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Frequency and prognostic significance of HPV DNA in sentinel lymph nodes of patients with cervical cancer
original article
Annals of Oncology 18: 1513–1517, 2007 doi:10.1093/annonc/mdm192
Frequency and prognostic significance of HPV DNA in sentinel lymph nodes of patients with cervical cancer C. Coutant1,2, E. Barranger2, A. Cortez3, D. Dabit3, S. Uzan2, J. F. Bernaudin1 & E. Darai2* 1
Departments of Histology and Tumor Biology; 2Gynecology and Obstetrics; 3Pathology, Hoˆpital Tenon, Assistance Publique des Hoˆpitaux de Paris, CancerEst, Universite´ Pierre et Marie Curie Paris VI, Paris, France
Received 9 February 2007; revised 26 March 2007; revised 6 April 2007; accepted 11 April 2007
introduction There are about 471 000 new cases of cervical cancer worldwide each year and 233 000 deaths [1]. Human papillomavirus (HPV), and especially types 16 and 18, is an established cause of cervical carcinoma [2, 3]. Lymph node status is a major prognostic factor in this setting, and a decision criterion for adjuvant therapy [4–6]. Despite the lack of apparent lymph node involvement, some patients have recurrences and shorter survival [5, 7]. Holmgren et al. suggested that histologically undetectable or ‘occult’ metastases in the lymphatic system could explain some recurrences [8]. HPV DNA screening by means of the polymerase chain reaction (PCR) has been proposed as a method to detect occult metastases [3, 8]. Pelvic and para-aortic lymph node dissection remains the most accurate way of assessing lymph node status in this setting but is associated with significant morbidity [7]. The sentinel lymph node (SN) procedure has emerged as a less aggressive alternative [7, 9, 10]. Moreover, serial sectioning and immunohistochemical analysis of SN can detect micrometastasis [7, 9, 10]. There are no published data on the prognostic relevance of HPV DNA detection by PCR in sentinel *Correspondence to: Prof. E. Daraı¨, Department of Gynecologic and Obstetrics, Hoˆpital Tenon, 4 rue de la Chine, 75020 Paris, France. Tel: +33-156-016-849; Fax: +33-156016-062; E-mail: [email protected]
ª 2007 European Society for Medical Oncology
nodes of women with cervical cancer. This study was designed to determine the frequency of HPV DNA detection by PCR in SN, and its relation to the clinical characteristics and outcome of women with cervical cancer.
patients and methods patients From July 2001 to September 2005, 59 patients referred to our institution with cervical cancer were included in the study. Disease stage was determined as recommended by the International Federation of Gynaecology and Obstetrics (FIGO) [11]. All patients gave their informed consent for sentinel node procedure. Follow-up of patients included a physical examination every 3 months for the first post therapeutic year then every 6 months for the four additional years.
methods surgery. Patients with FIGO stage Ia to Ib1 disease underwent initial laparoscopic pelvic lymphadenectomy and vaginal or laparoscopic radical hysterectomy. Patients with FIGO stage Ib2 or II disease underwent laparoscopic pelvic and para-aortic lymphadenectomy followed by concurrent chemoradiotherapy. SN procedure. SN procedure was performed in accordance with previous studies using combined radiocolloid and colorimetric technique [7, 9, 12, 13]. The position of each SN relative to the major pelvic vessels, and the count rate, were recorded.
original article
could explain some recurrences. HPV DNA screening by means of the polymerase chain reaction (PCR) has been proposed as a method to detect occult metastases. This study was designed to determine the frequency of HPV DNA detection by PCR in sentinel lymph node (SN), and its relation to the clinical characteristics and outcome of women with cervical cancer. Patients and methods: The primary cervical tumor and SN were tested for HPV DNA by means of PCR in 59 patients. Results: Fifteen (25.4%) of the 59 women undergoing the SN procedure had an involved SN. HPV DNA was more frequent in positive SN than in negative SN (P < 0.0001). Seven patients had a recurrence, after a mean delay of 17 months (range: 10–26). One of seven patients with a recurrence had an involved SN. HPV DNA was detected in an SN of one of seven patients with recurrence and nine (19.5%) of 46 patients without recurrence (not significant). Conclusion: In women with cervical cancer, HPV DNA screening of sentinel nodes might help to identify patients at risk of lymph node metastases and recurrence. Key words: cervical cancer, sentinel lymph node, human papillomavirus, polymerase chain reaction
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Background: It has been suggested that histologically undetectable or ‘occult’ metastases in the lymphatic system
original article
5#-GGTCGGTGGACCGGTCGATG-3’ and 5#-GCAATGTAGGTGTATCTCCA-3#, 5#-CCTTGGACGTAAATTTTTGG-3’ and 5#-CACGCACACGCTTGGCAGGT-3#, 5#-GGGATTGTTACAAAGCTACC-3’ and 5#-GTAGTTGAGGACAACTGAC-3#, 5#-GGAGAGCCAAGGACAGGTAC-3’ and 5#-CAACTTCATCCACGTTCAACC-3#.
statistical analysis Statistical analysis was performed using StatView Software version 5.0 (SAS Institute Inc, Cary, North Carolina, USA). Comparisons between continuous and dichotomous variables were based on the Mann–Whitney U-test. Comparisons between dichotomous variables were based on the Pearson chi-square test or Fisher’s test. A P-value <0.05 was considered to denote a significant difference
results epidemiological and histological characteristics of the population The median age of the 59 patients was 50.1 years (range 29–77 years). The FIGO stages were Ia, Ib1, Ib2, IIa and IIb in six (10.2%), 23 (39%), eight (13.6%), 12 (20.3%) and 10 cases (17%), respectively (see Table 1). The detection rate of SN was 85% and bilateral SN were identified in 40.7% of cases. One-hundred-and ten SN were detected (mean per patient: 1.9, range 1–5). Seventy-five per cent of SN were located in the external iliac area and 9% near the obturator. All 59 women underwent systematic pelvic lymphadenectomy, and a mean of 9.3 nodes were removed per patient (range 2–18). Fifteen women underwent both laparoscopic pelvic and para-aortic lymphadenectomy, and a mean of 13.2 para-aortic nodes per patient were removed (range 4–25).
