, these factors may have contributed to delayed histologic confirmation and appropriate clinical management despite persistent cytologic abnormalities.
Diagnostic pitfalls associated with delayed diagnosis
Diagnostic timelines of four patients with human papillomavirus (HPV)–independent cervical malignancies, illustrating the interval between the initial abnormal cervical cytology and the first histologic diagnosis. Each horizontal lane represents one case, plotted by months from the initial abnormal cytology, with the shaded band indicating the diagnostic delay. Cytologic and histologic findings are shown along each timeline, and HPV test results are displayed below. Symbols indicate cervical cytology, HPV test results (negative or positive), tissue sampling, first histologic diagnosis, and definitive surgery, as shown in the legend. Abnl, abnormal cervical cytology from a referring institution; ADC, adenocarcinoma; AEC, atypical endocervical cells; AGC, atypical glandular cells; AIS, adenocarcinoma in situ; ASC-H, atypical squamous cells, cannot exclude HSIL; ASC-US, atypical squamous cells of undetermined significance; Bx, biopsy; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; SCC, squamous cell carcinoma; TH, total hysterectomy.
Clinical course of each case
Representative cytologic, histologic, and p16 immunohistochemical findings from cases 1–3 are shown in Fig. 2.
Cytologic, histologic, and p16 immunohistochemical findings in human papillomavirus–independent cervical malignancies (HPV-IDCM) (cases 1–3). Each row represents one case, and the columns show cervical cytology (Papanicolaou stain) (A, D, G), histology (hematoxylin and eosin) (B, E, H), and p16 immunohistochemistry (C, F, I). (A–C) Case 1: HPV-independent gastric-type adenocarcinoma (ADC). (D–F) Case 2: HPV-independent gastric-type ADC. (G–I) Case 3: HPV-independent squamous cell carcinoma.
Case 1
A 71-year-old female presented with atypical endocervical cells on cervical cytology. Subsequent punch biopsy and conization were nondiagnostic, and HPV testing remained repeatedly negative. Persistent cytologic abnormalities, including repeated ASC-H results, prompted further evaluation. Total hysterectomy was performed 11 months after the initial abnormal cytologic finding revealed HPV-independent gastric-type adenocarcinoma involving the endocervix.
Case 2
A 77-year-old female was referred after abnormal cervical cytology. Initial conization demonstrated no significant lesion and HPV testing was negative. Follow-up cytology demonstrated atypical glandular cells, but additional evaluation was delayed. Repeat conization 22 months after the initial abnormal cytologic finding identified invasive HPV-independent gastric-type adenocarcinoma. The patient subsequently underwent hysterectomy and adjuvant chemoradiotherapy.
Case 3
A 79-year-old female had persistent abnormal cytology for more than 2 years, with repeated HSIL findings followed by LSIL. Repeated HPV testing remained negative. Despite multiple abnormal cytologic findings, cervical biopsy performed 30 months after the initial HSIL result established the diagnosis of invasive squamous cell carcinoma. The tumor was p16-negative and classified as HPV-independent squamous cell carcinoma.
Case 4
A 62-year-old female was referred after abnormal cervical cytology detected during routine health screening. Initial biopsy showed only low-grade squamous intraepithelial lesion with koilocytic change. Follow-up cytology repeatedly demonstrated atypical glandular cells, prompting conization 15 months after the initial abnormal cytologic finding. Histologic examination revealed HPV-independent adenocarcinoma in situ with focal mucin 6 (MUC6) expression and negative p16 staining. Subsequent hysterectomy confirmed HPV-independent gastric-type adenocarcinoma. The repeatedly positive high-risk HPV (HPV16) result was attributed to a concurrent HPV-associated squamous lesion, whereas the gastric-type adenocarcinoma was confirmed as HPV-independent based on characteristic gastric-type morphology, negative p16 staining, and MUC6 positivity in the glandular tumor. Coexistence of HPV-associated squamous and HPV-independent glandular lesions likely delayed recognition of the latter. Representative findings from case 4, including the concurrent HPV-associated squamous lesion and HPV-independent gastric-type adenocarcinoma, are shown in Fig. 3.
