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The accuracy of the sentinel lymph node concept in early stage squamous cell vulvar carcinoma
Gynecologic Oncology 116 (2010) 473–477
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
The accuracy of the sentinel lymph node concept in early stage squamous cell vulvar carcinoma Jakub Radziszewski a,⁎, Magdalena Kowalewska b, Tomasz Jedrzejczak c, Izabella Kozlowicz-Gudzinska c, Anna Nasierowska-Guttmejer d, Mariusz Bidzinski e, Janusz A. Siedlecki b a
Department of Brachytherapy, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland Department of Molecular Biology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland Department of Nuclear Medicine and Oncological Endocrinology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland d Central Clinical Hospital of the Ministry of Internal Affairs and Administration, Woloska 137, 02-507 Warsaw, Poland e Department of Gynecological Oncology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland b c
a r t i c l e
i n f o
Article history: Received 4 July 2009 Available online 17 November 2009 Keywords: Vulvar carcinoma Sentinel lymph node Patent blue V Technetium-99m
a b s t r a c t Objective. The purpose of the study was to determine the feasibility and accuracy of the sentinel lymph node (SLN) identification in vulvar carcinoma patients. Methods. Sixty-two patients with clinical early stage vulvar cancer underwent SLN detection procedure, followed by a complete inguinofemoral lymphadenectomy. The SLN was identified intraoperatively using lymphoscintigraphy with technetium-99m as well as patent blue V staining. The resected lymph nodes (LN) were submitted for histological examination by hematoxylin–eosin staining (H–E) and cytokeratin immunohistochemistry (IHC) and examined by the reverse transcriptase-polymerase chain reaction (RTPCR) assay. Results. A total of 109 inguinal LN were dissected in 56 patients. SLNs were identified in 76% groins with patent blue V and in 99% with the use of Tc-99m. The accuracy differed significantly (p b 0.0001). An H–E examination combined with IHC revealed 7 false-negative SLNs. The sensitivity of this method was 73% (95% CI, 64% to 81%) and the negative predictive value for a negative SLN finding was 92% (95% CI, 87% to 97%). The RT-PCR assay showed 8 false-negative SLNs. The sensitivity of the RT-PCR-based assay was 83% (95% CI, 75% to 90%) and the negative predictive value for a negative SLN was 88% (95% CI, 82% to 94%). The two diagnostic methods were found not to differ significantly. Conclusions. In SLN mapping, the Tc-99m colloid lymphoscintigraphy is superior to the blue dye staining. Our data do not support the concept of the SLN identification as a highly accurate procedure in predicting the inguinofemoral LN status in patients with early stage vulvar cancer. © 2009 Elsevier Inc. All rights reserved.
Introduction Vulvar cancer accounts for 2.5% to 5% of all gynaecological malignancies. In 2005, 406 new cases of vulvar cancer were diagnosed and 169 deaths due to this malignancy were noted in Poland [1]. During last years, an increasing trend in morbidity from vulvar cancer is observed, explained by the extending life span of the female population and the increasing prevalence of the human papillomavirus (HPV) infections. Cancer of the vulva usually affects women over the age of 60 years [2], the majority of whom are seriously compromised by other diseases, such as coronary heart disease, arterial hypertension and diabetes. Approximately 90% of vulvar malignancies are squamous cell carcinomas. Vulvar cancer spreads by local invasion and via the lymphatics to the inguinal and femoral
⁎ Corresponding author. Fax: +48 22 644 01 18. E-mail address: [email protected] (J. Radziszewski). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.10.072
lymph nodes, followed the deep pelvic nodes [2]. Hematogenous spread appears to be uncommon in the early stages of the disease. Radical vulvectomy and bilateral inguinal and femoral node dissection, with care taken to ensure tumour-free margins (more than 8 mm) in early stage vulvar cancer, i.e. stages T1 (tumour confined to the vulva or vulva and perineum, 2 cm or less in greatest dimension, and with stromal invasion greater than 1 mm) and T2 (tumour confined to the vulva or vulva and perineum, more than 2 cm and less than 4 cm in greatest dimension), remain a standard surgical treatment [3]. Although the 5-year survival rates in early stage cancer after radical surgery are relatively good (98% for stage I and 85% for stage II according to Gynaecologic Oncology Group [4]), the morbidity associated with this procedure is significant. The most common complications include postoperative wound healing problems, usually associated with infections and chronic lymphedema of the lower extremities, which must negatively influence the immune system function. Attempts to reduce leg lymphedema are ongoing, but unsuccessful so far [5]. These and other complications often prolong
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hospitalization time (median postoperative hospitalization time varies from 2 to 6 weeks) and delay introduction of the adjuvant therapy when needed. There is a general agreement that this aggressive treatment is a correct policy for patients with metastases in inguinal nodes. However, in early stages (I and II), the probability of a positive inguinal lymph node finding is only 10–26% [6,7]. Thus, approximately 80% of patients in stage I and II vulvar cancer undergo unnecessary lymphadenectomy. Novel diagnostic tests with an increased sensitivity are highly desirable in order to better identify patients without metastases, who should not undergo lymphadenectomy. Clinical examination does not present acceptable sensitivity and specificity, up to 24% of patients with clinically normal lymph nodes have metastases, and more than 20% of patients with enlarged nodes in clinical examination are found metastases-free in histopathologic examination [8]. Numerous non-invasive and minimally invasive tests, i.e. ultrasonography with or without fine needle aspiration, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), are inadequate. The sensitivity of these tests in determining the groin node status ranges from 45% to 86%, with negative likelihood ratio ranging from 0.12 to 0.6 [9–11]. The sentinel lymph node (SLN) biopsy provides an alternative procedure. The SN concept was proposed in 1977 by Cabanas for the management of