Volume 165 Number 2
viding instruction for Kegel exercises, I hope that our groups' efforts, under the primary direction of Dr. Jean Wyman, will allow us to answer that question, but the answer is 4 to 5 years away, I think the worst way to instruct patients is to ask them to interrupt the urinary stream repeatedly during micturition, I have three primary aversions to this timehonored technique, First, many women, and most incontinent women, cannot interrupt the stream, and asking them to do what they are unable to do simply accentuates their feelings of helplessness and hopelessness, Second, I don't think that the way you improve the bladder's storage ability is by interfering with its
Kegel exercise performance after verbal instruction
emptying function, A contracting sphincter in the midst of normal micturition is a pathologic condition known as vesicosphincter dyssynergia, and I don't think we should encourage patients to practice this technique, Remember, we can entrain our bladders to adopt bad habits in addition to good ones. Finally, when the completely normal woman interrupts the urine stream, the urethral closure pressure becomes positive and flow stops several seconds before the detrusor muscle contraction is suppressed. This detrusor contraction against a voluntary outlet obstruction several times during every voiding episode may result in reflux and eventually upper tract injury.
Vulvar squamous cell carcinoma and papillomaviruses: Two separate entities? Willie A. Andersen, MD,. Douglas W. Franquemont, MD," John Williams," Peyton T. Taylor, MD,c and Christopher P. Crum, MD··b,c Charlottesville, Virginia Vulvar squamous precancers (vulvar intraepithelial neoplasia) are associated with sexual factors, cigarette smoking, and human papillomaviruses. However, epidemiologic studies of invasive carcinoma of the vulva have produced conflicting evidence for these associations, in part because of a strong association with vulvar inflammatory disease (dystrophies) in older women. We analyzed a series of 42 vulvar invasive carcinomas for papillomavirus nucleic acids by deoxyribonucleic acid-deoxyribonucleic acid in situ hybridization and correlated their presence with age, smoking history, and morphologic type. The carcinomas were divided into well-differentiated, moderately and poorly differentiated, and intraepithelial-like growth patterns, the latter composed of nests of invasive neoplastic epithelium with preserved cell polarity, similar to intraepithelial disease. Of the lesions studied, 28% were human papillomavirus deoxyribonucleic acid-positive. Intraepithelial-like neoplasms segregated in women with a younger mean age (64 versus 73 years) than that of women with conventional squamous cell carcinoma and they more frequently had a history of Cigarette smoking (88% versus 28%). Moreover, intraepithelial-like lesions contained human papillomavirus nucleic acids more frequently (67% versus 13%) when analyzed by in situ hybridization. These observations confirm the diverse nature of vulvar squamous cell carcinoma and may explain in part why conflicting results are obtained from studies investigating the role of sexual and viral factors in the geneSis of vulvar cancer. They suggest that many invasive vulvar cancers may not be linked to papillomaviruses. (AM J OBSTET GVNECOL 1991 ;165:329-36.)
Key words: Vulvar carcinoma, human papillomavirus, vulvar intraepithelial neoplasia From the Departments of Pathology,' Microbiology,' Obstetrics and Gynecology," University of Virginia Health Sciences Center. Supported in part by grants from the American Cancer Society (MV395) and the National Cancer Institute. Dr Crum is a recipient of a Physician Scientist Award from the National Institute of Allergy and Infectious Disease (AI00628). Presented at the Fifty-third Annual Meeting of the South Atlantic Association of Obstetricians and Gynecologists, Hot Springs, Virginia, January 27-30,1991. Reprint requests: Christopher P. Crum, MD, Women's and Perinatal Pathology Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. 6/6/30071
Invasive carcinoma of the vulva is a rare disease with an incidence rate approximately one tenth that of its counterpart in the cervix. I, 2 Traditionally the disease has predominated in women in their seventh and eighth decades and is rarely observed in women younger than age 30 years. 2 This pattern of disease has changed slightly in recent years, and investigators have focused on three components of this disease spectrum that may shed light on the increasingly younger age of women seen with vulvar neoplasia. The first is the oc-
329
330 Andersen et al.
August 1991 Am J Obstet Gynecol
Fig. 1. Histologic subgroups studied. A, Well-differentiated keratinizing squamous cell carcinoma of the vulva; B, moderately to poorly differentiated squamous cell carcinoma of the vulva; C, intraepithelial-like squamous cell carcinoma of the vulva. Note the well-circumscribed nesting pattern of the tumor cells, similar to a precursor (vulvar intraepithelial neoplasia) lesion. The adjacent desmoplastic stromal changes (arrows) indicate that this is invasive cancer and not a tangentially sectioned vulvar intraepithelial neoplasia.
currence of vulvar precancel's, or vulvar intraepithelial neoplasia, which are occurring more frequently in younger women and carry some risk of evolving into cancer as a function of increasing age.3-6 The second is the human papiIlomavirus (HPV), which has been increasingly associated with cervical precancerous lesions and linked with vulvar intraepithelial neoplasms as wel1. 7 The third is the subset of squamous carcinomas, which are separated from their noninvasive counterparts by several decades , are infrequently associated with vulvar intraepithelial neoplasia, and appear to be more associated with vulvar inflammatory conditions than with sexually transmitted diseases ."·10 The potential discrepancy between sexual factors and vulvar cancer has been magnified by epidemiologic studies of vulvar carcinoma. Although vulvar neoplasia has been linked to sexual factors by some authors, ll · n and a case-control study of vulvar carcinoma identified preexisting genital warts and smoking as singificant risk factors, II others have found little relationship between sexual factors and invasive vulvar carcinoma. Mabuchi et al. 9 failed to associate eady age of coitus, first mal'riage and multiple miscarriages, or venereal factors with vulvar cancer.9 They observed a relationship to prior history of vulvar inflammation and urogenital cancer, whereas the relationship to prior cervical cancer (six cases of 149) was of borderline significance." Those studies suggest that, from an epidemiologic perspective, vulvar cancer may encompass more than
one distinct group. Additional evidence that vulvar carcinomas have a diverse pathogenesis comes from two morphologic studies that examine the epithelium adjacent to invasive vulvar cancers. Both Buscema et al. 8 and Zaino et al. lO noted that in only approximately one fourth of invasive cancers was there evidence of a classic vulvar intraepithelial neoplasm (carcinoma in situ) in the adjacent epithelium. In contrast, more than one half of cases were associated with epithelial hyperplasia or atrophy and inflammation , with or without cytologic atypia (lichen sclerosis, squamous hyperplasia, atypia).9. 10 These findings suggested that a significant proportion of vulvar carcinomas did not evolve from a long-standing precancerous lesion. It is important that cancer developed in only a fraction of women with chronic inflammatory diseases of the vulva. 14 One recent study" corroborated the above observations, noting a variable detection rate of HPV nucleic acids in vulvar cancer. It is important that these authors" observed a strong relationship between smoking and women with HPV-related vulvar precancel's versus squamous carcinomas, the majority of which had an associated vulvar intraepithelial neoplasia. Moreoever, another study has reported an absence of H PV nucleic acids in a small series of vulvar carcinomas associated with chronic inflammatory vulvar disease (lichen sclerosis) rather than with vulvar intraepithelial neoplasia. 16 From this it would appear that different subsets of cancers exist, and an association with vulvar
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331
Fig. 2. Presence of HPV nucleic acids in intraepithelial-like invasive carcinoma of the vulva. A, Sharply demarcated nests of invasive carcinoma with preserved cellular polarity and basal palisaded nuclei. B, Serial section after hybridization with a biotinylated HPV type 16/18 mixed probe. Nuclear staining is present in the more centrally located superficial epithelial cells (arrows).