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Table 1. Epidemiological, histological characteristics and histology of sentinel and non sentinel node of the 59 women with cervical cancer Total (n = 59)
Characteristics Mean age, years (range) FIGO stage Ia Ib1 Ib2 II Histology Squamous cell carcinoma Adenocarcinoma Histological grade Well differentiated Moderately differentiated Poorly differentiated Not graded Tumour size (cm) Mean (range) <2 cm 2–4 cm >4 cm LVSI Parametrial invasion Sentinel lymph nodes (SN) Nb of SN removed Nb of patient with positive SN [nb of SN]. Nb of patients with macrometastases in H&S [nb of SN]. Nb of patients with micrometastases in H&S [nb of SN]. Nb of patients with micrometastases in H&S [nb of SN]. Nb of patients with isolated tumors [nb of SN]. Non sentinel lymph nodes (non SN) Mean nb of non-SN (range) Nb of patients with positive non-SN Pelvic lymph nodes (PLN) = SN + non-SN SN+/non-SN+ SN+/non-SN2 SN–/non-SN2 SN–/non-SN2 False-negative rate
50.1 (29–77) 6 23 8 22
(10.2%) (39%) (13.6%) (37.3%)
45 (76.3%) 14 (23.7%) 40/52 10/52 2/52 7
(76.9%) (19.2%) (3.9%) (11.9%)
3.1 16 25 18 12/59 5/41
(0.3–6.3) (27.1%) (42.4%) (30.5%) (20.3%) (12.2%)
110 15 (25.4%) [20 (18%)] 12 [10]. 3 [5]. 0 2 [3].
9.3 (2–18) 6 (10.2%) 4 (6.8%) 11 (18.6%) 2 (3.4%) 42 (71.2%) 11.8%
SN = sentinel node; LVSI = lymphovascular space involvement; Nb = number; H&S = hematoxylin and eosin; IHC = immunohistochemistry.
histology of lymph nodes and HPV detection in cervical tumor and lymph nodes Fifteen (25.4%) of the 59 women had at least one positive SN. Twenty (18.2%) of the 110 SN were positive. Fifty-one cervical tumors were tested for HPV DNA, and 29 (57%) were positive. HPV-16, -18 and -31 DNA was detected in 22 cases (75.9%), five cases (17.2%) and two cases (6.9%), respectively (Table 1). HPV DNA was detected in 10 (50%) of 20 positive SN (seven of 15 patients) and in five (5.6%) of 90 negative SN (five of 44 patients). HPV DNA was more frequent in positive SN than in negative SN (P < 0.0001).
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histopathology and HPV detection. SN analysis was performed in accordance with previous studies [9]. In brief, SNs were cut perpendicular to the long axis. All SNs were submitted to intra-operative imprint cytology with a rapid May–Gru¨nwald–Giemsa method. Each half-SN was sectioned at 3mm intervals, fixed in formalin and paraffin embedded. Each 3-mm section was analysed at four additional levels of 150 lm, and four parallel sections; one was used for hematoxylin and eosin (H&E) staining, and H&E-negative sections were examined by immunohistochemistry (IHC) with an anticytokeratin antibody cocktail (Cytokeratin AE1-AE3, Dako Corporation, Glostrup, Denmark). Non-SNs were totally submitted and blocked individually following 3 mm distances and H&E staining. Micrometastasis was defined as a single focus of metastatic disease per node, measuring no more than 2 mm. The presence of single non-cohesive tumor cells was also recorded. SNs were considered positive when they contained macrometastases, micrometastases or isolated tumor cells. In accordance with previous studies [14, 15]., HPV DNA was detected in the primary tumor and lymph node by E6-specific PCR. To detect HPV-16, -18, and -31 DNA, we used primer pairs spanning shorter DNA sequences of the E6 open reading frame described by Resnick et al. [14] and Baay et al. [15]. The oligonucleotides used as primers for E6 HPV-16, 18, 31 and b globin DNA were respectively:
Annals of Oncology
original article
Annals of Oncology
In patients with positive SN, HPV DNA was detected in six (50%) of 12 positive SN from patients with HPV-positive tumors and in two (40%) of five positive SN from patients with HPV-negative tumors (P = NS). In patients with negative SN, HPV DNA was detected in three (13.6%) of 22 SN from patients with HPV-positive tumors and in two (11.1%) of 18 SN from patients with HPVnegative tumors (P = NS). HPV DNA was detected in six of the 12 SN containing macrometastases, in two of the five SN containing micrometastases (H&S staining), and in two of three SN containing isolated tumor cells (P = NS).
with recurrence had one positive SN. HPV DNA was detected in SN in one (14%) of seven patients with recurrence and in nine (19.5%) of 46 patients without recurrence (P = NS). In univariate analysis, parametrial involvement was the only factor associated with the risk of recurrence (P = 0.04). Among the seven women with recurrences, five had had an HPV DNA-positive primary cervical tumor (HPV-16 in three, HPV-18 in two). The rate of recurrence tended to be higher in women with HPV-18 DNA in their primary cervical tumor (Table 4).