penile cancer using lymphography. He reported that the lymphatic drainage of the penis merges into a group of lymph nodes called “sentinel lymph nodes” [12]. Nowadays, there are two methods of intraoperative SLN identification. Both were introduced in melanomas, the first by Morton et al. [13], who used blue dye injections to visualize the lymphatic flow pathways, and the second by Alex et al. [14], who implemented a direct localization using radioactive isotopes. Currently, the SLN identification technique is part of routine practice in the management of patients with early stages of breast cancer and melanoma. In vulvar carcinoma, sentinel lymph node identification using technetium-99m-labelled nanocolloid (alone or with blue dye) is the most accurate method, with a sensitivity of 97% and a negative likelihood ratio of 0.12, as reported by Selman et al. [8] based on pooled data published between 1979 and 2004. Positive non-sentinel nodes in the presence of negative SLN (i.e. false-negative SLNs) were reported in just a few published case studies [15]. The available data suggest that SLN detection procedure should only be performed in patients with small vulvar carcinoma, when tumour size is less than 4 cm in diameter with clinically negative groin nodes, and without infiltration to adjacent organs. The aim of this study was to evaluate the accuracy of sentinel lymph node identification with technetium-99m-labeled nanocolloid and blue dye. Since the predictive value of the SLN detection depends on the accuracy of the histopathologic investigation, we have examined lymph nodes by a combined staining with H–E and IHC, as well as with the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the cancer-associated marker, carbonic anhydrase 9 (CA9). CA9 is found in many tumours (with an exception of those of the gastrointestinal tract) but not in the corresponding normal tissues. CA9 expression is associated with hypoxia, a typical phenomenon for solid tumours [16]. Materials and methods Post-operative material from 62 consecutive patients treated at the Departments of Gynaecological Oncology and Brachytherapy of the Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw (MSCMCC) between January 2002 and December 2006 was studied (i.e. a time intended for the realization of a research grant from the Polish Ministry of Science and Higher Education). The inclusion criteria comprised the WHO (Zubrod)
overall performance status I–II, histopathologically confirmed invasive squamous cell carcinoma of the vulva and perineum, tumour 4 cm or less in greatest dimension, and with stromal invasion greater than 1 mm, clinical stage T1-2, N0, M0 and no history of previous treatment for either vulvar cancer or any other malignancy. Before final qualification patients underwent a thorough medical examination, and a number of diagnostic investigations, including a chest X-ray and abdominal ultrasound, being part of a routine examination before surgery at the MSCMCC were performed. A groins ultrasound examination was performed, and if there were suspicious nodes found, fine needle aspiration LN biopsies were done. Patients with positive nodes were excluded from the study. The study was approved by the Independent Ethics Committee of MSCMCC, and all patients gave their written informed consent before inclusion in the study. For all the eligible patients, the standardized protocol included three intradermal injections of Tc-99m isotope (technetium-99 colloid albumin, activity 1.2 mCi), applied in the direct vicinity of the vulvar tumour 24 h before surgery. The injections were given by the operating surgeon. One hour after isotope administration, a scintigraphic examination of the inguinal lymph nodes was performed in order to assess the areas of isotope accumulation. Scintigraphy was performed with a double-headed high-resolution VARICAM gamma probe (ELSCINT) with a low-energy collimator. The data were registered on a 256 × 256 matrix, zoom 1, over 10 min (approximately 100,000 counts from each head). The injection site was covered with leaden shielding (0.5 cm thick). EXPERT software (ELSCINT) was used for the processing of the results and figures. All patients were operated by a team of six surgeons/gynaecologists with over 10 years of experience, from the Departments of Brachytherapy and Gynaecological Oncology of the MSCMCC. In order to visualize the sentinel lymph node, a 2.5% solution of patent blue V was injected three times intradermally in the same way and points as the isotope in the direct vicinity of the vulvar tumour 10 min before skin incision. Inguinal lymph node excision was performed as the first step of the procedure. The sentinel nodes were identified with a handheld gamma probe equipped with a radiation meter (navigator gamma positioning system (GPS)) in the following sequence of measurements: (1) above the skin of the inguinal area and (2) after skin incision to identify the sentinel nodes “in vivo”. The blue and/or hot nodes were recognized as sentinels. After removal of the sentinels (either one or multiple nodes over which we detected the highest isotope accumulation), radioactivity of the wound itself was measured and noted as the so-called “background uptake”. The surgery was continued with radical excision of the entire group of inguinal lymph nodes and vulva, in accordance with the present MSCMCC standards of treatment of T1-2, N0-2, M0 squamous carcinoma of the vulva. In the operating theatre, all tissue specimens (i.e. SLNs and the corresponding inguinal LNs) were immediately divided into two parts. The cutting line was either through the hilus of the groin LN or over the hottest/blue spot in the SLN. One part of each tissue specimen was divided into 4–6 slices. Histopathologic examination was performed with routine hematoxylin and eosin staining (H–E) in half of the slices and with IHC staining in the rest of them. For immunohistochemical purposes, anti-human cytokeratin MAb clone CKMNF 116, which binds keratins 5, 6, 17 and 19 (Dako, Copenhagen, Denmark), was used to identify cells of epithelial origin in lymph node specimens. The other part of each specimen was analyzed for the expression of the cancer-associated CA9 marker by RT-PCR with specific primers (17). The sentinel lymph nodes were examined independently of the remaining lymph nodes. The accordance between the different methods of the nodal status assessment was verified using the McNemar test. The sensitivity and likelihood ratio were also calculated.