intraepithelial neoplasia may identify a group epidemiologically distinct from other invasive cancers. In this study, we analyzed a series of vulvar resections from 42 women with squamous cell carcinoma of the vulva. Our goal was to determine the frequency of detection of HPV nucleic acids and whether this parameter could be related to age, morphologic growth pattern, smoking history, and clinical behavior, We show that HPV nucleic acids are preferentially associated with a specific morphologic subtype of vulvar carcinoma and that this subtype is significantly associated with a younger age population and smoking history visa-vis other squamous cell carcinomas of the vulva. Material and methods
Hypotheses to be tested. We intended to determine the proportion of conventional invasive cancers that were not associated with HPV nucleic acids and whether a subset of such patients would be distinguished by virtue of one or more morphologic, clinical, or behavioral parameters from the patients whose lesions were associated with HPV. Patient selection and clinical data. From January 1978 to December 1987 the pathologic records of all women evaluated and treated for vulvar squamous cell carcinoma at the University of Virginia Health Sciences Center were reviewed, Retrospective chart review was performed and pertinent clinical data abstracted and entered for statistical analysis. Data obtained included age, smoking history, clinical stage of carcinoma, and survival. All histologic material was retrieved where
possible, and cases with paraffin-embedded tissue were selected. Histologic analysis. Cases were segregated into three morphologic categories: (1) Well-differentiated keratinizing (11 cases), (2) moderately and poorly differentiated (19 cases), and (3) intraepithelial-like (12 cases) squamous cell carcinoma. The latter contained areas of invasive neoplasm with a growth pattern similar to that of intraepithelial neoplasia (i.e., the cells were arranged in concentric nesting patterns with peripheral palisaded nuclei; Figs. 1 and 2). Analysis for HPV nucleic acids. Serial sections from each tumor were analyzed for HPV nucleic acids by DNA-DNA in situ hybridization with biotinylated probes (for HPV types 6111, 16118, 31/33/35; VIRATYPE, Life Technologies, Gaithersburg, Md.), Because the goal was to determine whether HPV nucleic acids were present rather than determine specific type, hybridizations were carried out under conditions similar to those recommended by the manufacturer, with posthybridization washes performed at 42° C. Under these conditions, HPV type 16118 DNA probes produced positive hybridization signals with formalinfixed paraffin-embedded HeLa cells, which contain 10 to 20 copies of HPV type 18 DNA. Statistical analysis. Statistical analysis was performed with a p value of 0,05 or less for statistical significance. Ordinal data were analyzed with comparison of median values with the Mann-Whitney U test; ratio data were analyzed with the Student t test; nominal data were analyzed with either X2 or Fisher's exact test. Kaplan-
332
Andersen et al.
August 1991 Am J Obstet Gynecol
Table I. Clinical characteristics correlated with histologic type Squamous cell carcinoma
IL
Age (yr) Mean Median Range Gravidity Median Range Race White Black Smoking Yes No Not known
Total
63.5 64 44-82
72.3 73 48-86
70.8 72 53-90
69.4 70 44-90
2 0-8
3 1-9
2 0-7
2 0-9
8 4
10 1
18 1
36 6
7 1 4
2 6 3
3 7 9
12 14 16
IL, Intraepithelial-like squamous cell carcinoma; WDK, welldifferentitated keratinizing squamous cell carcinoma; MP, moderately to poorly differentiated squamous cell carcinoma.
Meier actuarial survival curves were compared with the Z scores. Results
Clinical data. Forty-two cases were selected on the basis of the availability of material for histologic and viral analysis. Thirty-six were white and six black, and the ages ranged from 44 to 90 years with a mean of 69.1 ± 11.3 years and a median of 70 years. Gravidity ranged from 0 to 9 with a median of 3 (Table I). From chart review it could be determined that 12 of the 42 women had a history of current or past tobacco use, with 14 known definitely to have never smoked. The smoking history of the remaining 16 patients could not be determined (Table I). Tumor stage at initial presentation was as follows: International Federation of Gynecology and Obstetrics classification (FICO) stage I, eight patients; stage II, 13 patients; stage III, 15 patients; and stage IV, six patients. Thirty-two women were treated by surgery alone, three by a combination of surgery and radiation, and the remaining seven by radiation alone (Table II). Patients treated by surgery alone included six with radical vulvectomy, two with radical vulvectomy and unilateral groin node dissection, 22 with radical vulvectomy and bilateral groin node dissection, one with radical vulvectomy and abdominoperineal resection of the rectum and perineum, and one with radical vulvectomy and abdominoperineal resection ofthe rectum and perineum and unilateral groin node dissection (Table II).
Of the 28 patients with either unilateral or bilateral groin node dissections (with or without irradiation), 17 were found to have negative lymph nodes, seven had positive ipsilateral groin nodes, three had positive bilateral groin nodes, and one had a positive retroperitoneal (Cloquet's) node. The adjusted actuarial survival rate for the 42 patients at 5 years was 81.4% (±6.4%). The FIGO stage I patients had an adjusted actuarial survival rate of 100% at 5 years, with 91.7% (± 7.9%),72.2% (± 11.8%), and 60.0% (± 21. 9%), adjusted actuarial survival rates for stages II, III, and IV, respectively (Fig. 3). Histologic analysis. Of the 42 cases, 11 were welldifferentiated keratinized tumors, 19 were moderately to poorly differentiated, and 12 were intraepitheliallike. Coexisting vulvar intraepithelial neoplasia was found in none, none, and 33% of the above groups, respectively. Clinical features that correlate with these subtypes are summarized in Table I. Patients in the intraepithelial-like subgroup were significantly younger (p == 0.04) than the other groups. In addition, a significantly higher proportion of the intraepitheIiallike subgroup reported a history of smoking than that of the other two groups (p = 0.01). Race and gravidity were not associated significantly with histologic subtype. HPV DNA analysis. Twelve of 42 cases were positive for the presence of HPV DNA. Although the precise HPV type could not be determined because of the low stringency of hybridization, the strongest signals in each case were obtained with the HPV type 16118 or HPV type 31/33/35 probes or both. The association of HPV DNA with histologic subtype is summarized in Table III and illustrated in Fig. 2. The intraepitheliallike carcinomas were significantly associated with HPV DNA (p = 0.002) vis-a-vis the other types. The likelihood of a lesion being HPV -positive was also associated with higher gravidity (p == 0.05) but not with a younger age (p = 0.2). There was no correlation between survival and HPV DNA positivity. There was, however, bias in the groups because there was a statistically significant unequal distribution of treatment types between the HPV-positive and HPV-negative patients. Ninety percent (27/30) of the HPV-negative cases were treated by surgery alone in contrast with 6 of 12 (50%) HPV-positive cases that were treated with either radiation and surgery or radiation alone (p == 0.01). This bias was also reflected in an unequal distribution of treatment types as associated with histologic subgroup. Although 26 of 30 (86%) of the cases of well-differentiated keratinizing or moderately to poorly differentiated tumors were treated with surgery alone, only 6 of 12 (50%) of the intra-
Vulvar squamous cell carcinoma and papillomaviruses
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333
%NED
40
20 O~--------~--------r-------~r-------~--------~
o
2
5
4
3
YEARS STAGE I (N
= 8)
ST AGE IV (N = 6)
STAGE II (N
= 13)
STAGE III (N = 15)
ALL (N = 42)
Fig. 3. Actuarial adjusted survival squamous cancer of the vulva, University of Virginia Health
Sciences Center.
