112
LETTERS
to
the
EDITOR
HPV in cervical
smears
SIR,-We would respond to several points that your correspondents (May 30, p 1352) raise about our report (April 18, p 959). Our main point was that when HPV16 DNA levels in smears were high, CIN3 was almost always present in biopsy samples, irrespective of cytological grade. The patients had either severe, moderate, or at least two consecutive mild smears and would be referred for colposcopy under present guidelines. Most patients were seen within 6 months of referral smear. Our data strongly suggest that women with a single smear containing a high level of HPV16 DNA are also very likely to have CIN3 and should therefore be referred immediately. This would be an important complementary addition to the use of cytology alone in screening. We did not suggest any changes to the current management guidelines in the absence of high levels of HPV 16 DNA. However, the use of HPV determinants to improve the specificity of cytology reports is an important topic for future work. One concern was patients diagnosed with CIN3 who were negative for HPV 16. We have subsequently analysed all specimens reported in our paper for high levels of HPV types 6,11,18,31, and 33 on use of type-specific primers and, when negative for all of these, for strong signals due to other unidentified types with consensus primers. The details of this study, which forms part of a wider survey, will be presented elsewhere, but some of the findings are relevant here. A total of 14 patients had CIN3 that was not predicted by high levels of HPV16 in smears. Further examination showed that none had high levels of HPV6,11, or 18; 6 had HPV31; 2 had HPV33; 2 were positive by consensus primers; and 4 were negative or had low levels for all types. This should be compared with the group of 40 women diagnosed with CINl, HPV infection only, or less, in whom a high level of HPV6 was found in 1, HPV16 in 2, HPV18 in 2, HPV31 in 4, HPV33 in 1, and other types in 4; the remaining 26 (65%) being negative or at low levels for all types. Thus other types may be of use in screening, but they are not as specific as HPV16 for the identification of CIN3. A much larger study is underway to clarify this issue. Dr Herrington and colleagues report data showing a weaker relation than ours. Their failure to obtain clear-cut results may be related to the different methodology adopted, notably in the use of consensus primers and undefined standards for quantification. In our experience, the use of consensus primers in PCR can result in competition between non-specifically primed human DNA with HPV DNA and between different types of HPV DNA in individual clinical specimens, and the apparent level of HPV DNA of any particular type may be distorted after amplification. For this reason, we have used type-specific primers for quantification of HPV-typespecific DNA levels and have reserved consensus primers for qualitative demonstration of the presence of HPV types other than those specific types tested for. Currently we are typing and quantifying HPV DNA in specimens positive by consensus primers with type-specific primers. Our standards were amplified in the presence of 100 ng human DNA.
The careful DNA extraction we used was essential to establish the relation between levels of HPV 16 DNA in smears and presence of CIN3 in biopsy specimens. For this approach to be of general use simpler procedures are clearly needed. Computer hardware is available for a semi-automated PCR procedure and, having established a relation, we have been able to simplify the method of DNA extraction while maintaining the predictive value of the test. The results summarised in the table show that virtually identical COMPARISON OF HPV16 DNA LEVELS BY PCR FOR DIFFERENT DNA EXTRACTION METHODS
previously used, freeze/thaw, 4 cycles of freezing at -20°C and temperature; boil, for 10 min; NP40, 0 45% ; Tween20, 0-45%; proteinase K (PK), 200 g/mi at 50°C for 60 min and 95°C for 10 min. ++=high (100 fg/100 ng DNA); + = mtermediate (2to 100 fg), nd=not done. ± = low (0 4 to < 2 fg), - = negative (
LETTERS
to
the
EDITOR
HPV in cervical
smears
SIR,-We would respond to several points that your correspondents (May 30, p 1352) raise about our report (April 18, p 959). Our main point was that when HPV16 DNA levels in smears were high, CIN3 was almost always present in biopsy samples, irrespective of cytological grade. The patients had either severe, moderate, or at least two consecutive mild smears and would be referred for colposcopy under present guidelines. Most patients were seen within 6 months of referral smear. Our data strongly suggest that women with a single smear containing a high level of HPV16 DNA are also very likely to have CIN3 and should therefore be referred immediately. This would be an important complementary addition to the use of cytology alone in screening. We did not suggest any changes to the current management guidelines in the absence of high levels of HPV 16 DNA. However, the use of HPV determinants to improve the specificity of cytology reports is an important topic for future work. One concern was patients diagnosed with CIN3 who were negative for HPV 16. We have subsequently analysed all specimens reported in our paper for high levels of HPV types 6,11,18,31, and 33 on use of type-specific primers and, when negative for all of these, for strong signals due to other unidentified types with consensus primers. The details of this study, which forms part of a wider survey, will be presented elsewhere, but some of the findings are relevant here. A total of 14 patients had CIN3 that was not predicted by high levels of HPV16 in smears. Further examination showed that none had high levels of HPV6,11, or 18; 6 had HPV31; 2 had HPV33; 2 were positive by consensus primers; and 4 were negative or had low levels for all types. This should be compared with the group of 40 women diagnosed with CINl, HPV infection only, or less, in whom a high level of HPV6 was found in 1, HPV16 in 2, HPV18 in 2, HPV31 in 4, HPV33 in 1, and other types in 4; the remaining 26 (65%) being negative or at low levels for all types. Thus other types may be of use in screening, but they are not as specific as HPV16 for the identification of CIN3. A much larger study is underway to clarify this issue. Dr Herrington and colleagues report data showing a weaker relation than ours. Their failure to obtain clear-cut results may be related to the different methodology adopted, notably in the use of consensus primers and undefined standards for quantification. In our experience, the use of consensus primers in PCR can result in competition between non-specifically primed human DNA with HPV DNA and between different types of HPV DNA in individual clinical specimens, and the apparent level of HPV DNA of any particular type may be distorted after amplification. For this reason, we have used type-specific primers for quantification of HPV-typespecific DNA levels and have reserved consensus primers for qualitative demonstration of the presence of HPV types other than those specific types tested for. Currently we are typing and quantifying HPV DNA in specimens positive by consensus primers with type-specific primers. Our standards were amplified in the presence of 100 ng human DNA.
The careful DNA extraction we used was essential to establish the relation between levels of HPV 16 DNA in smears and presence of CIN3 in biopsy specimens. For this approach to be of general use simpler procedures are clearly needed. Computer hardware is available for a semi-automated PCR procedure and, having established a relation, we have been able to simplify the method of DNA extraction while maintaining the predictive value of the test. The results summarised in the table show that virtually identical COMPARISON OF HPV16 DNA LEVELS BY PCR FOR DIFFERENT DNA EXTRACTION METHODS
previously used, freeze/thaw, 4 cycles of freezing at -20°C and temperature; boil, for 10 min; NP40, 0 45% ; Tween20, 0-45%; proteinase K (PK), 200 g/mi at 50°C for 60 min and 95°C for 10 min. ++=high (100 fg/100 ng DNA); + = mtermediate (2to 100 fg), nd=not done. ± = low (0 4 to < 2 fg), - = negative (