J. Radziszewski et al. / Gynecologic Oncology 116 (2010) 473–477 Table 1 SLN identification with isotope and blue dye. Identification method
Numbers of SLNs identified/left groins
Numbers of SLNs identified/right groins
Total numbers of SLNs identified/all groins analyzed
Patent blue V Isotope
41/52 51/51
41/56 55/56
82/108 (76%) 106/107 (99%)
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from the study. Using the highly sensitive RT-PCR method, metastases were found in 38 of 107 SLNs and in 46 of 107 LNs in the monomial groins. The rate of false-negative sentinel lymph nodes was 17% (8/46) and was statistically significant (p = 0.0047). The sensitivity of detection of SLN involvement by this method was nearly 10% higher than by the combined H–E and ICH analysis, but the negative predictive value of the two methods differed less than 4% (Table 2).
Results
Discussion
A total of 62 patients with stage I and II vulvar carcinoma (age 37 to 94 years, median age 68 years) were qualified for the surgical treatment and enrolled in the study. However, six patients were excluded from the final analysis, as they failed to meet all study entry criteria (for instance, fine needle aspiration (FNA) revealed metastases in the inguinal lymph nodes). Fifty-six patients were operated on according to the protocol described above. Fifty-one patients had bilateral inguinofemoral lymphadenectomies, in 5 patients a half of the vulva and unilateral lymph nodes were removed. In 32% of patients, the primary tumour was in stage T1, in the remaining 68%— in stage T2. The accuracy of the sentinel lymph node procedure (using the isotope and dye methods) was analyzed in 56 patients. For SLN identification, lymphoscintigraphy was performed in 51 left and 56 right groins, and patent blue V staining—in 52 left and 56 right groins. The identification procedure with radioisotope was successful in 106 out of 107 cases (51 in the left groin, 55 in the right groin). Unfortunately in one case, the 56th right groin, SLN could not be detected due to technical problems with the GPS navigator. Using blue dye, 82 SLNs were visualized in 108 groins (41 in the left groin, 41 in the right groin). All specimens recognized as SLN consisted of the lymphatic tissue. The isotope-based method showed a much higher identification rate (99%) than the blue dye method (76%). The difference is statistically significant (p b 0.0001) (Table 1). No complications associated with the SLN procedures were observed. LNs from 109 groins of 56 patients with vulvar carcinoma were analyzed after histopathologic examination by both routine H–E and IHC staining. In 19 cases (17%), metastases were found in LNs recognized as sentinels. In 26 cases, metastases were found in monomial inguinofemoral LNs; therefore, seven cases of falsenegative SLNs were obtained (27%). Sensitivity and the negative prognostic values of the SLN status are shown in Table 2. Finally, we employed RT-PCR in order to assess the predictive value of sentinels. We have used several cancer-associated markers (i.e. β-hCG, mammaglobin A, EGFR, SCCA; data not shown) from which we eventually chose carbonic anhydrase 9 (CA9). Due to technical problems, specimens obtained from two patients were lost
In vulvar carcinoma, sentinel lymph node procedure seems to be the method of choice because of the lymphatic spread of this cancer and easy access to the regional lymph nodes. As mentioned above, none of non-invasive methods is accurate enough to predict the status of inguinal lymph nodes, especially in the early stages of the disease. According to the review of the multiple studies on the accuracy of the different techniques of diagnosing the inguinal lymph node status, the sentinel node identification using Tc-99m appears to be the most promising test to exclude inguinal metastases and to potentially reduce the excessive radical surgery [8]. The first data on lymphatic mapping of SLN in vulvar cancer using dye staining (patent blue) and lymphoscintigraphy were published by Levenback et al. [18] in 1994 and by Decesare et al. [19] in 1997, respectively. The two methods were first combined by de Hullu and colleagues [20] in 1998. We confirm the leading role of Tc-99m isotope in the SLN identification, which in our study had a high sensitivity of 99%. The sensitivity of dye staining with patent blue V at the level of 75% observed in our research is highly insufficient for SLN identification in vulvar cancer and cannot be used alone. These observations are consistent with literature data [20,21]. Levenback et al. [22] reported that the assessment of SLN status in squamous cell vulvar cancer predicts the absence/presence of metastases in the other non-sentinel LNs. This observation was confirmed by further research [19,23–28]. In the published data, the cases of false-negative SLNs are rare [29,30], but the number of patients in these studies was low. Although in some patients with vulvar carcinoma routine inguinofemoral lymphadenectomy may be of no benefit, a routine desist from performing radical lymphadenectomy in stages I and II of vulvar carcinoma is still not considered as a standard procedure. Groin relapse following a negative SLN biopsy is of concern and suggests a need of long-term follow-up [31]. For safety of the SLN biopsy, it is extremely important that the results obtained in the institution planning to implement this procedure should be comparable to the published data. Our results are inconsistent with the data reported by authors from the reference centers in other countries. The 74% sensitivity of SLN examination with the routine hematoxylin–eosin staining combined with immunohistochemistry means that in one fourth of the patients the existing
Table 2 The results of sentinel lymph node status examination. Method
Number of patients with LNs uninvolved Number of patients with LN involvement Number of SLNs with metastases Number of LNs with metastases in monomial groins Number of false-negative SLNs Sensitivity 95% CI Negative predictive value 95% CI