Table II. Clinical correlation stage versus treatment Stage Treatment
I
II
Radical vulvectomy Radical vulvectomy, unilateral groin node dissection Radical vulvectomy, bilateral groin node dissection Abdominoperineal resection of rectum, radical vulvectomy Abdominoperineal resection of rectum, radical vulvectomy, unilateral groin node dissection External beam irradiation therapy and radical vulvectomy, bilateral groin node dissection Radical vulvectomy, node biopsy, external beam irradiation therapy Radiation therapy only
2 2
4
4
9
TOTAL
epithelial-like group were managed exclusively b.y surgery. Comment
Although it has been assumed that a large proportion of invasive cervical carcinomas evolve from intraepithelial precursor lesions, the data in support of this mechanism for the development of vulvar cancer have been conflicting. Most compelling has been the marked disparity in the mean ages of women with vulvar intraepitheJial neoplasia versus invasive cancer. [·3 Moreover, several studies have reported that the majority of vulvar carcinomas are not associated temporally or topographically with typical precursor lesions such as
III
IV
Total
6
2 22
9
2
8
13
5 15
2 6
7
42
vulvar intraepithelial neoplasia, but with hyperplastic or inflammatory epithelial changes (dystrophies) with various grades of cytologic atypia. B. [0 Because papillomaviruses and sexual factors have been closely associated with vulvar intraepithelial neoplasia, attention has naturally focused on the relationship between these factors and invasive cancer. However, both sexual and nonsexual causes have been proposed. For example, Mabuchi et al. 9 observed little relationship between invasive vulvar carcinoma and a history of condylomata. In contrast, these authors" found a relationship between vulvar carcinoma and occupational exposures, caffeine consumption, and smoking in younger women. A different perspective
334 Andersen et al.
August 1991 Am J Obstet Gyneco1
Table III. Correlation of histology with clinical and viral data Ever smoked Histologic group
WDK MP IL
No.
Mean age (yr)
II 19 12
73 72 64*
No.
2/8 3110 7/8
I
%
HPV+ (%)
25 30 88*
9 16 67*
WDK, Well-differentiated keratinizing squamous cell carcinoma; MP, moderately to poorly differentiated squamous cell carcinoma; fL, intraepithelial-like squamous cell carcinoma; HPV +, human papilloma virus-DNA positive. *Significant (p < 0.05, Student's t test).
was put forth by Brinton et al. lI who reported a powerful association between a history of condylomata or smoking and both vulvar intraepithelial neoplasia and invasive vulvar carcinoma. These authors lI concluded that sexually transmitted agents occupied a central place in the pathogenesis of vulvar cancer. A simple resolution of the disparity between the conclusions provided by the studies described here is problematic. However, it is noteworthy that the population studied by Mabuchi et al. 9 included a higher proportion of women older than 55 years of age than that of Brinton et al. l1 Thus it is likely that Mabuchi et al." studied a population of which a larger proportion did not have associated intraepithelial disease. Indirect support for this is the much stronger association of vulvar neoplasia with smoking by Brinton et al. l1 in that this risk factor has been associated both with vulvar intraepithelial neoplasia and a younger age population. The association between papillomaviruses and vulvar neoplasia has likewise been inconsistent and controversial. Although HPV type 16 nucleic acids have been strongly associated with intraepithelial neoplasia in several studies, the association between HPV nucleic acids and invasive carcinoma is weak. 7.';.'7 Twiggs'7 observed that only 17% of invasive vulvar carcinomas were positive by in situ hybridization, similar to the results of this study. A possible link between HPV positivity and smoking in vulvar carcinoma has been observed. '5 Combined with the strong association between smoking and vulvar intraepithelial neoplasia, this suggests that one population of vulvar cancers may be identified by a strong smoking history, coexisting vulvar precursors, a younger mean age, and the presence of papillomavirus nucleic acids, implying in turn the role of sexual factors. This study supported this with the additional observation that most vulvar carcinomas associated with HPV nucleic acids were recognizable by the presence of certain morphologic features that resembled intraepithelial disease, even when a coexisting precursor (VIN) lesion was not identified. In contrast, evidence has begun to accumulate that cancers that are not associated with vulvar intraepithelial neoplasia, and specifically those associated with vul-
val' inflammatory conditions such as lichen sclerosis, are infrequently associated with HPV nucleic acids. 16 Combined, these data strongly suggest at least two different pathogenetic mechanisms for vulvar squamous cell carcinoma. It remains to be confirmed whether pathologic analysis will segregate the majority of HPV-related vulvar neoplasms from those that are not. However, the observations in this report combined with the negative association between coexisting lichen sclerosis and HPV-related carcinomas support the use of both viral and histologic parameters in epidemiologic studies of vulvar carcinomas. Such studies may be instrumental in placing the role of viral sexual factors in proper perspective and in targeting other risk factors associated with a large proportion of vulvar carcinomas in older women. REFERENCES 1. Zaino RJ. Carcinoma of the vulva, urethra, and Bartholin's glands. In: Wilkinson Ej, ed. Pathology of the vulva and vagina. New York: Churchill Livingstone, 1987:II953. 2. Henson D, Tarone R. An epidemiologic study of cancer of the cervix, vagina, and vulva based upon the Third National Cancer Survey in the United States. AMj OBSTET GYNECOL 1977; 129:525-32. 3. Friedrich EG, Wilkinson Ej, Fu YS. Carcinoma in situ of the vulva: a continuing challenge. AM j OBSTET GYNECOL 1980; 136:830. 4. jones RW, McClean MR. Carcinoma in situ of the vulva: a review of 31 treated and five untreated cases. Obstet Gynecol 1986;68:499. 5. Crum CP, Liskow A, Petras P, Keng WC, Frick HC. Vulvar intraepithelial neoplasia (severe atypia and carcinoma in situ): a clinicopathologic analysis of 41 cases. Cancer 1984;54: 1429-34. 6. Chafe W, Richards A, Morgan L, et al. Unrecognized invasive carcinoma vulvar intraepithelial neoplasia (VIN). Gynecol Oncol 1980;31:154. 7. Gross G, Hagedorn M, Ikenberg H, et al. Bowenoid papulosis: presence of human papillomavirus (HPV) structural antigens and of HPV 16-related DNA sequences. Arch Dermatol 1985;121:858. 8. Buscemaj, Sternj, WoodruffjD. The significance of the histologic alterations adjacent to invasive vulvar carcinoma. AMj OBSTET GYNECOL 1980;137:902. 9. Mabuchi K, Bross DS, Kessler II. Epidemiology of cancer of the vulva. Cancer 1985;55:1843-8. 10. Zaino Rj, Husseinzadeh N, Nahhas W, et al. Epithelial
Vulvar squamous cell carcinoma and papillomaviruses
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11. 12. 13. 14. 15. 16.
17.