Case 4: a concurrent human papillomavirus (HPV)–associated squamous lesion masking a coexisting HPV-independent gastric-type adenocarcinoma (ADC). (A, B) HPV-associated squamous lesion (low-grade squamous intraepithelial lesion [LSIL], HPV-16+). (C–F) HPV-independent gastric-type ADC. Cervical cytology showing atypical squamous cells of undetermined significance (A) and biopsy showing LSIL with koilocytosis, consistent with productive high-risk HPV-16 infection (B). Cytology showing atypical glandular cells (C), invasive gastric-type endocervical ADC (D), negative p16 immunohistochemistry (non-block-type) in tumor cells (E), and positive mucin 6 (MUC6) immunohistochemistry (F). The positive HPV test reflected the concurrent squamous lesion and masked the coexisting HPV-independent ADC, which was confirmed by negative p16 and positive MUC6 immunostaining.
DISCUSSION
In this study, four patients with HPV-IDCM experienced delayed diagnosis despite persistent abnormal cytologic findings. The median interval from the initial abnormal cytologic finding to definitive diagnosis was 18.5 months, and all patients underwent repeated cytologic examinations before histologic confirmation. Recurrent diagnostic pitfalls included persistently negative HPV test results, nondiagnostic biopsy or conization, delayed tissue evaluation despite persistent cytologic abnormalities, and concurrent HPV-associated lesions. These findings highlight the diagnostic challenges of HPV-IDCM and reinforce the continuing value of cytology in the era of HPV-based cervical cancer screening.
HPV-IDCM are a rare but clinically important subset of cervical neoplasms. Although squamous cell carcinoma remains the predominant cervical cancer, the proportion of adenocarcinoma has increased over recent decades [19,20]. Gastric-type adenocarcinoma is the most common HPV-independent adenocarcinoma and the predominant HPV-independent glandular neoplasm encountered in practice [7,8]. Since the introduction of the 2020 WHO classification, HPV-IDCM have been recognized as a distinct group with unique pathogenesis, aggressive behavior, and poorer outcomes than HPV-associated cervical cancers [1,21,22]. Gastric-type adenocarcinoma is associated with advanced-stage disease and inferior survival [8,23], whereas HPV-independent squamous cell carcinoma also demonstrates unfavorable clinicopathologic features [4,5]. These tumors also present substantial diagnostic challenges. Gastric-type adenocarcinomas often arise deep within the endocervical canal and may be missed by colposcopy or limited biopsy. More importantly, because they develop independently of HPV infection, they can escape HPV-based screening. Consequently, HPV-IDCM remain an important diagnostic blind spot despite widespread primary HPV screening [24].
A key finding was persistent abnormal cytologic findings despite repeatedly negative HPV test results. Three of four patients remained HPV-negative throughout the diagnostic course. Although HPV testing is the most sensitive method for detecting HPV-associated cervical neoplasms and has substantially improved cervical cancer screening [10], HPV-IDCM remain an important exception [25,26]. Repeated negative HPV results may falsely reassure clinicians and patients, especially when cytologic abnormalities are mild or histologic findings are inconclusive. Although the retrospective design precluded determining the direct influence of HPV results on clinical decision-making, persistent HPV negativity may have favored continued surveillance rather than more aggressive evaluation in several cases. These findings emphasize that persistent abnormal cytologic findings warrant careful evaluation, even when HPV test results are repeatedly negative.
Another important finding was the frequent discordance between cytologic and histologic findings. Three patients had persistent glandular or endocervical cytologic abnormalities despite nondiagnostic or discordant biopsy results. This discrepancy is particularly relevant in HPV-independent glandular neoplasms, especially gastric-type adenocarcinoma. Unlike HPV-associated cervical neoplasms, gastric-type adenocarcinomas often arise deep within the endocervical canal and exhibit an infiltrative growth pattern that is difficult to sample by routine punch biopsy or limited excision [14,15]. Consequently, histologic evaluation may yield false-negative or nonspecific findings despite persistent cytologic abnormalities [16,25]. In our cohort, abnormal cytologic findings preceded definitive histologic diagnosis by several months, illustrating the limitations of tissue sampling. One patient also had a concurrent HPV-associated squamous lesion that may have obscured recognition of the underlying HPV-independent glandular neoplasm. Notably, review identified atypical or malignant cells in cytology specimens from all four patients, indicating that delayed diagnosis resulted from limitations in histologic confirmation and subsequent clinical evaluation rather than failure of cytologic detection. These findings reinforce the value of cytology as an early indicator of HPV-IDCM, even when biopsy is nondiagnostic or HPV testing is negative.