relation between HPV status of the primary cervical tumor or SN, and clinical and histological prognostic factors No relation was found between HPV DNA detection in the primary tumor and any of the clinical or histological prognostic factors. Except for mean age, that was lower in patients with positive SN (P = 0.01), no relation was found between positive SN and clinical or histological prognostic factors (Table 2). Moreover, no relation was found between HPV DNA detection in SN and clinical or histological prognostic factors (Table 3). Fifty-three of the 59 patients were followed up, for a mean of 31.6 months (range: 10–60). Seven patients had a recurrence, after a mean of 17 months (range: 10–26). HPV DNA had been detected in the primary cervical tumor in five (71.5%) of seven patients with recurrence, versus 22 (52%) of 42 patients without recurrence (P = NS). One (14.3%) of seven patients
This study shows that sentinel node metastatic status correlates with nodal HPV DNA status determined by PCR. No relation was found between clinical or histological prognostic parameters and the presence of HPV DNA in the sentinel node or the primary cervical tumor. HPV DNA was detected in 50 and 5.6% of positive and negative SN, respectively. The frequency of sentinel node HPV positivity did not differ according to the existence of macrometastasis, micrometastasis, or isolated tumor cells. There are no other published data on HPV DNA expression in sentinel nodes from patients with cervical cancer. However, our results are in keeping with previous studies in which the frequency of HPV DNA detection in metastatic pelvic lymph nodes, diagnosed by standard H&S staining, varied from 42 to 100% of cases [3, 16–26]. Our results also confirm those of Hernadi et al. [16] and Chan et al. [17], demonstrating the
Characteristics Mean age, years (range) FIGO stage Ia1 to Ib1 Ib2 to IIb Histology Squamous cell carcinoma Adenocarcinoma Histological grade Well differentiated Moderately differentiated Poorly differentiated Tumour size (cm) Mean (range) <2 cm 2–4 cm >4 cm LVSI Parametrial invasion HPV DNA in primary cervical tumor HPV DNA in SN
Positive SN n = 15
Negative SN n = 44
43.9 (31–62)
52.2 (29–77)
5 (33.3%) 10 (66,7%)
24 (54.5%) 20 (45.5%)
10 (66.7%) 5 (33.3%)
35 (79.5%) 9 (20.5%)
11/15 (73.3%) 3/15 (20%) 1/15 (66.7%)
29/37 (78.4%) 7/37 (19.9%) 1/37 (2.7%)
0.01 NS
NS
NS (0.3–6.3) (20%) (33.3%) (46.7%) (40%) (28.6%) (60%)
7/15 (46.7%)
2.9 13 20 11 10/38 3/34 21/38
(0.3–6) (29.5%) (45.5%) (25%) (26.3%) (8.8%) (55.2%)
6/44 (13.6%)
SN = sentinel node; LVSI = lymphovascular space involvement.
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NS NS NS 0.02
HPV DNA negative SN n=4
Characteristics
HPV DNA positive SN n = 12
Mean age, years (range) FIGO stage Ia1 to Ib1 Ib2 to IIb Histology Squamous cell carcinoma Adenocarcinoma Differentiation grade Low (grade 1) Intermediate (grade 2) High (grade 3) Tumour size (cm) Mean (range) < 2 cm 2–4 cm > 4 cm LVSI Parametrial invasion Positive SN HPV DNA in primary cervical cancer
50,3 (31–77)
50.1 (29–77)
7 (58.3%) 5 (41,7%)
22 (46.8%) 25 (53.2%)
6 (50%) 6 (50%)
39 (83%) 8 (17%)
P
NS
3.6 3 5 7 4/10 2/7 9/15
Table 3. Relation between HPV DNA detection in SN and clinical and histological prognostic factors P
NS NS
0.04
NS 7/10 (70%) 3/10 (30%) 0
33/41 (78.6%) 7/41 (16.6%) 1/41 (4.8%)
3.1 (0.6–6) 3 (25%) 6 (50%) 3 (25%) 2/10 (20%) 0/7 7/12 (58.3%) 8/12 (66.7%)
3.1 13 19 15 12/38 5/29 8/47 22/41
NS (0.3–6.3) (27.7%) (40.4%) (31.9%) (31.6%) (14.7%) (17%) (53.7%)
NS NS 0.01 NS
SN = sentinel node; LVSI = lymphovascular space involvement.
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Table 2. Relation between SN involvement and clinical and histological prognostic factors
discussion
original article
Annals of Oncology
Table 4. Relation of recurrence with HPV DNA detection and clinicalhistological prognostic factors Characteristics
Women with follow-up (n = 53)
P
48.9 (30–69)
NS NS
3 (42.9%) 4 (57.1%) NS 4 (57.1%) 3 (42.9%) NS 5 (71.4%) 1 (14.3%) 1 (14.3%) NS 3.1 2 3 2 3 3
(0.5-6) (28.6%) (42.9%) (28.6%) (42.9%) (42.9%)
NS 0.04 NS
1 (14.3%) NS 1 (14.3%)
5/7 (71.5%)
NS
3/2
NS
1/7 (14%) 0/1 1/6 (17%) 1/0
NS NS
LVSI = lymphovascular space involvement; H&S = hematoxylin and eosin; IHC = immunohistochemistry.
relation between lymph node involvement and HPV status. In contrast, Baay et al. [18] found no relation between HPV DNA detection and lymph node status. However, these authors analysed only one node from the obturator region of each patient, whereas we found that the most common site of SN was the medial external iliac region (68%) and that only 9% of SN were located in the obturator area, in keeping with other studies [9, 19]. The low rate of negative SN with detectable HPV DNA (5.6%) contrasts somewhat with previous reports of HPV DNA detection in 23–60% of tumor-free pelvic lymph nodes. The high reported frequency of HPV DNA detection in negative lymph nodes could be explained by differences in histological analysis. Indeed, previous studies used the standard H&S technique, and this can miss some metastases detected by the SN method based on systematic multiple sectioning and immunohistochemical labeling. Indeed, most of the positive SN
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Mean age, years (range) 48.6 (29–77) FIGO stage Ia1 to Ib1 27 (50.9%) Ib2 to IIb 26 (49.1%) Histology Squamous cell carcinoma 40 (75.5%) Adenocarcinoma 13 (24.5%) Differentiation grade Well differentiated 37 (69.8%) Moderately differentiated 9 (17%) Poorly differentiated 2 (3.8%) Tumor size (cm) Mean (range) 2.9 (0.3–6) <2 cm 16 (30.2%) 2–4 cm 22 (41.5%) >4 cm 15 (28.3%) LVSI 12 (22.6%) Parametrial invasion 5 (9.4%) Sentinel node (SN) Number of patients with 14 (26.4%) positive SN Pelvic lymph node (PLN) Number of patients with 16 (30.2%) positive PLN HPV DNA status HPV DNA in primary 27/49 cervical cancer Type of HPV: HPV 16 / HPV 18 HPV DNA in SN 10/53 (18.9%) Positive SN Negative SN Type of HPV: HPV 16 / HPV 18
Women with recurrence (n = 7)