like lesions) and that the intraepithelial-like subtype is associated with a younger mean age and a positive smoking history. The results of this study are only as good as the sensitivity of the assay for the HPV DNA. The in situ hybridization assay used to detect HPV DNA in this article is the only method that allows precise microscopic detection of HPV nucleic acids within cells. The major drawback of the in situ test is its reduced sensitivity, relative to Southern blot or dot blot hybridization in the detection of latently HPV -infected tumors. Latently infected tumors or adjacent normal epithelium would likely receive a negative score in the in situ assay. Therefore the absence of HPV DNA in vulvar squamous cell carcinoma does not exclude an HPV etiologic factor. It is also important to note that negative HPV DNA results only mean that the test is negative for the HPV types evaluated. The morphologic interpretation and proposed histologic criteria of intraepithelial-like invasive squamous cell carcinoma of the vulva in this article includes nests of invasive neoplastic epithelium with preserved cell polarity, similar to intraepithelial disease. This definition seems to create some confusion and uncertainty for the following reasons: (1) Vulvar intraepithelial neoplasia represents a continuous morphologic spectrum of epithelial cellular abnormality and maturation. Vulvar intraepithelial neoplasia is divided into types I, II, and III. Do the authors mean the intraepitheliallesion mimics vulvar intraepithelial neoplasia type III? It would seem difficult to classify the intraepithelial-like invasive squamous cell carcinoma into a separate distinct morphologic entity on the basis of their proposed criteria. (2) It is also known that invasive squamous cell carcinoma of the vulva is composed of a heterogeneous morphologic pattern of neoplasia, even within the same tumor. Focal areas of vulvar invasive squamous cell carcinoma may fit into the author's criteria of intraepithelial-like growth pattern, but other areas of the same
alterations in proximity to invasive squamous carcinoma of the vulva. lnt j Gynecol Pathol 1982; 1: 173. Brinton LA, Nasca PC, Mallin K, Baptiste MS, Wilbanks GD, Richart RM. Case-control study of cancer of the vulva. Obstet Gynecol 1990;75:859. japaze H, Garcia-Bunuel R, WoodruffjD. Primary vulvar neoplasia: a review of in situ and invasive carcinoma, 1935-1972. Obstet Gynecol 1977;49;404-11. Franklin EW, Rutledge FD. Epidemiology of epidermoid carcinoma of the vulva. Obstet Gynecol 1972;39: 165-72. McAdams Aj, Kistner RW. The relationship of chronic vulvar disease, leukoplakia, and carcinoma in situ to carcinoma of the vulva. Cancer 1958; 11:740-75. Liao SY, Wilczynski SP, Bloss jD, Peake M, Berman M. Clinical and histopathologic features of vulvar carcinomas analyzed for HPV status. Lab Invest 1990;335A. Neill SM, Lessanaleibowitch M, Pelisse M, Moyalbarracco M. Lichen sclerosis, invasive squamous cell carcinoma, and human papillomavirus. AM j OBSTET GYNECOL 1990; 162: 1633-4. Twiggs L, Okagaki T, Clark B, Fukushima M, Ostrow R, Faras A. A clinical, histopathologic,and molecular biologic investigation of vulvar intraepithelial neoplasia. lnt j Gynecol Pathol 1988;7:48.
Discussion T, GIVEN, JR" Norfolk, Virginia. Dr. Andersen et al. address the association of the HPV and invasive squamous cell carcinoma of the vulva and correlate this with age, smoking history, and morphology. For my comments I am indebted to the expertise of two of my colleagues at the Eastern Virginia Medical School, Dr. Ken Somers, Professor of Microbiology and Immunology, and Dr. J eng G. Hsiu, Associate Professor of Pathology. The primary goal of the authors was to determine by the use of in situ hybridization the frequency of association of the HPV and the correlation of this occurrence with specific morphologic types of vulvar cancer as well as age and smoking history. The evidence presented is interpreted to show that HPV DNA is preferentially associated with a specific morphologic subtype of vulvar carcinoma (intraepithelialDR. FRED
Jt
NORMAL CELLS PAPILLOMAVIRUS.. or. INFECTIOY. M .UTAGENS Z~·::~~!,!~c' j::O. ~.IMMUNE t •• H[RP'ES SI"~LEX ~'8 ~CIN I :::0 ! ~ VIRUS INFECTIONS. \ . ~ ./... SYSTEM S"OKINOI UJ : rn ./
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INVASIVE CARCINOMA Fig. 1. Proposed model for synergistic effects in human genital cancer. (From zur Hausen H. Lancet 1982;2:1371. By permission.)
336 Andersen et al.
tumor may show a totally different morphologic growth pattern. How will the authors clarify such squamous cell carcinomas that comprise different patterns? The data on smoking cause me some concern and that concern is confounding. This was well pointed out in an editorial. I There may be many other initiating events or promoting factors in the cause of vulvar cancer, just as there are in carcinoma of the cervix. This is certainly well illustrated in the model proposed by Dr. zur Hausen (Fig. 1).2 The author's data certainly seem to show some association between the papillomavirus and intraepithelial-like carcinoma of the vulva. This seems to occur at a somewhat younger age; however, it remains to be seen whether survival is affected by being able to isolate this particular subgroup of patients. Is the biologic behavior of the intraepithelial-like lesion described by the authors different? This study should be repeated with an even more sensitive technique, specifically, polymerase chain reaction amplification of HPV DNA with primers that recognize highly conserved regions within the HPV genome. 3 Until these data are presented, negative results should be interpreted with caution. There should also be adjustment for confounding, such as the sexual activity, number of partners, exposure to passive smoke and caffeine if they want to truly see the effects of smoking per se on the epidemiology of carcinoma of the vulva. We believe this study should be continued in much more detail and with a larger number of patients, perhaps requiring a multiinstitutional protocol to confirm these preliminary results. I have three questions for Dr. Andersen: 1. Do you believe that you will be able to classify the intraepithelial-like invasive squamous cell carcinoma of the vulva in a heterogenous type of neoplasm? 2. Do you believe that with a much larger series the group with the intraepithelial-like growth pattern will have a different survival rate than other invasive squamous cell carcinomas of the vulva? 3. Are you convinced of the significance of the smoking data with a series this small? REFERENCES 1. Layde PM, ed. Smoking and cervical cancer: cause or co-
incidence? JAMA 1989;261:1631. 2. zur Hausen H. Human genital cancer: synergism between two virus infections or synergism between a virus infection and initiating events? Lancet 1982;2:1370. 3. Bauer HM, Ting Y, Greer CE, et al. Genital human papillomavirus infection in female university student as determined by a PCR-based method. JAMA 1991;265:472. DR. MATTHEW O. BURRELL, Atlanta, Georgia. Did the authors have any cases with preexisting or coexisting lichen sclerosis? In Atlanta we have had four patients in whom early invasive carcinoma has developed in preexisting lichen sclerosis.
August 1991 Am J Obstet Gynecol
DR. ANDERSEN (Closing). We looked at the surrounding epithelium in most cases and four of 12 with an intraepithelial-like pattern had associated vulvar intraepithelial neoplasia. Eight of the remaining 28 were associated with some other form of vulva dystrophy. We had one of the 42 in whom an invasive cancer developed while being folowed up for vulvar dystrophy. Therefore I think vulva dystrophies demand careful attention and follow-up. To address Dr. Given's comments and concerns, it is Dr. Crum's belief that the intraepithelial-like pattern can be distinguished by the orientation of the cells in circumscribed nests with retention of cell polarity and palisaded nuclei at the periphery (Figs. 1, C, and 2, A). This arrangement of cells is similar to that seen in vulvar intraepithelial neoplasms, irrespective of grade, but cannot be compared directly with them gradewise because this pattern is being described in invasive carcinomas. In all the intraepithelial-like lesions, this growth pattern was clearly present in an invasive component by virtue of the stromal reaction around the nests (Fig. 1, C). We agree that lesions may be heterogeneous and contain more than one pattern, but the intraepitheliallike pattern was classified separately because of its distinctive appearance. The association of HPV nucleic acids with intraepithelial-like neoplasms may be fortuitous or reflect the fact that invasive carcinomas within this growth pattern are more likely to have derived from vulvar intraepithelial neoplasms. As you know, a high percentage of the latter are known to contain genital papiIIomavirus nucleic acids. Whereas a variety of other techniques have been used to search for HPV DNA in human cells, we believed in this retrospective study that the in situ hybridization technique was the most appropriate because it provided for direct localization of HPV nucleic acids in the cells of the neoplasm. The polymerase chain reaction, although superior in sensitivity, does not establish the location of the viral nucleic acids detected and can be plagued by false-positive results. I would reemphasize that the hybridization conditions used by Dr. Crum permitted detection of not only HPV DNAs identical to those in the probes, but more distantly related HPV types as well, and the sensitivity was such that as few as 10 to 20 copies of HPV DNA could be detected in an individual cell. Our smoking data were based purely on chart review and in many instances we could not tell whether the patients had ever smoked. The issue of smoking exposure is quite complex and although in our small series with small numbers we can show a statistical association between smoking and intraepithelial-like histologic pattern as well as the presence of HPV, clearly this is only a preliminary observation that requires much more sophisticated epidemiologic study to confirm this potential association.
viding instruction for Kegel exercises, I hope that our groups' efforts, under the primary direction of Dr. Jean Wyman, will allow us to answer that question, but the answer is 4 to 5 years away, I think the worst way to instruct patients is to ask them to interrupt the urinary stream repeatedly during micturition, I have three primary aversions to this timehonored technique, First, many women, and most incontinent women, cannot interrupt the stream, and asking them to do what they are unable to do simply accentuates their feelings of helplessness and hopelessness, Second, I don't think that the way you improve the bladder's storage ability is by interfering with its
Kegel exercise performance after verbal instruction
emptying function, A contracting sphincter in the midst of normal micturition is a pathologic condition known as vesicosphincter dyssynergia, and I don't think we should encourage patients to practice this technique, Remember, we can entrain our bladders to adopt bad habits in addition to good ones. Finally, when the completely normal woman interrupts the urine stream, the urethral closure pressure becomes positive and flow stops several seconds before the detrusor muscle contraction is suppressed. This detrusor contraction against a voluntary outlet obstruction several times during every voiding episode may result in reflux and eventually upper tract injury.