H–E + IHC
RT-PCR
35 (62.5%) 21 (35.5%) 19/109 (17%) 26/109 (24%) 7/26 (27% ), p b 0.0082 73.08%, 64.75–81.40% 92.22%, 87. 19–97.25%
33 (61%) 21 (38%) 38/107 (35%) 46/107 (43%) 8/46 (17%), p b 0.0047 82.61%, 75.43–89.79% 88.41%, 82.34–94.47%
H–E, hematoxylin–eosin staining; IHC, cytokeratin immunohistochemistry; LN, lymph node; SLN, sentinel lymph node; CI, confidence interval.
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metastases were not detected and those patients may not receive an optimal treatment. In addition, a false-negative ratio of 6% is unacceptable, especially in light of the results of surveys of vulvar cancer patients who have undergone complete inguinofemoral lymphadenectomy. The majority of these surveys would not advise to introduce the SLN procedure with the 5% rate of missing a lymph node metastasis [32]. In the RT-PCR method, sensitivity reached 83% and false-negative SLNs ratio was 10% lower. As expected, the RT-PCR test detected more micrometastases, namely in 43% of examined groins versus 24% detected by the combined H–E and IHC examination. As we have previously shown, CA9 marker is useful in the tumour cell detection in the lymph nodes of vulvar carcinoma patients by RT-PCR [17]. The impact on survival and recurrence rate of these micrometastases needs to be evaluated in further studies. Several possible reasons for the inconsistency concerning the rate of false-negative SLNs between our results and other published data can be ruled out. The quality of isotope and diagnostic equipment (Neoprobe gamma camera) were standardized and fulfilled regulations. The injections of isotope were made according to the recommendations of other authors [23,25]. The handling mistake during tissue collection, marking and transport were possible, but unlikely to be responsible for such a high rate of false-negative results, especially as the whole procedure was carefully monitored once the first false-negative result was obtained. Histopathologic H–E and IHC examinations were performed by an experienced pathologist. Our RTPCR assay was found to be very sensitive and all the control requirements were met [17]. It is highly probable that the main factor responsible for the high false-negative SLN number was the surgeons' experience. Although all of the operations were performed by surgeons and gynaecologists with at least 15-year experience, the SLN detection procedure, according to the protocol used, was performed only a few times by each of them. The SLN biopsy is a technically challenging and demanding procedure that requires attention to detail. The “learning curve” is defined as the optimum number of sentinel node biopsy cases combined with groin dissection that the surgeon should perform to obtain a certain experience. There is no definite consensus on how many procedures surgeons need to perform before they pass the learning curve [33]. Morton et al. [34] commended that usually a surgeon must perform at least 30 cases to achieve accurate staging of the regional nodal basin in melanoma. It is worth noticing that the sentinel node procedure routinely applied in other malignancies (e.g. breast cancer) is not performed in Poland by gynaecologists. Therefore, we can suspect that in our study, the learning curve has not been achieved. Because the incidence of vulvar carcinoma in Poland is rather low compared to other malignancies, it is questionable how much time it will take before each of the gynaecologists, even in a reference centre, will pass the learning curve. In the MSCMCC, we needed 4 years to enroll 62 eligible patients. Unfortunately, according to the operating surgeons, the low number of patients with vulvar cancer in their experience could not allow to draw conclusions on the improvement of the patients' management in the MSCMCC. One additional aspect has not been addressed so far, in countries with low population density, some patients have to travel long distances to be treated in one of the few reference centers. Some of vulvar carcinoma patients may not be willing to or able to take this opportunity and eventually choose to be treated in the nearest community hospital. It is questionable if any of the gynaecologists working in the community hospital will pass the learning curve. Results of a multicenter observational study on SN detection show low rate groin recurrence, excellent survival and minimal treatment-related morbidity in patients with negative SN. However, the authors suggest that the sentinel node dissection should be performed by a quality-controlled multidisciplinary team [35]. Papers describing the accuracy of sentinel lymph node procedure in squamous cell carcinoma of the
vulva were already published but negative data only in case reports. Our study points problems that may occur if this procedure would be wide implemented. Conclusion We conclude that the most accurate method of identification of the sentinel lymph node in vulvar carcinoma is preoperative lymphoscintigraphy (administration of Tc-99m) and intraoperative biopsy under gamma probe. The dye staining alone is insufficient for SLN identification. The RT-PCR detection of CA9 can be recommended for further systematic examination of its use for the detection of vulvar carcinoma cells in the lymph nodes. The RT-PCR test presents higher sensitivity than histopathologic examination with combined H–E and cytokeratin IHC stainings. In our settings, the SLN procedure cannot be recommended as a reliable procedure to avoid radical inguinofemoral lymphadenectomy in early stages of vulvar cancer because of high false-negative SLN rate. In summary, further investigation is needed, especially to assess indispensable conditions to gain the learning curve expertise. Conflict of interest statement The authors declare that there are no conflicts of interest.