in our study contained micrometastases or isolated tumor cells. Hence, our results suggest that HPV DNA detection in pelvic lymph nodes might be a marker of metastatic involvement and that HPV DNA-positive lymph nodes might qualify for histological analysis similar to that applied to SN, leading to better detection of so-called ‘occult’ metastasis. It has been suggested that lymph nodes should not only be screened for HPV DNA, but also for HPV RNA and CK 19 mRNA, or be quantified by real-time PCR [17, 20]. We found no relationship between SN status, and classical clinical or pathological prognostic parameters such as FIGO stage, histological grade, and lymphovascular space involvement. No relation was found either between the HPV DNA status of pelvic lymph nodes and prognostic factors. Moreover, no relation was noted between the HPV DNA status of pelvic lymph nodes and the risk of recurrence. This could be explained by the fact that nearly half the positive SN were HPV DNA-negative, and also by the limited sample size. The lack of a strict relation between HPV DNA and SN involvement could be related to elimination of viral DNA molecules by scavenger lymphocytes and to HPV clearance from lymph nodes [17, 18, 21, 22]. The prognostic significance of HPV DNA detection in pelvic lymph nodes is controversial. For Pilch et al. [23]. and Hernardi et al. [16], lymph node HPV DNA status is a significant predictor of recurrent disease. Moreover, Burnett et al. [24] detected HPV DNA in five of six patients with recurrence, and Kobayashi et al. [25] in all nine patients with clinical relapse despite pelvic node non-involvement. In contrast, Hording et al. [26] detected HPV DNA in pelvic nodes in two of 10 patients with recurrence and in three of eight patients in remission. These discrepancies could be related to the small sample sizes. We detected HPV DNA (mainly HPV-16) in 57% of cervical cancers, in keeping with a reported prevalence of HPV DNA in cervical cancers ranging from 48 to 100% [3, 18, 22]. Unger et al. [27]. suggested that the presence of HPV DNA in primary cervical cancer was associated with poor survival. In contrast, we found no relation between HPV DNA and the FIGO stage or other classical prognostic factors. Our data are in line with those of van Bommel et al. [6], showing no difference in survival between women with and without HPV DNA in their cervical tumors. The high frequency of HPV-16 in our study is in line with epidemiological studies showing its predominance in Europe and North America [1]. Like Barnes et al. [28], we found that HPV-18 tended to be associated with a high risk of recurrence: HPV-18 DNA was found in the primary tumors of two of the five patients who recurred, and in only two of the 22 patients who remained in remission. No relationship was found between HPV DNA detection in the primary cervical tumor and in the sentinel node. Indeed, HPV DNA was detected in positive SN of two of three patients with negative primary tumors. These results are in keeping with those of Fu¨le et al. [21], showing the lack of any close correlation between metastasis and viral detection in lymph nodes. Beyer-Finkler et al. [29] pointed out that lymph node HPV DNA might originate from disintegrating tumor cells, being conveyed passively by lymphatic flow or actively by phagocytic cells. Fu¨le et al. showed that, in highly heterogeneous advanced-stage tumors with numerous genetic
Annals of Oncology
acknowledgements Charles COUTANT was a fellow from ‘Fondation pour la Recherche Me´dicale’. We are particularly grateful to ACTT (‘Ami du Centre des Tumeurs de Tenon’).
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12. Roca I, Caresia AP, Gil-Moreno A et al. Usefulness of sentinel lymph node detection in early stages of cervical cancer. Eur J Nucl Med Mol Imaging 2005; 32: 1210–1216. 13. Kraft O, Sevcik L, Klat J, Koliba P, Curik R, Kriozva H. Detection of sentinel lymph nodes in cervical cancer. A comparison of two protocols. Nucl Med Rev Cent East Eur 2006; 9: 65–68. 14. Resnick RM, Cornelissen MT, Wright DK et al. Detection and typing of human papillomavirus in archival cervical cancer specimens by DNA amplification with consensus primers. J Natl Cancer Inst 1990; 82: 1477–1484. 15. Baay MF, Quint WG, Koudstaal J et al. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 1996; 34: 745–747. 16. Hernadi Z, Szarka K, Sapy T et al. The prognostic significance of HPV-16 genome status of the lymph nodes, the integration status and p53 genotype in HPV-16 positive cervical cancer: a long term follow up. Bjog 2003; 110: 205–209. 17. Chan PK, Yu MM, Cheung TH et al. Detection and quantitation of human papillomavirus DNA in primary tumour and lymph nodes of patients with early stage cervical carcinoma. J Clin Virol 2005; 33: 201–205. 18. Baay MF, Koudstaal J, Hollema H et al. Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer. J Clin Pathol 1997; 50: 960–961. 19. Barranger E, Coutant C, Cortez A et al. Sentinel node biopsy is reliable in early-stage cervical cancer but not in locally advanced disease. Ann Oncol 2005; 16: 1237–1242. 20. Lukaszuk K, Liss J, Wozniak I et al. HPV and histological status of pelvic lymph node metastases in cervical cancer: a prospective study. J Clin Pathol 2004; 57: 472–476. 21. Fule T, Csapo Z, Mathe M et al. Prognostic significance of high-risk HPV status in advanced cervical cancers and pelvic lymph nodes. Gynecol Oncol 2006; 100: 570–578. 22. Sapy T, Hernadi Z, Konya J, Lukacsko L. Poor clinical outcome in early stage cervical cancer with human papillomavirus-18 positive lymph nodes. Eur J Obstet Gynecol Reprod Biol 2000; 90: 93–95. 23. Pilch H, Gunzel S, Schaffer U et al. The presence of HPV DNA in cervical cancer: correlation with clinico-pathologic parameters and prognostic significance: 10 years experience at the Department of Obstetrics and Gynecology of the Mainz University. Int J Gynecol Cancer 2001; 11: 39–48. 24. Burnett AF, Barnes WA, Johnson JC et al. Prognostic significance of polymerase chain reaction detected human papillomavirus of tumors and lymph nodes in surgically treated stage IB cervical cancer. Gynecol Oncol 1992; 47: 343–347. 25. Kobayashi Y, Yoshinouchi M, Tianqi G et al. Presence of human papilloma virus DNA in pelvic lymph nodes can predict unexpected recurrence of cervical cancer in patients with histologically negative lymph nodes. Clin Cancer Res 1998; 4: 979–983. 26. Hording U, Ravn V, Knudsen J, Visfeldt J. The use of polymerase chain reaction to detect metastatic cancer cells within lymph nodes in stage I cervical carcinoma. Int J Gynecol Pathol 1995; 14: 339–343. 27. Unger ER, Vernon SD, Thoms WW et al. Human papillomavirus and diseasefree survival in FIGO stage Ib cervical cancer. J Infect Dis 1995; 172: 1184–1190. 28. Barnes W, Woodworth G, Waggoner S et al. Rapid dysplastic transformation of human genital cells by human papillomavirus type 18. Gynecol Oncol 1990; 38: 343–346. 29. Beyer-Finkler E, Girardi F, Sillem M, Pfister H. Human papillomavirus DNA in genital cancers, metastases, and lymph nodes. Intervirology 1995; 38: 173–180. 30. Levenback C, Coleman RL, Burke TW et al. Lymphatic mapping and sentinel node identification in patients with cervix cancer undergoing radical hysterectomy and pelvic lymphadenectomy. J Clin Oncol 2002; 20: 688–693.