These findings should not be interpreted as evidence against HPV-based screening. HPV testing remains the cornerstone of cervical cancer prevention and contemporary screening strategies [10]. However, HPV-IDCM represent a small but clinically important group that may not be detected by HPV testing alone [27]. In our cohort, persistent cytologic abnormalities consistently provided the earliest indication of underlying malignancy. These observations support the continuing value of cervical cytology as a complementary test rather than a replacement for HPV testing [16]. In healthcare systems where repeated cytologic follow-up and timely diagnostic evaluation are feasible, persistent cytologic abnormalities may facilitate earlier detection of HPV-IDCM. Because this case series specifically included patients with persistent abnormal cytologic findings, it characterizes the diagnostic pathways of cytology-positive delayed cases and does not address the overall diagnostic performance of cervical cytology for HPV-IDCM.
The low proportion of eligible cases (1.9%) likely reflects the clinical presentation of HPV-IDCM. These tumors are often diagnosed only after symptom onset, when cytology and histologic evaluation occur during the same diagnostic episode. Consequently, few patients have a measurable interval between the first abnormal cytologic finding and definitive histologic diagnosis within a single institution. This observation is consistent with a nationwide Swedish registry study showing that HPV-negative cervical cancers occurred predominantly in older females with advanced-stage disease and that most HPV-negative results were obtained near the time of diagnosis [28]. Although HPV-negative cervical cancers are not synonymous with HPV-independent tumors because HPV-negative results may also reflect sampling or assay limitations [29], both observations highlight a diagnostic limitation of HPV-based screening alone. Together with evidence that cytology–high-risk HPV cotesting improves detection of cervical adenocarcinoma compared with either modality alone [30], our findings support careful evaluation of persistent cytologic abnormalities, even when HPV test results are negative.
This study has several clinical implications. Persistent abnormal cytologic findings should not be dismissed solely because HPV test results are negative, particularly when abnormalities recur or remain unexplained by histologic evaluation. Careful integration of cytologic, clinical, and histologic findings may facilitate earlier recognition of HPV-IDCM and reduce diagnostic delay. When persistent abnormal cytology is accompanied by repeatedly negative HPV test results or nondiagnostic biopsy, HPV-IDCM should be considered, and additional evaluation, including endocervical curettage, deeper or repeat sampling, or conization, should be pursued instead of continued routine surveillance.
A major strength of this study is its analysis of real-world diagnostic pathways in a rare and underrecognized group of cervical neoplasms. However, this study has several limitations, including the retrospective design, small sample size, and multicenter heterogeneity. Additionally, only cases with linked cytologic and histologic records within the same institution were included because cross-institutional linkage was unavailable; more complete screening histories would likely identify additional delayed cases. An HPV-associated comparison group was also unavailable, precluding assessment of the relative magnitude of diagnostic delay. Future studies incorporating cross-institutional data linkage and an HPV-associated comparison group would help clarify the true frequency and relative magnitude of diagnostic delay in HPV-IDCM. Accordingly, the present findings should be regarded as observations from a small case series, and larger cohort studies are needed to confirm their broader clinical applicability. Despite these limitations, the findings consistently demonstrate that persistent abnormal cytologic findings may provide an important clue to HPV-IDCM. In conclusion, abnormal cytologic findings should not be disregarded solely because HPV test results are negative. Continued attention to persistent cytologic abnormalities may be important for timely diagnosis of HPV-IDCM in the era of HPV-based screening.
Notes
Ethics Statement
All procedures performed in the current study were approved by the Institutional Review Board (IRB) of Inje University Sanggye Paik Hospital (IRB No. SGPAIK2024-04-011; approved April 30, 2024) in accordance with the 1964 Helsinki Declaration of Helsinki and its later amendments. Formal written informed consent was not required with a waiver by the appropriate IRB.
Availability of Data and Material
The datasets generated or analyzed during this study are available from the corresponding author upon reasonable request.
Code Availability
Not applicable.
Author Contributions
Conceptualization: HEP, HRJ, HJK. Data curation: HEP, HRJ, HJK. Formal analysis: HEP, HRJ, HJK. Investigation: HEP, HRJ, HJK. Funding acquisition: HEP, HRJ, HJK. Methodology: HEP, HRJ, HJK. Project administration: HEP, HRJ, HJK. Supervision: HJK. Validation: HEP, HRJ, HJK. Writing—original draft: HEP. Writing—review and editing: HEP, HRJ, HJK. Approval of final manuscript: all authors.
Conflicts of Interest
The authors declare that they have no potential conflicts of interest.
Funding Statement
This study was supported in part by the Korean Society for Cytopathology (Grant No. 23-01), Republic of Korea.
Acknowledgments
The authors gratefully acknowledge the cooperation and scholarly discussions provided by members of the Korean Society for Cytopathology during the course of this study.