1231
HPV-16 and cervical intraepithelial
neoplasia SIR,-Dr Cuzick and his colleagues (April 18, p 959) report that detecting human papillomavirus type 16 (HPV-16) in mild or moderately dyskaryotic cervical smears is an indicator of high-grade cervical intraepithelial neoplasia. Any test that offers greater
specificity
in
identifying
those
at
risk of
having
or
developing
cervical carcinoma is to be welcomed; however, caution should be exercised in suggesting that HPV typing provides this solution. Evidence is accumulating that HPV-negative carcinomas carry a worse prognosis than HPV positive cancers,l,2 and our studies suggest one explanation. HPV positive cancers express the virally encoded E6 protein which can enhance degradation of the tumour-suppressor gene product p53. We have shown that HPV negative anogenital cancers show evidence of clonal somatic mutation within p53,3-5 suggesting that expression of a mutant p53 protein contributes to the development of HPV negative tumours whereas in HPV positive cancers expression of E6 protein substitutes for mutations within the p53 gene. This difference between HPV positive and negative cancers may be of critical significance since expression ofE6 seems to lead to loss of functional p53, whereas somatic mutation results in the expression of an altered p53 protein which may gain positive transforming activity in addition to losing wild type function. It is therefore possible that the more aggressive nature of HPV negative carcinomas may be due, at least in part, to the expression of a mutant p53 protein. The frequency of p53 mutation in HPV-negative cervical intraepithelial neoplasia is unknown at present, but such alterations may be already present in the precursor lesions of patients who are likely to have a more aggressive carcinoma and are thus in most need of early detection. Until more of these facts are known we should be wary of being falsely reassured by HPV-16 negative mild or
moderate dyskaryosis. TIM CROOK ALAN FARTHING KAREN VOUSDEN
Ludwig Insitute for Cancer Research, St Mary’s Hospital Medical School, London W2 1PG, UK
1. Riou G, Favre M, Jeannel D, Bourhis J, Le Doussal V, Orth G. Association between poor prognosis in early-stage invasive cervical carcinomas and non-detection of HPV DNA. Lancet 1990; 335: 1171-74. 2. Higgins GD, Davy M, Roder D, Uzelin DM, Phillips GE, Burrell CJ. Increased age and mortality associated with cervical carcinomas negative for human papillomavirus. Lancet 1991; 338: 910-13. 3. Crook T, Wrede D, Vousden KH. p53 point mutation in HPV negative human cervical carcinoma cell lines. Oncogene 1991; 6: 873-75. 4. Crook T, Wrede D, Tidy J, Scholefield J, Crawford L, Vousden KH. Status of c-myc, p53 and retinoblastoma genes in human papillomavirus positive and negative squamous cell carcinomas of the anus. Oncogene 1991; 6: 1251-57. 5. Crook T, Wrede D, Tidy JA, Mason WP, Evans DI, Vousden KH. Clonal p53 mutation in primary cervical cancer: association with human-papillomavirusnegative tumours. Lancet 1992; 339: 1070-73.
Testing histologically negative lymph nodes for papillomavirus when evaluating metastasis in cervical
SIR,-Lymph-node positivity strongly
cancer
correlates with risk of
recurrence in cervical cancer, but about 50% of patients with recurrence after radical surgery have had histologically negative
lymph nodes
and clear resection
margins.’