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aberrations, the loss of HPV DNA did not affect the proliferative capacity of cell clones [21]. Our study confirms the relevance of the SN procedure for assessing lymph node status in women with cervical cancer. The detection rate (85%) was slightly lower than that obtained elsewhere with dual tracer detection [9, 30], possibly owing to the inclusion of women with locally advanced cervical cancer in whom the detection rate is lower than in women with earlier stages [19]. However, some limitations of the present study have to be underlined such as the small sample size and the low rate of HPV-DNA detection in both primary tumors and sentinel nodes. In conclusion, this study suggests that the combination of the sentinel node procedure and HPV DNA screening of the excised nodes might contribute to better evaluation of lymph node status in women with cervical cancer and, hence, better identification of women who have an increased risk of recurrence. Further studies with larger populations are required to evaluate the prognostic relevance of HPV DNA detection in pelvic lymph nodes and in primary cervical tumors.
original article
Annals of Oncology 18: 1513–1517, 2007 doi:10.1093/annonc/mdm192
Frequency and prognostic significance of HPV DNA in sentinel lymph nodes of patients with cervical cancer C. Coutant1,2, E. Barranger2, A. Cortez3, D. Dabit3, S. Uzan2, J. F. Bernaudin1 & E. Darai2* 1
Departments of Histology and Tumor Biology; 2Gynecology and Obstetrics; 3Pathology, Hoˆpital Tenon, Assistance Publique des Hoˆpitaux de Paris, CancerEst, Universite´ Pierre et Marie Curie Paris VI, Paris, France
Received 9 February 2007; revised 26 March 2007; revised 6 April 2007; accepted 11 April 2007
introduction There are about 471 000 new cases of cervical cancer worldwide each year and 233 000 deaths [1]. Human papillomavirus (HPV), and especially types 16 and 18, is an established cause of cervical carcinoma [2, 3]. Lymph node status is a major prognostic factor in this setting, and a decision criterion for adjuvant therapy [4–6]. Despite the lack of apparent lymph node involvement, some patients have recurrences and shorter survival [5, 7]. Holmgren et al. suggested that histologically undetectable or ‘occult’ metastases in the lymphatic system could explain some recurrences [8]. HPV DNA screening by means of the polymerase chain reaction (PCR) has been proposed as a method to detect occult metastases [3, 8]. Pelvic and para-aortic lymph node dissection remains the most accurate way of assessing lymph node status in this setting but is associated with significant morbidity [7]. The sentinel lymph node (SN) procedure has emerged as a less aggressive alternative [7, 9, 10]. Moreover, serial sectioning and immunohistochemical analysis of SN can detect micrometastasis [7, 9, 10]. There are no published data on the prognostic relevance of HPV DNA detection by PCR in sentinel *Correspondence to: Prof. E. Daraı¨, Department of Gynecologic and Obstetrics, Hoˆpital Tenon, 4 rue de la Chine, 75020 Paris, France. Tel: +33-156-016-849; Fax: +33-156016-062; E-mail: [email protected]
ª 2007 European Society for Medical Oncology
nodes of women with cervical cancer. This study was designed to determine the frequency of HPV DNA detection by PCR in SN, and its relation to the clinical characteristics and outcome of women with cervical cancer.
patients and methods patients From July 2001 to September 2005, 59 patients referred to our institution with cervical cancer were included in the study. Disease stage was determined as recommended by the International Federation of Gynaecology and Obstetrics (FIGO) [11]. All patients gave their informed consent for sentinel node procedure. Follow-up of patients included a physical examination every 3 months for the first post therapeutic year then every 6 months for the four additional years.
methods surgery. Patients with FIGO stage Ia to Ib1 disease underwent initial laparoscopic pelvic lymphadenectomy and vaginal or laparoscopic radical hysterectomy. Patients with FIGO stage Ib2 or II disease underwent laparoscopic pelvic and para-aortic lymphadenectomy followed by concurrent chemoradiotherapy. SN procedure. SN procedure was performed in accordance with previous studies using combined radiocolloid and colorimetric technique [7, 9, 12, 13]. The position of each SN relative to the major pelvic vessels, and the count rate, were recorded.
