GOG protocol #171 was initiated in 1998 when the criteria of AGC diagnosis for patient enrollment was based on the 1991 TBS classification and the conventional study Pap smears were used. HPV testing in a liquid based cytology specimen was added as a study amendment in 2003 to incorporate advances in scientific understanding and the clinical management of women with cervical lesions. The protocol was closed for accrual in 2005.
Between September 28, 1998 and October 10, 2005, 592 women with a cytologic diagnosis of AGC were enrolled in the study. One-hundred eighty-nine women were excluded from the study. The reasons for exclusions were as the follows: withdrew consent (n=3); lack of histological evaluation of the transformation zone (n=110); and unsatisfactory study Pap smears (n=76). The clinical characteristics and histologic diagnosis of the 403 evaluable women are presented in . The ages ranged from 20 to 86 with a median of 43 years old. The distribution by race and ethnicity is illustrated in .
Clinical Characteristics and Histology Diagnosis
also provides the distribution of benign and neoplastic cervical lesions observed in this cohort. Of 403 patients enrolled in the study, 111 (28%) women had a SCL, 74 (18%) had significant squamous lesions (SLs), and 37 (9%) had significant glandular lesions (GLs). Among those with a SCL (n=111), 67% were SLs, including CIN2 (n=14), CIN3 (n=58) and squamous cell carcinoma (n=2), and 33% were GLs, including AIS (n=23) and invasive adenocarcinoma (n=14). The incidence of invasive carcinoma in the study was 4%. There were five women whose malignancy was found outside of the cervix. These malignant tumors involved the endometrium (n=3), ovary (n=1) and fallopian tube (n=1). All of these women with extra-cervical malignancy were diagnosed during the evaluation of AGC, either by endometrial biopsy or abnormal physical and radiographic abnormalities. All five women received a total hysterectomy and bilateral salpingo-oophorectomy. The cervix of each of these women was histologically confirmed to lack evidence of a significant lesion. The study was designed to focus only on significant lesions of the cervix, thus, these cases were coded as negative for the purposes of the CA-IX and HPV analyses
illustrates the four CA-IX immunohistochemical staining patterns. Diffuse or focal CA-IX immunoreactivity in the atypical cells (pattern A) and focal strong or diffuse immunoreactivity in normal looking endocervical cells (pattern B) were classified as positive. CA-IX immunoreactivity that was focal and weak in normal endocervical cells (pattern C) or negative (pattern D) was classified as negative. Pattern A was easily discriminated from pattern C and D. The rate of agreement among any two of the three reviewers reached 97% on pattern A and 100% on pattern D before the arbitrating consensus review. Five cases were upgraded from pattern C to pattern A, and five cases from pattern C to pattern B, but only two cases were downgraded from pattern B to pattern C after the consensus review.
The intent of the initial study was to determine the accuracy of CA-IX expression exclusively as an indicator of the presence of a SCL. Thus, the data shown in the upper panel (#1) of depict the results of CA-IX testing for all of the specimens (n=403). Positive staining for CA-IX protein expression was observed in 118 (29%) conventional study Pap smear specimens. Among these positive cases, 83 (70%) had a SCL, including 48 of 74 (65%) SLs, and 35 of 37 (95%) GLs. Among 35 insignificant cervical lesions with positive CA-IX immunoreactivity, two were CIN1, one was atypia and one was glandular hyperplasia. Thus, positive CA-IX immunoreactivity in a conventional Pap smear had an overall sensitivity and specificity of 75% and 88%, respectively with a FNR of 10%, for detecting a SCL. The sensitivity for CA-IX detection in GLs was 95% and in SLs was 65%. ().
Biomarker Test (CA-IX, HPV, and CA-IX + HPV) Result by Histologic Diagnosis
Diagnostic Accuracy of CA-IX, HPV, and CA-IX + HPV
A direct comparison between CA-IX expression and HPV detection was performed on those specimens where a LBC specimen was available for HPV analysis (n=122). For these cases, 31 (25%) were positive for CA-IX expression. Among these CA-IX positive cases, 25 (80%) had a SCL, including 16 of 28 (57%) SLs, and 9 of 10 (90%) GLs. Thus, the overall sensitivity of SCLs, SLs or GLs was 66%, 57% or 90% respectively; with a specificity of 93% and a FNR of 14%. The comparative analysis of CA-IX and HPV is given below and in and .
The HC2 method of HPV testing was performed on 122 cases. Patient ages ranged from 20 to 71, with a median age of 36 for the HPV positive group and 45 for the HPV negative group (). H-HPV DNA was detected in 48 (39%) of LBC specimens (, #2). Among these positive cases, 37 (77%) had a SCL, including 27 of 28 (96%) SLs and 10 of 10 (100%) GLs. This provided an overall sensitivity of 97%, 96% and 100% for SCLs, SLs and GLs, respectively; with a specificity of 87% and a FNRof 1% (). There were 11 cases in the insignificant lesion category and among these, four were diagnosed as CIN1 and one was atypia.
112 of 122 cases tested for the presence of HPV by the HC2 method were also processed for HPV genotyping, using the PCR-based Roche LINEAR ARRAY (RLA) kit. Sixty-five cases (58%) were positive for H-HPV. Among these positive cases, 36 (54%) had SCLs, including 26/27 (96%) SLs, and 10 of 10 (100%) GLs. Thus, the PCR-based HPV genotyping method for detecting SCLs gives an overall sensitivity and specificity of 97% and 61%, respectively, and a FNR of 2%. In terms of the rates of positive HPV detection, the PCR-based RLA and HC2 methods did show some differences, with the RLA method detecting more positives (39% versus 13%) in the insignificant lesion category and less (97% versus 100%) in the SCL category, respectively. The comparative analysis of HC2 testing and PCR-based RLA genotyping is given in .
The Comparison of Hybrid capture 2 (HC2) and PCR-based Roche Linear Array (RLA) HPV detection
A compilation of the genotyping data revealed that approximately 49% of both benign lesions and SCLs contained multiple HPV types (). There was no apparent difference with respect to whether the lesion was positive or negative by the HC2 method of detection (data not shown). The distribution of the specific H-HPV types in the cervical lesions is shown in .
Detection of High Risk HPV types by PCR-Based Roche Linear Array Testing
High-Risk HPV Genotypes using the Roche Linear Array Kit
The combined accuracy of CA-IX and HPV testing
The data were also evaluated based on the combined CA-IX and HPV testing (HC2 method) performed in 122 cases. For the combination of CA-IX with HPV, a case was negative if there was a negative result for both CA-IX and HPV. If either or both were positive, the case was then called positive. The combined CA-IX and HPV testing had an overall sensitivity and specificity of 97% and 80% and a FNR of 1%. Details are given in .