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
The accuracy of the sentinel lymph node concept in early stage squamous cell vulvar carcinoma Jakub Radziszewski a,⁎, Magdalena Kowalewska b, Tomasz Jedrzejczak c, Izabella Kozlowicz-Gudzinska c, Anna Nasierowska-Guttmejer d, Mariusz Bidzinski e, Janusz A. Siedlecki b a
Department of Brachytherapy, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland Department of Molecular Biology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland Department of Nuclear Medicine and Oncological Endocrinology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland d Central Clinical Hospital of the Ministry of Internal Affairs and Administration, Woloska 137, 02-507 Warsaw, Poland e Department of Gynecological Oncology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland b c
a r t i c l e
i n f o
Article history: Received 4 July 2009 Available online 17 November 2009 Keywords: Vulvar carcinoma Sentinel lymph node Patent blue V Technetium-99m
a b s t r a c t Objective. The purpose of the study was to determine the feasibility and accuracy of the sentinel lymph node (SLN) identification in vulvar carcinoma patients. Methods. Sixty-two patients with clinical early stage vulvar cancer underwent SLN detection procedure, followed by a complete inguinofemoral lymphadenectomy. The SLN was identified intraoperatively using lymphoscintigraphy with technetium-99m as well as patent blue V staining. The resected lymph nodes (LN) were submitted for histological examination by hematoxylin–eosin staining (H–E) and cytokeratin immunohistochemistry (IHC) and examined by the reverse transcriptase-polymerase chain reaction (RTPCR) assay. Results. A total of 109 inguinal LN were dissected in 56 patients. SLNs were identified in 76% groins with patent blue V and in 99% with the use of Tc-99m. The accuracy differed significantly (p b 0.0001). An H–E examination combined with IHC revealed 7 false-negative SLNs. The sensitivity of this method was 73% (95% CI, 64% to 81%) and the negative predictive value for a negative SLN finding was 92% (95% CI, 87% to 97%). The RT-PCR assay showed 8 false-negative SLNs. The sensitivity of the RT-PCR-based assay was 83% (95% CI, 75% to 90%) and the negative predictive value for a negative SLN was 88% (95% CI, 82% to 94%). The two diagnostic methods were found not to differ significantly. Conclusions. In SLN mapping, the Tc-99m colloid lymphoscintigraphy is superior to the blue dye staining. Our data do not support the concept of the SLN identification as a highly accurate procedure in predicting the inguinofemoral LN status in patients with early stage vulvar cancer. © 2009 Elsevier Inc. All rights reserved.
Introduction Vulvar cancer accounts for 2.5% to 5% of all gynaecological malignancies. In 2005, 406 new cases of vulvar cancer were diagnosed and 169 deaths due to this malignancy were noted in Poland [1]. During last years, an increasing trend in morbidity from vulvar cancer is observed, explained by the extending life span of the female population and the increasing prevalence of the human papillomavirus (HPV) infections. Cancer of the vulva usually affects women over the age of 60 years [2], the majority of whom are seriously compromised by other diseases, such as coronary heart disease, arterial hypertension and diabetes. Approximately 90% of vulvar malignancies are squamous cell carcinomas. Vulvar cancer spreads by local invasion and via the lymphatics to the inguinal and femoral
⁎ Corresponding author. Fax: +48 22 644 01 18. E-mail address: [email protected] (J. Radziszewski). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.10.072
lymph nodes, followed the deep pelvic nodes [2]. Hematogenous spread appears to be uncommon in the early stages of the disease. Radical vulvectomy and bilateral inguinal and femoral node dissection, with care taken to ensure tumour-free margins (more than 8 mm) in early stage vulvar cancer, i.e. stages T1 (tumour confined to the vulva or vulva and perineum, 2 cm or less in greatest dimension, and with stromal invasion greater than 1 mm) and T2 (tumour confined to the vulva or vulva and perineum, more than 2 cm and less than 4 cm in greatest dimension), remain a standard surgical treatment [3]. Although the 5-year survival rates in early stage cancer after radical surgery are relatively good (98% for stage I and 85% for stage II according to Gynaecologic Oncology Group [4]), the morbidity associated with this procedure is significant. The most common complications include postoperative wound healing problems, usually associated with infections and chronic lymphedema of the lower extremities, which must negatively influence the immune system function. Attempts to reduce leg lymphedema are ongoing, but unsuccessful so far [5]. These and other complications often prolong
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hospitalization time (median postoperative hospitalization time varies from 2 to 6 weeks) and delay introduction of the adjuvant therapy when needed. There is a general agreement that this aggressive treatment is a correct policy for patients with metastases in inguinal nodes. However, in early stages (I and II), the probability of a positive inguinal lymph node finding is only 10–26% [6,7]. Thus, approximately 80% of patients in stage I and II vulvar cancer undergo unnecessary lymphadenectomy. Novel diagnostic tests with an increased sensitivity are highly desirable in order to better identify patients without metastases, who should not undergo lymphadenectomy. Clinical examination does not present acceptable sensitivity and specificity, up to 24% of patients with clinically normal lymph nodes have metastases, and more than 20% of patients with enlarged nodes in clinical examination are found metastases-free in histopathologic examination [8]. Numerous non-invasive and minimally invasive tests, i.e. ultrasonography with or without fine needle aspiration, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), are inadequate. The sensitivity of these tests in determining the groin node status ranges from 45% to 86%, with negative likelihood ratio ranging from 0.12 to 0.6 [9–11]. The sentinel lymph node (SLN) biopsy provides an alternative procedure. The SN concept was proposed in 1977 by Cabanas for the management of penile cancer using lymphography. He reported that the lymphatic drainage of the penis merges into a