original article
could explain some recurrences. HPV DNA screening by means of the polymerase chain reaction (PCR) has been proposed as a method to detect occult metastases. This study was designed to determine the frequency of HPV DNA detection by PCR in sentinel lymph node (SN), and its relation to the clinical characteristics and outcome of women with cervical cancer. Patients and methods: The primary cervical tumor and SN were tested for HPV DNA by means of PCR in 59 patients. Results: Fifteen (25.4%) of the 59 women undergoing the SN procedure had an involved SN. HPV DNA was more frequent in positive SN than in negative SN (P < 0.0001). Seven patients had a recurrence, after a mean delay of 17 months (range: 10–26). One of seven patients with a recurrence had an involved SN. HPV DNA was detected in an SN of one of seven patients with recurrence and nine (19.5%) of 46 patients without recurrence (not significant). Conclusion: In women with cervical cancer, HPV DNA screening of sentinel nodes might help to identify patients at risk of lymph node metastases and recurrence. Key words: cervical cancer, sentinel lymph node, human papillomavirus, polymerase chain reaction
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Background: It has been suggested that histologically undetectable or ‘occult’ metastases in the lymphatic system
original article
5#-GGTCGGTGGACCGGTCGATG-3’ and 5#-GCAATGTAGGTGTATCTCCA-3#, 5#-CCTTGGACGTAAATTTTTGG-3’ and 5#-CACGCACACGCTTGGCAGGT-3#, 5#-GGGATTGTTACAAAGCTACC-3’ and 5#-GTAGTTGAGGACAACTGAC-3#, 5#-GGAGAGCCAAGGACAGGTAC-3’ and 5#-CAACTTCATCCACGTTCAACC-3#.
statistical analysis Statistical analysis was performed using StatView Software version 5.0 (SAS Institute Inc, Cary, North Carolina, USA). Comparisons between continuous and dichotomous variables were based on the Mann–Whitney U-test. Comparisons between dichotomous variables were based on the Pearson chi-square test or Fisher’s test. A P-value <0.05 was considered to denote a significant difference
results epidemiological and histological characteristics of the population The median age of the 59 patients was 50.1 years (range 29–77 years). The FIGO stages were Ia, Ib1, Ib2, IIa and IIb in six (10.2%), 23 (39%), eight (13.6%), 12 (20.3%) and 10 cases (17%), respectively (see Table 1). The detection rate of SN was 85% and bilateral SN were identified in 40.7% of cases. One-hundred-and ten SN were detected (mean per patient: 1.9, range 1–5). Seventy-five per cent of SN were located in the external iliac area and 9% near the obturator. All 59 women underwent systematic pelvic lymphadenectomy, and a mean of 9.3 nodes were removed per patient (range 2–18). Fifteen women underwent both laparoscopic pelvic and para-aortic lymphadenectomy, and a mean of 13.2 para-aortic nodes per patient were removed (range 4–25).
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Table 1. Epidemiological, histological characteristics and histology of sentinel and non sentinel node of the 59 women with cervical cancer Total (n = 59)
Characteristics Mean age, years (range) FIGO stage Ia Ib1 Ib2 II Histology Squamous cell carcinoma Adenocarcinoma Histological grade Well differentiated Moderately differentiated Poorly differentiated Not graded Tumour size (cm) Mean (range) <2 cm 2–4 cm >4 cm LVSI Parametrial invasion Sentinel lymph nodes (SN) Nb of SN removed Nb of patient with positive SN [nb of SN]. Nb of patients with macrometastases in H&S [nb of SN]. Nb of patients with micrometastases in H&S [nb of SN]. Nb of patients with micrometastases in H&S [nb of SN]. Nb of patients with isolated tumors [nb of SN]. Non sentinel lymph nodes (non SN) Mean nb of non-SN (range) Nb of patients with positive non-SN Pelvic lymph nodes (PLN) = SN + non-SN SN+/non-SN+ SN+/non-SN2 SN–/non-SN2 SN–/non-SN2 False-negative rate
50.1 (29–77) 6 23 8 22
(10.2%) (39%) (13.6%) (37.3%)
45 (76.3%) 14 (23.7%) 40/52 10/52 2/52 7
(76.9%) (19.2%) (3.9%) (11.9%)
3.1 16 25 18 12/59 5/41
(0.3–6.3) (27.1%) (42.4%) (30.5%) (20.3%) (12.2%)
110 15 (25.4%) [20 (18%)] 12 [10]. 3 [5]. 0 2 [3].
9.3 (2–18) 6 (10.2%) 4 (6.8%) 11 (18.6%) 2 (3.4%) 42 (71.2%) 11.8%
SN = sentinel node; LVSI = lymphovascular space involvement; Nb = number; H&S = hematoxylin and eosin; IHC = immunohistochemistry.
histology of lymph nodes and HPV detection in cervical tumor and lymph nodes Fifteen (25.4%) of the 59 women had at least one positive SN. Twenty (18.2%) of the 110 SN were positive. Fifty-one cervical tumors were tested for HPV DNA, and 29 (57%) were positive. HPV-16, -18 and -31 DNA was detected in 22 cases (75.9%), five cases (17.2%) and two cases (6.9%), respectively (Table 1). HPV DNA was detected in 10 (50%) of 20 positive SN (seven of 15 patients) and in five (5.6%) of 90 negative SN (five of 44 patients). HPV DNA was more frequent in positive SN than in negative SN (P < 0.0001).
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histopathology and HPV detection. SN analysis was performed in accordance with previous studies [9]. In brief, SNs were cut perpendicular to the long axis. All SNs were submitted to intra-operative imprint cytology with a rapid May–Gru¨nwald–Giemsa method. Each half-SN was sectioned at 3mm intervals, fixed in formalin and paraffin embedded. Each 3-mm section was analysed at four additional levels of 150 lm, and four parallel sections; one was used for hematoxylin and eosin (H&E) staining, and H&E-negative sections were examined by immunohistochemistry (IHC) with an anticytokeratin antibody cocktail (Cytokeratin AE1-AE3, Dako Corporation, Glostrup, Denmark). Non-SNs were totally submitted and blocked individually following 3 mm distances and H&E staining. Micrometastasis was defined as a single focus of metastatic disease per node, measuring no more than 2 mm. The presence of single non-cohesive tumor cells was also recorded. SNs were considered positive when they contained macrometastases, micrometastases or isolated tumor cells. In accordance with previous studies [14, 15]., HPV DNA was detected in the primary tumor and lymph node by E6-specific PCR. To detect HPV-16, -18, and -31 DNA, we used primer pairs spanning shorter DNA sequences of the E6 open reading frame described by Resnick et al. [14] and Baay et al. [15]. The oligonucleotides used as primers for E6 HPV-16, 18, 31 and b globin DNA were respectively:
Annals of Oncology
original article
Annals of Oncology
In patients with positive SN, HPV DNA was detected in six (50%) of 12 positive SN from patients with HPV-positive tumors and in two (40%) of five positive SN from patients with HPV-negative tumors (P = NS). In patients with negative SN, HPV DNA was detected in three (13.6%) of 22 SN from patients with HPV-positive tumors and in two (11.1%) of 18 SN from patients with HPVnegative tumors (P = NS). HPV DNA was detected in six of the 12 SN containing macrometastases, in two of the five SN containing micrometastases (H&S staining), and in two of three SN containing isolated tumor cells (P = NS).