This may in part be
explained by the fact that lymph-node metastasis tends to be assessed by histological examination alone. Fuchs et aF have shown that human papillomavirus (HPV) type 16 DNA can occasionally be detected in histologically negative lymph nodes by Southern blot hybridisation, and it was suggested that HPV DNA might be a useful marker of node metastasis when the original tumour contains HPV DNA. However, this is not easy because Southern blot hybridisation requires fresh-frozen tissue. We have developed a nested polymerase chain reaction (PCR) method for looking at formalin-fixed, paraffin-embedded tissues retrospectively. We investigated 350 histologically normal lymph nodes from fourteen patients treated for stage Ib/IIb invasive cervical cancer between January, 1983, and December, 1987, at Nagoya University Hospital. As negative controls, lymph nodes from ten patients with ovarian or endometrial cancer were also examined. The nested PCR
protocol was forty cycles (melting 94°C, 1.5min; annealing 40°C, 15 min; elongation 60°C, 2 min) with two consensus primers to HPV 16 and 18 as the first step3 and another forty cycles (94°C, 30 s; 55°C, 30 s; 70°C, 2 min) with four primers (positions 104-123 and 208-227 in type 16and 106-125 and 274-293 in type 185) as the second step. After amplification, PCR products were separated on 2% agarose gel and stained with ethidium bromide. The products were further confirmed by Southern blot hybridisation with probes (positions 141-180 in type 16and 151-190 in type 18). HPV DNA was detected in nine of the cervical cancers, and when we examined negative lymph nodes from these patients by nested PCR we found HPV DNA in the lymph nodes of five of them; in all five cases HPV type in lymph nodes and tumour was the same. Two of these five pateints had had a recurrence (pelvic cavity, lung) but the other three had no recurrences. Since all five patients had had pelvic radiotherapy after radical hysterectomy and lymphadenectomy the three patients may have been protected from recurrence. These findings show that nested PCR is a very useful method to evaluate early lymph node involvement retrospectively in HPVpositive cases, and we recommend it for the detection of micrometastasis as well as the evaluation of prognosis.
Department of Obstetrics and Gynaecology and Virus Laboratory, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine,
Nagoya 466, Japan
AKIHIRO NAWA YUKIHIRO NISHIYAMA FUMITAKA KIKKAWA OSAMU KURAUCHI SETSUKO GOTO NOBUHIKO SUGANUMA SIGEHIKO MIZUTANI YUTAKA TOMODA
1. Linda MS, Thomas WB, Elvio GS, et al. Prognostic factor in stage Ib squamous cervical cancer patients with low risk for recurrence. Obstet Gynecol 1991; 77: 271-75. 2. Fuchs PG, Girardi F, Pfister H. Human papillomavirus 16 DNA in cervical cancers and in lymph nodes of cervical cancer patients: a diagnostic marker for early metastasis? Int J Cancer 1989; 43: 41-44. 3. Yoshikawa H, Kawana T, Kitagawa K, et al. Amplification and typing of multiple cervical cancer-associated human papillomavirus DNAs using a single pair of primers. Int J Cancer 1990; 45: 990-92. 4. Seedorf K, Krämmer G, Durst M, et al. Human papillomavirus type 16 DNA sequence. Virology 1985; 145: 181-85. 5. Cole ST, Danos O. Nucleotide sequence and comparative analysis of the human papillomaviruses type 18 genome. J Mol Biol 1987; 193: 599-608.
Anaphylactoid response to intravenous acetylcysteine SIR,-Intravenous acetylcysteine (NAC) is the treatment of choice for a substantial paracetamol (acetaminophen) overdose. An anaphylactoid response to intravenous NAC has been described, usually beginning within 20 min of the start of treatment and resolving rapidly when treatment stops. It consists of one or more features of anaphylaxis, including bronchospasm, angio-oedema, rash, pruritus, and hypotension. By pooling reports to the UK National Poisons Information Service and the manufacturer (Duncan Flockhart), we compiled features of 38 cases in which this type of reaction developed after correct administration of the drug and we also collected 15 cases where an overdose had been given in error.’ The frequency of the anaphylactoid response was estimated at between 03% and 3% of courses of NAC correctly administered, whereas 11of the 15 cases who were given more than the recommended dose (often ten times too much!) had an anaphylactoid response. Hypotension was a much more prominent feature in patients given higher doses of NAC (6/11 reacting adversely to overdose vs 6/38). Conversely, features more specific to immune system activation (pruritus, angio-oedema, bronchospasm) tended to predominate in patients with an anaphylactoid response to conventional dosage. The hypotensive response to NAC may be distinct from the anaphylactoid response. It seems likely that this reaction is dose-dependent. While studying vasodilator mechanisms involving the vascular endothelium we investigated the action of NAC on isolated human and rat resistance arteries (internal diameter 273-391 )m). After contraction with 10 pmol/1 noradrenaline, cumulative addition of 01to 30 mmol/1 NAC induced a concentration-dependent relaxation (figure); the median concentration of NAC causing
HPV-16 and cervical intraepithelial
neoplasia SIR,-Dr Cuzick and his colleagues (April 18, p 959) report that detecting human papillomavirus type 16 (HPV-16) in mild or moderately dyskaryotic cervical smears is an indicator of high-grade cervical intraepithelial neoplasia. Any test that offers greater
specificity
in
identifying
those
at
risk of
having
or
developing
cervical carcinoma is to be welcomed; however, caution should be exercised in suggesting that HPV typing provides this solution. Evidence is accumulating that HPV-negative carcinomas carry a worse prognosis than HPV positive cancers,l,2 and our studies suggest one explanation. HPV positive cancers express the virally encoded E6 protein which can enhance degradation of the tumour-suppressor gene product p53. We have shown that HPV negative anogenital cancers show evidence of clonal somatic mutation within p53,3-5 suggesting that expression of a mutant p53 protein contributes to the development of HPV negative tumours whereas in HPV positive cancers expression of E6 protein substitutes for mutations within the p53 gene. This difference between HPV positive and negative cancers may be of critical significance since expression ofE6 seems to lead to loss of functional p53, whereas somatic mutation results in the expression of an altered p53 protein which may gain positive transforming activity in addition to losing wild type function. It is therefore possible that the more aggressive nature of HPV negative carcinomas may be due, at least in part, to the expression of a mutant p53 protein. The frequency of p53 mutation in HPV-negative cervical intraepithelial neoplasia is unknown at present, but such alterations may be already present in the precursor lesions of patients who are likely to have a more aggressive carcinoma and are thus in most need of early detection. Until more of these facts are known we should be wary of being falsely reassured by HPV-16 negative mild or
moderate dyskaryosis. TIM CROOK ALAN FARTHING KAREN VOUSDEN
Ludwig Insitute for Cancer Research, St Mary’s Hospital Medical School, London W2 1PG, UK
1. Riou G, Favre M, Jeannel D, Bourhis J, Le Doussal V, Orth G. Association between poor prognosis in early-stage invasive cervical carcinomas and non-detection of HPV DNA. Lancet 1990; 335: 1171-74. 2. Higgins GD, Davy M, Roder D, Uzelin DM, Phillips GE, Burrell CJ. Increased age and mortality associated with cervical carcinomas negative for human papillomavirus. Lancet 1991; 338: 910-13. 3. Crook T, Wrede D, Vousden KH. p53 point mutation in HPV negative human cervical carcinoma cell lines. Oncogene 1991; 6: 873-75. 4. Crook T, Wrede D, Tidy J, Scholefield J, Crawford L, Vousden KH. Status of c-myc, p53 and retinoblastoma genes in human papillomavirus positive and negative squamous cell carcinomas of the anus. Oncogene 1991; 6: 1251-57. 5. Crook T, Wrede D, Tidy JA, Mason WP, Evans DI, Vousden KH. Clonal p53 mutation in primary cervical cancer: association with human-papillomavirusnegative tumours. Lancet 1992; 339: 1070-73.
Testing histologically negative lymph nodes for papillomavirus when evaluating metastasis in cervical
SIR,-Lymph-node positivity strongly
cancer
correlates with risk of
recurrence in cervical cancer, but about 50% of patients with recurrence after radical surgery have had histologically negative
lymph nodes
and clear resection
margins.’