Vulvar squamous cell carcinoma and papillomaviruses: Two separate entities? Willie A. Andersen, MD,. Douglas W. Franquemont, MD," John Williams," Peyton T. Taylor, MD,c and Christopher P. Crum, MD··b,c Charlottesville, Virginia Vulvar squamous precancers (vulvar intraepithelial neoplasia) are associated with sexual factors, cigarette smoking, and human papillomaviruses. However, epidemiologic studies of invasive carcinoma of the vulva have produced conflicting evidence for these associations, in part because of a strong association with vulvar inflammatory disease (dystrophies) in older women. We analyzed a series of 42 vulvar invasive carcinomas for papillomavirus nucleic acids by deoxyribonucleic acid-deoxyribonucleic acid in situ hybridization and correlated their presence with age, smoking history, and morphologic type. The carcinomas were divided into well-differentiated, moderately and poorly differentiated, and intraepithelial-like growth patterns, the latter composed of nests of invasive neoplastic epithelium with preserved cell polarity, similar to intraepithelial disease. Of the lesions studied, 28% were human papillomavirus deoxyribonucleic acid-positive. Intraepithelial-like neoplasms segregated in women with a younger mean age (64 versus 73 years) than that of women with conventional squamous cell carcinoma and they more frequently had a history of Cigarette smoking (88% versus 28%). Moreover, intraepithelial-like lesions contained human papillomavirus nucleic acids more frequently (67% versus 13%) when analyzed by in situ hybridization. These observations confirm the diverse nature of vulvar squamous cell carcinoma and may explain in part why conflicting results are obtained from studies investigating the role of sexual and viral factors in the geneSis of vulvar cancer. They suggest that many invasive vulvar cancers may not be linked to papillomaviruses. (AM J OBSTET GVNECOL 1991 ;165:329-36.)
Key words: Vulvar carcinoma, human papillomavirus, vulvar intraepithelial neoplasia From the Departments of Pathology,' Microbiology,' Obstetrics and Gynecology," University of Virginia Health Sciences Center. Supported in part by grants from the American Cancer Society (MV395) and the National Cancer Institute. Dr Crum is a recipient of a Physician Scientist Award from the National Institute of Allergy and Infectious Disease (AI00628). Presented at the Fifty-third Annual Meeting of the South Atlantic Association of Obstetricians and Gynecologists, Hot Springs, Virginia, January 27-30,1991. Reprint requests: Christopher P. Crum, MD, Women's and Perinatal Pathology Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. 6/6/30071
Invasive carcinoma of the vulva is a rare disease with an incidence rate approximately one tenth that of its counterpart in the cervix. I, 2 Traditionally the disease has predominated in women in their seventh and eighth decades and is rarely observed in women younger than age 30 years. 2 This pattern of disease has changed slightly in recent years, and investigators have focused on three components of this disease spectrum that may shed light on the increasingly younger age of women seen with vulvar neoplasia. The first is the oc-
329
330 Andersen et al.
August 1991 Am J Obstet Gynecol
Fig. 1. Histologic subgroups studied. A, Well-differentiated keratinizing squamous cell carcinoma of the vulva; B, moderately to poorly differentiated squamous cell carcinoma of the vulva; C, intraepithelial-like squamous cell carcinoma of the vulva. Note the well-circumscribed nesting pattern of the tumor cells, similar to a precursor (vulvar intraepithelial neoplasia) lesion. The adjacent desmoplastic stromal changes (arrows) indicate that this is invasive cancer and not a tangentially sectioned vulvar intraepithelial neoplasia.
currence of vulvar precancel's, or vulvar intraepithelial neoplasia, which are occurring more frequently in younger women and carry some risk of evolving into cancer as a function of increasing age.3-6 The second is the human papiIlomavirus (HPV), which has been increasingly associated with cervical precancerous lesions and linked with vulvar intraepithelial neoplasms as wel1. 7 The third is the subset of squamous carcinomas, which are separated from their noninvasive counterparts by several decades , are infrequently associated with vulvar intraepithelial neoplasia, and appear to be more associated with vulvar inflammatory conditions than with sexually transmitted diseases ."·10 The potential discrepancy between sexual factors and vulvar cancer has been magnified by epidemiologic studies of vulvar carcinoma. Although vulvar neoplasia has been linked to sexual factors by some authors, ll · n and a case-control study of vulvar carcinoma identified preexisting genital warts and smoking as singificant risk factors, II others have found little relationship between sexual factors and invasive vulvar carcinoma. Mabuchi et al. 9 failed to associate eady age of coitus, first mal'riage and multiple miscarriages, or venereal factors with vulvar cancer.9 They observed a relationship to prior history of vulvar inflammation and urogenital cancer, whereas the relationship to prior cervical cancer (six cases of 149) was of borderline significance." Those studies suggest that, from an epidemiologic perspective, vulvar cancer may encompass more than
one distinct group. Additional evidence that vulvar carcinomas have a diverse pathogenesis comes from two morphologic studies that examine the epithelium adjacent to invasive vulvar cancers. Both Buscema et al. 8 and Zaino et al. lO noted that in only approximately one fourth of invasive cancers was there evidence of a classic vulvar intraepithelial neoplasm (carcinoma in situ) in the adjacent epithelium. In contrast, more than one half of cases were associated with epithelial hyperplasia or atrophy and inflammation , with or without cytologic atypia (lichen sclerosis, squamous hyperplasia, atypia).9. 10 These findings suggested that a significant proportion of vulvar carcinomas did not evolve from a long-standing precancerous lesion. It is important that cancer developed in only a fraction of women with chronic inflammatory diseases of the vulva. 14 One recent study" corroborated the above observations, noting a variable detection rate of HPV nucleic acids in vulvar cancer. It is important that these authors" observed a strong relationship between smoking and women with HPV-related vulvar precancel's versus squamous carcinomas, the majority of which had an associated vulvar intraepithelial neoplasia. Moreoever, another study has reported an absence of H PV nucleic acids in a small series of vulvar carcinomas associated with chronic inflammatory vulvar disease (lichen sclerosis) rather than with vulvar intraepithelial neoplasia. 16 From this it would appear that different subsets of cancers exist, and an association with vulvar
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Fig. 2. Presence of HPV nucleic acids in intraepithelial-like invasive carcinoma of the vulva. A, Sharply demarcated nests of invasive carcinoma with preserved cellular polarity and basal palisaded nuclei. B, Serial section after hybridization with a biotinylated HPV type 16/18 mixed probe. Nuclear staining is present in the more centrally located superficial epithelial cells (arrows).