with recurrence had one positive SN. HPV DNA was detected in SN in one (14%) of seven patients with recurrence and in nine (19.5%) of 46 patients without recurrence (P = NS). In univariate analysis, parametrial involvement was the only factor associated with the risk of recurrence (P = 0.04). Among the seven women with recurrences, five had had an HPV DNA-positive primary cervical tumor (HPV-16 in three, HPV-18 in two). The rate of recurrence tended to be higher in women with HPV-18 DNA in their primary cervical tumor (Table 4).
relation between HPV status of the primary cervical tumor or SN, and clinical and histological prognostic factors No relation was found between HPV DNA detection in the primary tumor and any of the clinical or histological prognostic factors. Except for mean age, that was lower in patients with positive SN (P = 0.01), no relation was found between positive SN and clinical or histological prognostic factors (Table 2). Moreover, no relation was found between HPV DNA detection in SN and clinical or histological prognostic factors (Table 3). Fifty-three of the 59 patients were followed up, for a mean of 31.6 months (range: 10–60). Seven patients had a recurrence, after a mean of 17 months (range: 10–26). HPV DNA had been detected in the primary cervical tumor in five (71.5%) of seven patients with recurrence, versus 22 (52%) of 42 patients without recurrence (P = NS). One (14.3%) of seven patients
This study shows that sentinel node metastatic status correlates with nodal HPV DNA status determined by PCR. No relation was found between clinical or histological prognostic parameters and the presence of HPV DNA in the sentinel node or the primary cervical tumor. HPV DNA was detected in 50 and 5.6% of positive and negative SN, respectively. The frequency of sentinel node HPV positivity did not differ according to the existence of macrometastasis, micrometastasis, or isolated tumor cells. There are no other published data on HPV DNA expression in sentinel nodes from patients with cervical cancer. However, our results are in keeping with previous studies in which the frequency of HPV DNA detection in metastatic pelvic lymph nodes, diagnosed by standard H&S staining, varied from 42 to 100% of cases [3, 16–26]. Our results also confirm those of Hernadi et al. [16] and Chan et al. [17], demonstrating the
Characteristics Mean age, years (range) FIGO stage Ia1 to Ib1 Ib2 to IIb Histology Squamous cell carcinoma Adenocarcinoma Histological grade Well differentiated Moderately differentiated Poorly differentiated Tumour size (cm) Mean (range) <2 cm 2–4 cm >4 cm LVSI Parametrial invasion HPV DNA in primary cervical tumor HPV DNA in SN
Positive SN n = 15
Negative SN n = 44
43.9 (31–62)
52.2 (29–77)
5 (33.3%) 10 (66,7%)
24 (54.5%) 20 (45.5%)
10 (66.7%) 5 (33.3%)
35 (79.5%) 9 (20.5%)
11/15 (73.3%) 3/15 (20%) 1/15 (66.7%)
29/37 (78.4%) 7/37 (19.9%) 1/37 (2.7%)
0.01 NS
NS
NS (0.3–6.3) (20%) (33.3%) (46.7%) (40%) (28.6%) (60%)
7/15 (46.7%)
2.9 13 20 11 10/38 3/34 21/38
(0.3–6) (29.5%) (45.5%) (25%) (26.3%) (8.8%) (55.2%)
6/44 (13.6%)
SN = sentinel node; LVSI = lymphovascular space involvement.
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NS NS NS 0.02
HPV DNA negative SN n=4
Characteristics
HPV DNA positive SN n = 12
Mean age, years (range) FIGO stage Ia1 to Ib1 Ib2 to IIb Histology Squamous cell carcinoma Adenocarcinoma Differentiation grade Low (grade 1) Intermediate (grade 2) High (grade 3) Tumour size (cm) Mean (range) < 2 cm 2–4 cm > 4 cm LVSI Parametrial invasion Positive SN HPV DNA in primary cervical cancer
50,3 (31–77)
50.1 (29–77)
7 (58.3%) 5 (41,7%)
22 (46.8%) 25 (53.2%)
6 (50%) 6 (50%)
39 (83%) 8 (17%)
P
NS
3.6 3 5 7 4/10 2/7 9/15
Table 3. Relation between HPV DNA detection in SN and clinical and histological prognostic factors P
NS NS
0.04
NS 7/10 (70%) 3/10 (30%) 0
33/41 (78.6%) 7/41 (16.6%) 1/41 (4.8%)
3.1 (0.6–6) 3 (25%) 6 (50%) 3 (25%) 2/10 (20%) 0/7 7/12 (58.3%) 8/12 (66.7%)
3.1 13 19 15 12/38 5/29 8/47 22/41
NS (0.3–6.3) (27.7%) (40.4%) (31.9%) (31.6%) (14.7%) (17%) (53.7%)
NS NS 0.01 NS
SN = sentinel node; LVSI = lymphovascular space involvement.