This may in part be
explained by the fact that lymph-node metastasis tends to be assessed by histological examination alone. Fuchs et aF have shown that human papillomavirus (HPV) type 16 DNA can occasionally be detected in histologically negative lymph nodes by Southern blot hybridisation, and it was suggested that HPV DNA might be a useful marker of node metastasis when the original tumour contains HPV DNA. However, this is not easy because Southern blot hybridisation requires fresh-frozen tissue. We have developed a nested polymerase chain reaction (PCR) method for looking at formalin-fixed, paraffin-embedded tissues retrospectively. We investigated 350 histologically normal lymph nodes from fourteen patients treated for stage Ib/IIb invasive cervical cancer between January, 1983, and December, 1987, at Nagoya University Hospital. As negative controls, lymph nodes from ten patients with ovarian or endometrial cancer were also examined. The nested PCR
protocol was forty cycles (melting 94°C, 1.5min; annealing 40°C, 15 min; elongation 60°C, 2 min) with two consensus primers to HPV 16 and 18 as the first step3 and another forty cycles (94°C, 30 s; 55°C, 30 s; 70°C, 2 min) with four primers (positions 104-123 and 208-227 in type 16and 106-125 and 274-293 in type 185) as the second step. After amplification, PCR products were separated on 2% agarose gel and stained with ethidium bromide. The products were further confirmed by Southern blot hybridisation with probes (positions 141-180 in type 16and 151-190 in type 18). HPV DNA was detected in nine of the cervical cancers, and when we examined negative lymph nodes from these patients by nested PCR we found HPV DNA in the lymph nodes of five of them; in all five cases HPV type in lymph nodes and tumour was the same. Two of these five pateints had had a recurrence (pelvic cavity, lung) but the other three had no recurrences. Since all five patients had had pelvic radiotherapy after radical hysterectomy and lymphadenectomy the three patients may have been protected from recurrence. These findings show that nested PCR is a very useful method to evaluate early lymph node involvement retrospectively in HPVpositive cases, and we recommend it for the detection of micrometastasis as well as the evaluation of prognosis.
Department of Obstetrics and Gynaecology and Virus Laboratory, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine,
Nagoya 466, Japan
AKIHIRO NAWA YUKIHIRO NISHIYAMA FUMITAKA KIKKAWA OSAMU KURAUCHI SETSUKO GOTO NOBUHIKO SUGANUMA SIGEHIKO MIZUTANI YUTAKA TOMODA
1. Linda MS, Thomas WB, Elvio GS, et al. Prognostic factor in stage Ib squamous cervical cancer patients with low risk for recurrence. Obstet Gynecol 1991; 77: 271-75. 2. Fuchs PG, Girardi F, Pfister H. Human papillomavirus 16 DNA in cervical cancers and in lymph nodes of cervical cancer patients: a diagnostic marker for early metastasis? Int J Cancer 1989; 43: 41-44. 3. Yoshikawa H, Kawana T, Kitagawa K, et al. Amplification and typing of multiple cervical cancer-associated human papillomavirus DNAs using a single pair of primers. Int J Cancer 1990; 45: 990-92. 4. Seedorf K, Krämmer G, Durst M, et al. Human papillomavirus type 16 DNA sequence. Virology 1985; 145: 181-85. 5. Cole ST, Danos O. Nucleotide sequence and comparative analysis of the human papillomaviruses type 18 genome. J Mol Biol 1987; 193: 599-608.
Anaphylactoid response to intravenous acetylcysteine SIR,-Intravenous acetylcysteine (NAC) is the treatment of choice for a substantial paracetamol (acetaminophen) overdose. An anaphylactoid response to intravenous NAC has been described, usually beginning within 20 min of the start of treatment and resolving rapidly when treatment stops. It consists of one or more features of anaphylaxis, including bronchospasm, angio-oedema, rash, pruritus, and hypotension. By pooling reports to the UK National Poisons Information Service and the manufacturer (Duncan Flockhart), we compiled features of 38 cases in which this type of reaction developed after correct administration of the drug and we also collected 15 cases where an overdose had been given in error.’ The frequency of the anaphylactoid response was estimated at between 03% and 3% of courses of NAC correctly administered, whereas 11of the 15 cases who were given more than the recommended dose (often ten times too much!) had an anaphylactoid response. Hypotension was a much more prominent feature in patients given higher doses of NAC (6/11 reacting adversely to overdose vs 6/38). Conversely, features more specific to immune system activation (pruritus, angio-oedema, bronchospasm) tended to predominate in patients with an anaphylactoid response to conventional dosage. The hypotensive response to NAC may be distinct from the anaphylactoid response. It seems likely that this reaction is dose-dependent. While studying vasodilator mechanisms involving the vascular endothelium we investigated the action of NAC on isolated human and rat resistance arteries (internal diameter 273-391 )m). After contraction with 10 pmol/1 noradrenaline, cumulative addition of 01to 30 mmol/1 NAC induced a concentration-dependent relaxation (figure); the median concentration of NAC causing