intraepithelial neoplasia may identify a group epidemiologically distinct from other invasive cancers. In this study, we analyzed a series of vulvar resections from 42 women with squamous cell carcinoma of the vulva. Our goal was to determine the frequency of detection of HPV nucleic acids and whether this parameter could be related to age, morphologic growth pattern, smoking history, and clinical behavior, We show that HPV nucleic acids are preferentially associated with a specific morphologic subtype of vulvar carcinoma and that this subtype is significantly associated with a younger age population and smoking history visa-vis other squamous cell carcinomas of the vulva. Material and methods
Hypotheses to be tested. We intended to determine the proportion of conventional invasive cancers that were not associated with HPV nucleic acids and whether a subset of such patients would be distinguished by virtue of one or more morphologic, clinical, or behavioral parameters from the patients whose lesions were associated with HPV. Patient selection and clinical data. From January 1978 to December 1987 the pathologic records of all women evaluated and treated for vulvar squamous cell carcinoma at the University of Virginia Health Sciences Center were reviewed, Retrospective chart review was performed and pertinent clinical data abstracted and entered for statistical analysis. Data obtained included age, smoking history, clinical stage of carcinoma, and survival. All histologic material was retrieved where
possible, and cases with paraffin-embedded tissue were selected. Histologic analysis. Cases were segregated into three morphologic categories: (1) Well-differentiated keratinizing (11 cases), (2) moderately and poorly differentiated (19 cases), and (3) intraepithelial-like (12 cases) squamous cell carcinoma. The latter contained areas of invasive neoplasm with a growth pattern similar to that of intraepithelial neoplasia (i.e., the cells were arranged in concentric nesting patterns with peripheral palisaded nuclei; Figs. 1 and 2). Analysis for HPV nucleic acids. Serial sections from each tumor were analyzed for HPV nucleic acids by DNA-DNA in situ hybridization with biotinylated probes (for HPV types 6111, 16118, 31/33/35; VIRATYPE, Life Technologies, Gaithersburg, Md.), Because the goal was to determine whether HPV nucleic acids were present rather than determine specific type, hybridizations were carried out under conditions similar to those recommended by the manufacturer, with posthybridization washes performed at 42° C. Under these conditions, HPV type 16118 DNA probes produced positive hybridization signals with formalinfixed paraffin-embedded HeLa cells, which contain 10 to 20 copies of HPV type 18 DNA. Statistical analysis. Statistical analysis was performed with a p value of 0,05 or less for statistical significance. Ordinal data were analyzed with comparison of median values with the Mann-Whitney U test; ratio data were analyzed with the Student t test; nominal data were analyzed with either X2 or Fisher's exact test. Kaplan-
332
Andersen et al.
August 1991 Am J Obstet Gynecol
Table I. Clinical characteristics correlated with histologic type Squamous cell carcinoma
IL
Age (yr) Mean Median Range Gravidity Median Range Race White Black Smoking Yes No Not known
Total
63.5 64 44-82
72.3 73 48-86
70.8 72 53-90
69.4 70 44-90
2 0-8
3 1-9
2 0-7
2 0-9
8 4
10 1
18 1
36 6
7 1 4
2 6 3
3 7 9
12 14 16
IL, Intraepithelial-like squamous cell carcinoma; WDK, welldifferentitated keratinizing squamous cell carcinoma; MP, moderately to poorly differentiated squamous cell carcinoma.
Meier actuarial survival curves were compared with the Z scores. Results
Clinical data. Forty-two cases were selected on the basis of the availability of material for histologic and viral analysis. Thirty-six were white and six black, and the ages ranged from 44 to 90 years with a mean of 69.1 ± 11.3 years and a median of 70 years. Gravidity ranged from 0 to 9 with a median of 3 (Table I). From chart review it could be determined that 12 of the 42 women had a history of current or past tobacco use, with 14 known definitely to have never smoked. The smoking history of the remaining 16 patients could not be determined (Table I). Tumor stage at initial presentation was as follows: International Federation of Gynecology and Obstetrics classification (FICO) stage I, eight patients; stage II, 13 patients; stage III, 15 patients; and stage IV, six patients. Thirty-two women were treated by surgery alone, three by a combination of surgery and radiation, and the remaining seven by radiation alone (Table II). Patients treated by surgery alone included six with radical vulvectomy, two with radical vulvectomy and unilateral groin node dissection, 22 with radical vulvectomy and bilateral groin node dissection, one with radical vulvectomy and abdominoperineal resection of the rectum and perineum, and one with radical vulvectomy and abdominoperineal resection ofthe rectum and perineum and unilateral groin node dissection (Table II).
Of the 28 patients with either unilateral or bilateral groin node dissections (with or without irradiation), 17 were found to have negative lymph nodes, seven had positive ipsilateral groin nodes, three had positive bilateral groin nodes, and one had a positive retroperitoneal (Cloquet's) node. The adjusted actuarial survival rate for the 42 patients at 5 years was 81.4% (±6.4%). The FIGO stage I patients had an adjusted actuarial survival rate of 100% at 5 years, with 91.7% (± 7.9%),72.2% (± 11.8%), and 60.0% (± 21. 9%), adjusted actuarial survival rates for stages II, III, and IV, respectively (Fig. 3). Histologic analysis. Of the 42 cases, 11 were welldifferentiated keratinized tumors, 19 were moderately to poorly differentiated, and 12 were intraepitheliallike. Coexisting vulvar intraepithelial neoplasia was found in none, none, and 33% of the above groups, respectively. Clinical features that correlate with these subtypes are summarized in Table I. Patients in the intraepithelial-like subgroup were significantly younger (p == 0.04) than the other groups. In addition, a significantly higher proportion of the intraepitheIiallike subgroup reported a history of smoking than that of the other two groups (p = 0.01). Race and gravidity were not associated significantly with histologic subtype. HPV DNA analysis. Twelve of 42 cases were positive for the presence of HPV DNA. Although the precise HPV type could not be determined because of the low stringency of hybridization, the strongest signals in each case were obtained with the HPV type 16118 or HPV type 31/33/35 probes or both. The association of HPV DNA with histologic subtype is summarized in Table III and illustrated in Fig. 2. The intraepitheliallike carcinomas were significantly associated with HPV DNA (p = 0.002) vis-a-vis the other types. The likelihood of a lesion being HPV -positive was also associated with higher gravidity (p == 0.05) but not with a younger age (p = 0.2). There was no correlation between survival and HPV DNA positivity. There was, however, bias in the groups because there was a statistically significant unequal distribution of treatment types between the HPV-positive and HPV-negative patients. Ninety percent (27/30) of the HPV-negative cases were treated by surgery alone in contrast with 6 of 12 (50%) HPV-positive cases that were treated with either radiation and surgery or radiation alone (p == 0.01). This bias was also reflected in an unequal distribution of treatment types as associated with histologic subgroup. Although 26 of 30 (86%) of the cases of well-differentiated keratinizing or moderately to poorly differentiated tumors were treated with surgery alone, only 6 of 12 (50%) of the intra-
Vulvar squamous cell carcinoma and papillomaviruses
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333
%NED
40
20 O~--------~--------r-------~r-------~--------~
o
2
5
4
3
YEARS STAGE I (N
= 8)
ST AGE IV (N = 6)
STAGE II (N
= 13)
STAGE III (N = 15)
ALL (N = 42)
Fig. 3. Actuarial adjusted survival squamous cancer of the vulva, University of Virginia Health
Sciences Center.