group of lymph nodes called “sentinel lymph nodes” [12]. Nowadays, there are two methods of intraoperative SLN identification. Both were introduced in melanomas, the first by Morton et al. [13], who used blue dye injections to visualize the lymphatic flow pathways, and the second by Alex et al. [14], who implemented a direct localization using radioactive isotopes. Currently, the SLN identification technique is part of routine practice in the management of patients with early stages of breast cancer and melanoma. In vulvar carcinoma, sentinel lymph node identification using technetium-99m-labelled nanocolloid (alone or with blue dye) is the most accurate method, with a sensitivity of 97% and a negative likelihood ratio of 0.12, as reported by Selman et al. [8] based on pooled data published between 1979 and 2004. Positive non-sentinel nodes in the presence of negative SLN (i.e. false-negative SLNs) were reported in just a few published case studies [15]. The available data suggest that SLN detection procedure should only be performed in patients with small vulvar carcinoma, when tumour size is less than 4 cm in diameter with clinically negative groin nodes, and without infiltration to adjacent organs. The aim of this study was to evaluate the accuracy of sentinel lymph node identification with technetium-99m-labeled nanocolloid and blue dye. Since the predictive value of the SLN detection depends on the accuracy of the histopathologic investigation, we have examined lymph nodes by a combined staining with H–E and IHC, as well as with the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the cancer-associated marker, carbonic anhydrase 9 (CA9). CA9 is found in many tumours (with an exception of those of the gastrointestinal tract) but not in the corresponding normal tissues. CA9 expression is associated with hypoxia, a typical phenomenon for solid tumours [16]. Materials and methods Post-operative material from 62 consecutive patients treated at the Departments of Gynaecological Oncology and Brachytherapy of the Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology in Warsaw (MSCMCC) between January 2002 and December 2006 was studied (i.e. a time intended for the realization of a research grant from the Polish Ministry of Science and Higher Education). The inclusion criteria comprised the WHO (Zubrod)
overall performance status I–II, histopathologically confirmed invasive squamous cell carcinoma of the vulva and perineum, tumour 4 cm or less in greatest dimension, and with stromal invasion greater than 1 mm, clinical stage T1-2, N0, M0 and no history of previous treatment for either vulvar cancer or any other malignancy. Before final qualification patients underwent a thorough medical examination, and a number of diagnostic investigations, including a chest X-ray and abdominal ultrasound, being part of a routine examination before surgery at the MSCMCC were performed. A groins ultrasound examination was performed, and if there were suspicious nodes found, fine needle aspiration LN biopsies were done. Patients with positive nodes were excluded from the study. The study was approved by the Independent Ethics Committee of MSCMCC, and all patients gave their written informed consent before inclusion in the study. For all the eligible patients, the standardized protocol included three intradermal injections of Tc-99m isotope (technetium-99 colloid albumin, activity 1.2 mCi), applied in the direct vicinity of the vulvar tumour 24 h before surgery. The injections were given by the operating surgeon. One hour after isotope administration, a scintigraphic examination of the inguinal lymph nodes was performed in order to assess the areas of isotope accumulation. Scintigraphy was performed with a double-headed high-resolution VARICAM gamma probe (ELSCINT) with a low-energy collimator. The data were registered on a 256 × 256 matrix, zoom 1, over 10 min (approximately 100,000 counts from each head). The injection site was covered with leaden shielding (0.5 cm thick). EXPERT software (ELSCINT) was used for the processing of the results and figures. All patients were operated by a team of six surgeons/gynaecologists with over 10 years of experience, from the Departments of Brachytherapy and Gynaecological Oncology of the MSCMCC. In order to visualize the sentinel lymph node, a 2.5% solution of patent blue V was injected three times intradermally in the same way and points as the isotope in the direct vicinity of the vulvar tumour 10 min before skin incision. Inguinal lymph node excision was performed as the first step of the procedure. The sentinel nodes were identified with a handheld gamma probe equipped with a radiation meter (navigator gamma positioning system (GPS)) in the following sequence of measurements: (1) above the skin of the inguinal area and (2) after skin incision to identify the sentinel nodes “in vivo”. The blue and/or hot nodes were recognized as sentinels. After removal of the sentinels (either one or multiple nodes over which we detected the highest isotope accumulation), radioactivity of the wound itself was measured and noted as the so-called “background uptake”. The surgery was continued with radical excision of the entire group of inguinal lymph nodes and vulva, in accordance with the present MSCMCC standards of treatment of T1-2, N0-2, M0 squamous carcinoma of the vulva. In the operating theatre, all tissue specimens (i.e. SLNs and the corresponding inguinal LNs) were immediately divided into two parts. The cutting line was either through the hilus of the groin LN or over the hottest/blue spot in the SLN. One part of each tissue specimen was divided into 4–6 slices. Histopathologic examination was performed with routine hematoxylin and eosin staining (H–E) in half of the slices and with IHC staining in the rest of them. For immunohistochemical purposes, anti-human cytokeratin MAb clone CKMNF 116, which binds keratins 5, 6, 17 and 19 (Dako, Copenhagen, Denmark), was used to identify cells of epithelial origin in lymph node specimens. The other part of each specimen was analyzed for the expression of the cancer-associated CA9 marker by RT-PCR with specific primers (17). The sentinel lymph nodes were examined independently of the remaining lymph nodes. The accordance between the different methods of the nodal status assessment was verified using the McNemar test. The sensitivity and likelihood ratio were also calculated.