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Table 2. Relation between SN involvement and clinical and histological prognostic factors
discussion
original article
Annals of Oncology
Table 4. Relation of recurrence with HPV DNA detection and clinicalhistological prognostic factors Characteristics
Women with follow-up (n = 53)
P
48.9 (30–69)
NS NS
3 (42.9%) 4 (57.1%) NS 4 (57.1%) 3 (42.9%) NS 5 (71.4%) 1 (14.3%) 1 (14.3%) NS 3.1 2 3 2 3 3
(0.5-6) (28.6%) (42.9%) (28.6%) (42.9%) (42.9%)
NS 0.04 NS
1 (14.3%) NS 1 (14.3%)
5/7 (71.5%)
NS
3/2
NS
1/7 (14%) 0/1 1/6 (17%) 1/0
NS NS
LVSI = lymphovascular space involvement; H&S = hematoxylin and eosin; IHC = immunohistochemistry.
relation between lymph node involvement and HPV status. In contrast, Baay et al. [18] found no relation between HPV DNA detection and lymph node status. However, these authors analysed only one node from the obturator region of each patient, whereas we found that the most common site of SN was the medial external iliac region (68%) and that only 9% of SN were located in the obturator area, in keeping with other studies [9, 19]. The low rate of negative SN with detectable HPV DNA (5.6%) contrasts somewhat with previous reports of HPV DNA detection in 23–60% of tumor-free pelvic lymph nodes. The high reported frequency of HPV DNA detection in negative lymph nodes could be explained by differences in histological analysis. Indeed, previous studies used the standard H&S technique, and this can miss some metastases detected by the SN method based on systematic multiple sectioning and immunohistochemical labeling. Indeed, most of the positive SN
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Mean age, years (range) 48.6 (29–77) FIGO stage Ia1 to Ib1 27 (50.9%) Ib2 to IIb 26 (49.1%) Histology Squamous cell carcinoma 40 (75.5%) Adenocarcinoma 13 (24.5%) Differentiation grade Well differentiated 37 (69.8%) Moderately differentiated 9 (17%) Poorly differentiated 2 (3.8%) Tumor size (cm) Mean (range) 2.9 (0.3–6) <2 cm 16 (30.2%) 2–4 cm 22 (41.5%) >4 cm 15 (28.3%) LVSI 12 (22.6%) Parametrial invasion 5 (9.4%) Sentinel node (SN) Number of patients with 14 (26.4%) positive SN Pelvic lymph node (PLN) Number of patients with 16 (30.2%) positive PLN HPV DNA status HPV DNA in primary 27/49 cervical cancer Type of HPV: HPV 16 / HPV 18 HPV DNA in SN 10/53 (18.9%) Positive SN Negative SN Type of HPV: HPV 16 / HPV 18
Women with recurrence (n = 7)
in our study contained micrometastases or isolated tumor cells. Hence, our results suggest that HPV DNA detection in pelvic lymph nodes might be a marker of metastatic involvement and that HPV DNA-positive lymph nodes might qualify for histological analysis similar to that applied to SN, leading to better detection of so-called ‘occult’ metastasis. It has been suggested that lymph nodes should not only be screened for HPV DNA, but also for HPV RNA and CK 19 mRNA, or be quantified by real-time PCR [17, 20]. We found no relationship between SN status, and classical clinical or pathological prognostic parameters such as FIGO stage, histological grade, and lymphovascular space involvement. No relation was found either between the HPV DNA status of pelvic lymph nodes and prognostic factors. Moreover, no relation was noted between the HPV DNA status of pelvic lymph nodes and the risk of recurrence. This could be explained by the fact that nearly half the positive SN were HPV DNA-negative, and also by the limited sample size. The lack of a strict relation between HPV DNA and SN involvement could be related to elimination of viral DNA molecules by scavenger lymphocytes and to HPV clearance from lymph nodes [17, 18, 21, 22]. The prognostic significance of HPV DNA detection in pelvic lymph nodes is controversial. For Pilch et al. [23]. and Hernardi et al. [16], lymph node HPV DNA status is a significant predictor of recurrent disease. Moreover, Burnett et al. [24] detected HPV DNA in five of six patients with recurrence, and Kobayashi et al. [25] in all nine patients with clinical relapse despite pelvic node non-involvement. In contrast, Hording et al. [26] detected HPV DNA in pelvic nodes in two of 10 patients with recurrence and in three of eight patients in remission. These discrepancies could be related to the small sample sizes. We detected HPV DNA (mainly HPV-16) in 57% of cervical cancers, in keeping with a reported prevalence of HPV DNA in cervical cancers ranging from 48 to 100% [3, 18, 22]. Unger et al. [27]. suggested that the presence of HPV DNA in primary cervical cancer was associated with poor survival. In contrast, we found no relation between HPV DNA and the FIGO stage or other classical prognostic factors. Our data are in line with those of van Bommel et al. [6], showing no difference in survival between women with and without HPV DNA in their cervical tumors. The high frequency of HPV-16 in our study is in line with epidemiological studies showing its predominance in Europe and North America [1]. Like Barnes et al. [28], we found that HPV-18 tended to be associated with a high risk of recurrence: HPV-18 DNA was found in the primary tumors of two of the five patients who recurred, and in only two of the 22 patients who remained in remission. No relationship was found between HPV DNA detection in the primary cervical tumor and in the sentinel node. Indeed, HPV DNA was detected in positive SN of two of three patients with negative primary tumors. These results are in keeping with those of Fu¨le et al. [21], showing the lack of any close correlation between metastasis and viral detection in lymph nodes. Beyer-Finkler et al. [29] pointed out that lymph node HPV DNA might originate from disintegrating tumor cells, being conveyed passively by lymphatic flow or actively by phagocytic cells. Fu¨le et al. showed that, in highly heterogeneous advanced-stage tumors with numerous genetic
Annals of Oncology
acknowledgements Charles COUTANT was a fellow from ‘Fondation pour la Recherche Me´dicale’. We are particularly grateful to ACTT (‘Ami du Centre des Tumeurs de Tenon’).
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aberrations, the loss of HPV DNA did not affect the proliferative capacity of cell clones [21]. Our study confirms the relevance of the SN procedure for assessing lymph node status in women with cervical cancer. The detection rate (85%) was slightly lower than that obtained elsewhere with dual tracer detection [9, 30], possibly owing to the inclusion of women with locally advanced cervical cancer in whom the detection rate is lower than in women with earlier stages [19]. However, some limitations of the present study have to be underlined such as the small sample size and the low rate of HPV-DNA detection in both primary tumors and sentinel nodes. In conclusion, this study suggests that the combination of the sentinel node procedure and HPV DNA screening of the excised nodes might contribute to better evaluation of lymph node status in women with cervical cancer and, hence, better identification of women who have an increased risk of recurrence. Further studies with larger populations are required to evaluate the prognostic relevance of HPV DNA detection in pelvic lymph nodes and in primary cervical tumors.
original article