Table II. Clinical correlation stage versus treatment Stage Treatment
I
II
Radical vulvectomy Radical vulvectomy, unilateral groin node dissection Radical vulvectomy, bilateral groin node dissection Abdominoperineal resection of rectum, radical vulvectomy Abdominoperineal resection of rectum, radical vulvectomy, unilateral groin node dissection External beam irradiation therapy and radical vulvectomy, bilateral groin node dissection Radical vulvectomy, node biopsy, external beam irradiation therapy Radiation therapy only
2 2
4
4
9
TOTAL
epithelial-like group were managed exclusively b.y surgery. Comment
Although it has been assumed that a large proportion of invasive cervical carcinomas evolve from intraepithelial precursor lesions, the data in support of this mechanism for the development of vulvar cancer have been conflicting. Most compelling has been the marked disparity in the mean ages of women with vulvar intraepitheJial neoplasia versus invasive cancer. [·3 Moreover, several studies have reported that the majority of vulvar carcinomas are not associated temporally or topographically with typical precursor lesions such as
III
IV
Total
6
2 22
9
2
8
13
5 15
2 6
7
42
vulvar intraepithelial neoplasia, but with hyperplastic or inflammatory epithelial changes (dystrophies) with various grades of cytologic atypia. B. [0 Because papillomaviruses and sexual factors have been closely associated with vulvar intraepithelial neoplasia, attention has naturally focused on the relationship between these factors and invasive cancer. However, both sexual and nonsexual causes have been proposed. For example, Mabuchi et al. 9 observed little relationship between invasive vulvar carcinoma and a history of condylomata. In contrast, these authors" found a relationship between vulvar carcinoma and occupational exposures, caffeine consumption, and smoking in younger women. A different perspective
334 Andersen et al.
August 1991 Am J Obstet Gyneco1
Table III. Correlation of histology with clinical and viral data Ever smoked Histologic group
WDK MP IL
No.
Mean age (yr)
II 19 12
73 72 64*
No.
2/8 3110 7/8
I
%
HPV+ (%)
25 30 88*
9 16 67*
WDK, Well-differentiated keratinizing squamous cell carcinoma; MP, moderately to poorly differentiated squamous cell carcinoma; fL, intraepithelial-like squamous cell carcinoma; HPV +, human papilloma virus-DNA positive. *Significant (p < 0.05, Student's t test).
was put forth by Brinton et al. lI who reported a powerful association between a history of condylomata or smoking and both vulvar intraepithelial neoplasia and invasive vulvar carcinoma. These authors lI concluded that sexually transmitted agents occupied a central place in the pathogenesis of vulvar cancer. A simple resolution of the disparity between the conclusions provided by the studies described here is problematic. However, it is noteworthy that the population studied by Mabuchi et al. 9 included a higher proportion of women older than 55 years of age than that of Brinton et al. l1 Thus it is likely that Mabuchi et al." studied a population of which a larger proportion did not have associated intraepithelial disease. Indirect support for this is the much stronger association of vulvar neoplasia with smoking by Brinton et al. l1 in that this risk factor has been associated both with vulvar intraepithelial neoplasia and a younger age population. The association between papillomaviruses and vulvar neoplasia has likewise been inconsistent and controversial. Although HPV type 16 nucleic acids have been strongly associated with intraepithelial neoplasia in several studies, the association between HPV nucleic acids and invasive carcinoma is weak. 7.';.'7 Twiggs'7 observed that only 17% of invasive vulvar carcinomas were positive by in situ hybridization, similar to the results of this study. A possible link between HPV positivity and smoking in vulvar carcinoma has been observed. '5 Combined with the strong association between smoking and vulvar intraepithelial neoplasia, this suggests that one population of vulvar cancers may be identified by a strong smoking history, coexisting vulvar precursors, a younger mean age, and the presence of papillomavirus nucleic acids, implying in turn the role of sexual factors. This study supported this with the additional observation that most vulvar carcinomas associated with HPV nucleic acids were recognizable by the presence of certain morphologic features that resembled intraepithelial disease, even when a coexisting precursor (VIN) lesion was not identified. In contrast, evidence has begun to accumulate that cancers that are not associated with vulvar intraepithelial neoplasia, and specifically those associated with vul-
val' inflammatory conditions such as lichen sclerosis, are infrequently associated with HPV nucleic acids. 16 Combined, these data strongly suggest at least two different pathogenetic mechanisms for vulvar squamous cell carcinoma. It remains to be confirmed whether pathologic analysis will segregate the majority of HPV-related vulvar neoplasms from those that are not. However, the observations in this report combined with the negative association between coexisting lichen sclerosis and HPV-related carcinomas support the use of both viral and histologic parameters in epidemiologic studies of vulvar carcinomas. Such studies may be instrumental in placing the role of viral sexual factors in proper perspective and in targeting other risk factors associated with a large proportion of vulvar carcinomas in older women. REFERENCES 1. Zaino RJ. Carcinoma of the vulva, urethra, and Bartholin's glands. In: Wilkinson Ej, ed. Pathology of the vulva and vagina. New York: Churchill Livingstone, 1987:II953. 2. Henson D, Tarone R. An epidemiologic study of cancer of the cervix, vagina, and vulva based upon the Third National Cancer Survey in the United States. AMj OBSTET GYNECOL 1977; 129:525-32. 3. Friedrich EG, Wilkinson Ej, Fu YS. Carcinoma in situ of the vulva: a continuing challenge. AM j OBSTET GYNECOL 1980; 136:830. 4. jones RW, McClean MR. Carcinoma in situ of the vulva: a review of 31 treated and five untreated cases. Obstet Gynecol 1986;68:499. 5. Crum CP, Liskow A, Petras P, Keng WC, Frick HC. Vulvar intraepithelial neoplasia (severe atypia and carcinoma in situ): a clinicopathologic analysis of 41 cases. Cancer 1984;54: 1429-34. 6. Chafe W, Richards A, Morgan L, et al. Unrecognized invasive carcinoma vulvar intraepithelial neoplasia (VIN). Gynecol Oncol 1980;31:154. 7. Gross G, Hagedorn M, Ikenberg H, et al. Bowenoid papulosis: presence of human papillomavirus (HPV) structural antigens and of HPV 16-related DNA sequences. Arch Dermatol 1985;121:858. 8. Buscemaj, Sternj, WoodruffjD. The significance of the histologic alterations adjacent to invasive vulvar carcinoma. AMj OBSTET GYNECOL 1980;137:902. 9. Mabuchi K, Bross DS, Kessler II. Epidemiology of cancer of the vulva. Cancer 1985;55:1843-8. 10. Zaino Rj, Husseinzadeh N, Nahhas W, et al. Epithelial
Vulvar squamous cell carcinoma and papillomaviruses
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11. 12. 13. 14. 15. 16.
17.