J. Radziszewski et al. / Gynecologic Oncology 116 (2010) 473–477 Table 1 SLN identification with isotope and blue dye. Identification method
Numbers of SLNs identified/left groins
Numbers of SLNs identified/right groins
Total numbers of SLNs identified/all groins analyzed
Patent blue V Isotope
41/52 51/51
41/56 55/56
82/108 (76%) 106/107 (99%)
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from the study. Using the highly sensitive RT-PCR method, metastases were found in 38 of 107 SLNs and in 46 of 107 LNs in the monomial groins. The rate of false-negative sentinel lymph nodes was 17% (8/46) and was statistically significant (p = 0.0047). The sensitivity of detection of SLN involvement by this method was nearly 10% higher than by the combined H–E and ICH analysis, but the negative predictive value of the two methods differed less than 4% (Table 2).
Results
Discussion
A total of 62 patients with stage I and II vulvar carcinoma (age 37 to 94 years, median age 68 years) were qualified for the surgical treatment and enrolled in the study. However, six patients were excluded from the final analysis, as they failed to meet all study entry criteria (for instance, fine needle aspiration (FNA) revealed metastases in the inguinal lymph nodes). Fifty-six patients were operated on according to the protocol described above. Fifty-one patients had bilateral inguinofemoral lymphadenectomies, in 5 patients a half of the vulva and unilateral lymph nodes were removed. In 32% of patients, the primary tumour was in stage T1, in the remaining 68%— in stage T2. The accuracy of the sentinel lymph node procedure (using the isotope and dye methods) was analyzed in 56 patients. For SLN identification, lymphoscintigraphy was performed in 51 left and 56 right groins, and patent blue V staining—in 52 left and 56 right groins. The identification procedure with radioisotope was successful in 106 out of 107 cases (51 in the left groin, 55 in the right groin). Unfortunately in one case, the 56th right groin, SLN could not be detected due to technical problems with the GPS navigator. Using blue dye, 82 SLNs were visualized in 108 groins (41 in the left groin, 41 in the right groin). All specimens recognized as SLN consisted of the lymphatic tissue. The isotope-based method showed a much higher identification rate (99%) than the blue dye method (76%). The difference is statistically significant (p b 0.0001) (Table 1). No complications associated with the SLN procedures were observed. LNs from 109 groins of 56 patients with vulvar carcinoma were analyzed after histopathologic examination by both routine H–E and IHC staining. In 19 cases (17%), metastases were found in LNs recognized as sentinels. In 26 cases, metastases were found in monomial inguinofemoral LNs; therefore, seven cases of falsenegative SLNs were obtained (27%). Sensitivity and the negative prognostic values of the SLN status are shown in Table 2. Finally, we employed RT-PCR in order to assess the predictive value of sentinels. We have used several cancer-associated markers (i.e. β-hCG, mammaglobin A, EGFR, SCCA; data not shown) from which we eventually chose carbonic anhydrase 9 (CA9). Due to technical problems, specimens obtained from two patients were lost
In vulvar carcinoma, sentinel lymph node procedure seems to be the method of choice because of the lymphatic spread of this cancer and easy access to the regional lymph nodes. As mentioned above, none of non-invasive methods is accurate enough to predict the status of inguinal lymph nodes, especially in the early stages of the disease. According to the review of the multiple studies on the accuracy of the different techniques of diagnosing the inguinal lymph node status, the sentinel node identification using Tc-99m appears to be the most promising test to exclude inguinal metastases and to potentially reduce the excessive radical surgery [8]. The first data on lymphatic mapping of SLN in vulvar cancer using dye staining (patent blue) and lymphoscintigraphy were published by Levenback et al. [18] in 1994 and by Decesare et al. [19] in 1997, respectively. The two methods were first combined by de Hullu and colleagues [20] in 1998. We confirm the leading role of Tc-99m isotope in the SLN identification, which in our study had a high sensitivity of 99%. The sensitivity of dye staining with patent blue V at the level of 75% observed in our research is highly insufficient for SLN identification in vulvar cancer and cannot be used alone. These observations are consistent with literature data [20,21]. Levenback et al. [22] reported that the assessment of SLN status in squamous cell vulvar cancer predicts the absence/presence of metastases in the other non-sentinel LNs. This observation was confirmed by further research [19,23–28]. In the published data, the cases of false-negative SLNs are rare [29,30], but the number of patients in these studies was low. Although in some patients with vulvar carcinoma routine inguinofemoral lymphadenectomy may be of no benefit, a routine desist from performing radical lymphadenectomy in stages I and II of vulvar carcinoma is still not considered as a standard procedure. Groin relapse following a negative SLN biopsy is of concern and suggests a need of long-term follow-up [31]. For safety of the SLN biopsy, it is extremely important that the results obtained in the institution planning to implement this procedure should be comparable to the published data. Our results are inconsistent with the data reported by authors from the reference centers in other countries. The 74% sensitivity of SLN examination with the routine hematoxylin–eosin staining combined with immunohistochemistry means that in one fourth of the patients the existing