like lesions) and that the intraepithelial-like subtype is associated with a younger mean age and a positive smoking history. The results of this study are only as good as the sensitivity of the assay for the HPV DNA. The in situ hybridization assay used to detect HPV DNA in this article is the only method that allows precise microscopic detection of HPV nucleic acids within cells. The major drawback of the in situ test is its reduced sensitivity, relative to Southern blot or dot blot hybridization in the detection of latently HPV -infected tumors. Latently infected tumors or adjacent normal epithelium would likely receive a negative score in the in situ assay. Therefore the absence of HPV DNA in vulvar squamous cell carcinoma does not exclude an HPV etiologic factor. It is also important to note that negative HPV DNA results only mean that the test is negative for the HPV types evaluated. The morphologic interpretation and proposed histologic criteria of intraepithelial-like invasive squamous cell carcinoma of the vulva in this article includes nests of invasive neoplastic epithelium with preserved cell polarity, similar to intraepithelial disease. This definition seems to create some confusion and uncertainty for the following reasons: (1) Vulvar intraepithelial neoplasia represents a continuous morphologic spectrum of epithelial cellular abnormality and maturation. Vulvar intraepithelial neoplasia is divided into types I, II, and III. Do the authors mean the intraepitheliallesion mimics vulvar intraepithelial neoplasia type III? It would seem difficult to classify the intraepithelial-like invasive squamous cell carcinoma into a separate distinct morphologic entity on the basis of their proposed criteria. (2) It is also known that invasive squamous cell carcinoma of the vulva is composed of a heterogeneous morphologic pattern of neoplasia, even within the same tumor. Focal areas of vulvar invasive squamous cell carcinoma may fit into the author's criteria of intraepithelial-like growth pattern, but other areas of the same
alterations in proximity to invasive squamous carcinoma of the vulva. lnt j Gynecol Pathol 1982; 1: 173. Brinton LA, Nasca PC, Mallin K, Baptiste MS, Wilbanks GD, Richart RM. Case-control study of cancer of the vulva. Obstet Gynecol 1990;75:859. japaze H, Garcia-Bunuel R, WoodruffjD. Primary vulvar neoplasia: a review of in situ and invasive carcinoma, 1935-1972. Obstet Gynecol 1977;49;404-11. Franklin EW, Rutledge FD. Epidemiology of epidermoid carcinoma of the vulva. Obstet Gynecol 1972;39: 165-72. McAdams Aj, Kistner RW. The relationship of chronic vulvar disease, leukoplakia, and carcinoma in situ to carcinoma of the vulva. Cancer 1958; 11:740-75. Liao SY, Wilczynski SP, Bloss jD, Peake M, Berman M. Clinical and histopathologic features of vulvar carcinomas analyzed for HPV status. Lab Invest 1990;335A. Neill SM, Lessanaleibowitch M, Pelisse M, Moyalbarracco M. Lichen sclerosis, invasive squamous cell carcinoma, and human papillomavirus. AM j OBSTET GYNECOL 1990; 162: 1633-4. Twiggs L, Okagaki T, Clark B, Fukushima M, Ostrow R, Faras A. A clinical, histopathologic,and molecular biologic investigation of vulvar intraepithelial neoplasia. lnt j Gynecol Pathol 1988;7:48.
Discussion T, GIVEN, JR" Norfolk, Virginia. Dr. Andersen et al. address the association of the HPV and invasive squamous cell carcinoma of the vulva and correlate this with age, smoking history, and morphology. For my comments I am indebted to the expertise of two of my colleagues at the Eastern Virginia Medical School, Dr. Ken Somers, Professor of Microbiology and Immunology, and Dr. J eng G. Hsiu, Associate Professor of Pathology. The primary goal of the authors was to determine by the use of in situ hybridization the frequency of association of the HPV and the correlation of this occurrence with specific morphologic types of vulvar cancer as well as age and smoking history. The evidence presented is interpreted to show that HPV DNA is preferentially associated with a specific morphologic subtype of vulvar carcinoma (intraepithelialDR. FRED
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NORMAL CELLS PAPILLOMAVIRUS.. or. INFECTIOY. M .UTAGENS Z~·::~~!,!~c' j::O. ~.IMMUNE t •• H[RP'ES SI"~LEX ~'8 ~CIN I :::0 ! ~ VIRUS INFECTIONS. \ . ~ ./... SYSTEM S"OKINOI UJ : rn ./
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INVASIVE CARCINOMA Fig. 1. Proposed model for synergistic effects in human genital cancer. (From zur Hausen H. Lancet 1982;2:1371. By permission.)
336 Andersen et al.
tumor may show a totally different morphologic growth pattern. How will the authors clarify such squamous cell carcinomas that comprise different patterns? The data on smoking cause me some concern and that concern is confounding. This was well pointed out in an editorial. I There may be many other initiating events or promoting factors in the cause of vulvar cancer, just as there are in carcinoma of the cervix. This is certainly well illustrated in the model proposed by Dr. zur Hausen (Fig. 1).2 The author's data certainly seem to show some association between the papillomavirus and intraepithelial-like carcinoma of the vulva. This seems to occur at a somewhat younger age; however, it remains to be seen whether survival is affected by being able to isolate this particular subgroup of patients. Is the biologic behavior of the intraepithelial-like lesion described by the authors different? This study should be repeated with an even more sensitive technique, specifically, polymerase chain reaction amplification of HPV DNA with primers that recognize highly conserved regions within the HPV genome. 3 Until these data are presented, negative results should be interpreted with caution. There should also be adjustment for confounding, such as the sexual activity, number of partners, exposure to passive smoke and caffeine if they want to truly see the effects of smoking per se on the epidemiology of carcinoma of the vulva. We believe this study should be continued in much more detail and with a larger number of patients, perhaps requiring a multiinstitutional protocol to confirm these preliminary results. I have three questions for Dr. Andersen: 1. Do you believe that you will be able to classify the intraepithelial-like invasive squamous cell carcinoma of the vulva in a heterogenous type of neoplasm? 2. Do you believe that with a much larger series the group with the intraepithelial-like growth pattern will have a different survival rate than other invasive squamous cell carcinomas of the vulva? 3. Are you convinced of the significance of the smoking data with a series this small? REFERENCES 1. Layde PM, ed. Smoking and cervical cancer: cause or co-
incidence? JAMA 1989;261:1631. 2. zur Hausen H. Human genital cancer: synergism between two virus infections or synergism between a virus infection and initiating events? Lancet 1982;2:1370. 3. Bauer HM, Ting Y, Greer CE, et al. Genital human papillomavirus infection in female university student as determined by a PCR-based method. JAMA 1991;265:472. DR. MATTHEW O. BURRELL, Atlanta, Georgia. Did the authors have any cases with preexisting or coexisting lichen sclerosis? In Atlanta we have had four patients in whom early invasive carcinoma has developed in preexisting lichen sclerosis.
August 1991 Am J Obstet Gynecol
DR. ANDERSEN (Closing). We looked at the surrounding epithelium in most cases and four of 12 with an intraepithelial-like pattern had associated vulvar intraepithelial neoplasia. Eight of the remaining 28 were associated with some other form of vulva dystrophy. We had one of the 42 in whom an invasive cancer developed while being folowed up for vulvar dystrophy. Therefore I think vulva dystrophies demand careful attention and follow-up. To address Dr. Given's comments and concerns, it is Dr. Crum's belief that the intraepithelial-like pattern can be distinguished by the orientation of the cells in circumscribed nests with retention of cell polarity and palisaded nuclei at the periphery (Figs. 1, C, and 2, A). This arrangement of cells is similar to that seen in vulvar intraepithelial neoplasms, irrespective of grade, but cannot be compared directly with them gradewise because this pattern is being described in invasive carcinomas. In all the intraepithelial-like lesions, this growth pattern was clearly present in an invasive component by virtue of the stromal reaction around the nests (Fig. 1, C). We agree that lesions may be heterogeneous and contain more than one pattern, but the intraepitheliallike pattern was classified separately because of its distinctive appearance. The association of HPV nucleic acids with intraepithelial-like neoplasms may be fortuitous or reflect the fact that invasive carcinomas within this growth pattern are more likely to have derived from vulvar intraepithelial neoplasms. As you know, a high percentage of the latter are known to contain genital papiIIomavirus nucleic acids. Whereas a variety of other techniques have been used to search for HPV DNA in human cells, we believed in this retrospective study that the in situ hybridization technique was the most appropriate because it provided for direct localization of HPV nucleic acids in the cells of the neoplasm. The polymerase chain reaction, although superior in sensitivity, does not establish the location of the viral nucleic acids detected and can be plagued by false-positive results. I would reemphasize that the hybridization conditions used by Dr. Crum permitted detection of not only HPV DNAs identical to those in the probes, but more distantly related HPV types as well, and the sensitivity was such that as few as 10 to 20 copies of HPV DNA could be detected in an individual cell. Our smoking data were based purely on chart review and in many instances we could not tell whether the patients had ever smoked. The issue of smoking exposure is quite complex and although in our small series with small numbers we can show a statistical association between smoking and intraepithelial-like histologic pattern as well as the presence of HPV, clearly this is only a preliminary observation that requires much more sophisticated epidemiologic study to confirm this potential association.
![[Vulvar Squamous cell carcinoma: Why monitor a lichen sclerosus].](/assets/img/hold.png)