Table 2 The results of sentinel lymph node status examination. Method
Number of patients with LNs uninvolved Number of patients with LN involvement Number of SLNs with metastases Number of LNs with metastases in monomial groins Number of false-negative SLNs Sensitivity 95% CI Negative predictive value 95% CI
H–E + IHC
RT-PCR
35 (62.5%) 21 (35.5%) 19/109 (17%) 26/109 (24%) 7/26 (27% ), p b 0.0082 73.08%, 64.75–81.40% 92.22%, 87. 19–97.25%
33 (61%) 21 (38%) 38/107 (35%) 46/107 (43%) 8/46 (17%), p b 0.0047 82.61%, 75.43–89.79% 88.41%, 82.34–94.47%
H–E, hematoxylin–eosin staining; IHC, cytokeratin immunohistochemistry; LN, lymph node; SLN, sentinel lymph node; CI, confidence interval.
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metastases were not detected and those patients may not receive an optimal treatment. In addition, a false-negative ratio of 6% is unacceptable, especially in light of the results of surveys of vulvar cancer patients who have undergone complete inguinofemoral lymphadenectomy. The majority of these surveys would not advise to introduce the SLN procedure with the 5% rate of missing a lymph node metastasis [32]. In the RT-PCR method, sensitivity reached 83% and false-negative SLNs ratio was 10% lower. As expected, the RT-PCR test detected more micrometastases, namely in 43% of examined groins versus 24% detected by the combined H–E and IHC examination. As we have previously shown, CA9 marker is useful in the tumour cell detection in the lymph nodes of vulvar carcinoma patients by RT-PCR [17]. The impact on survival and recurrence rate of these micrometastases needs to be evaluated in further studies. Several possible reasons for the inconsistency concerning the rate of false-negative SLNs between our results and other published data can be ruled out. The quality of isotope and diagnostic equipment (Neoprobe gamma camera) were standardized and fulfilled regulations. The injections of isotope were made according to the recommendations of other authors [23,25]. The handling mistake during tissue collection, marking and transport were possible, but unlikely to be responsible for such a high rate of false-negative results, especially as the whole procedure was carefully monitored once the first false-negative result was obtained. Histopathologic H–E and IHC examinations were performed by an experienced pathologist. Our RTPCR assay was found to be very sensitive and all the control requirements were met [17]. It is highly probable that the main factor responsible for the high false-negative SLN number was the surgeons' experience. Although all of the operations were performed by surgeons and gynaecologists with at least 15-year experience, the SLN detection procedure, according to the protocol used, was performed only a few times by each of them. The SLN biopsy is a technically challenging and demanding procedure that requires attention to detail. The “learning curve” is defined as the optimum number of sentinel node biopsy cases combined with groin dissection that the surgeon should perform to obtain a certain experience. There is no definite consensus on how many procedures surgeons need to perform before they pass the learning curve [33]. Morton et al. [34] commended that usually a surgeon must perform at least 30 cases to achieve accurate staging of the regional nodal basin in melanoma. It is worth noticing that the sentinel node procedure routinely applied in other malignancies (e.g. breast cancer) is not performed in Poland by gynaecologists. Therefore, we can suspect that in our study, the learning curve has not been achieved. Because the incidence of vulvar carcinoma in Poland is rather low compared to other malignancies, it is questionable how much time it will take before each of the gynaecologists, even in a reference centre, will pass the learning curve. In the MSCMCC, we needed 4 years to enroll 62 eligible patients. Unfortunately, according to the operating surgeons, the low number of patients with vulvar cancer in their experience could not allow to draw conclusions on the improvement of the patients' management in the MSCMCC. One additional aspect has not been addressed so far, in countries with low population density, some patients have to travel long distances to be treated in one of the few reference centers. Some of vulvar carcinoma patients may not be willing to or able to take this opportunity and eventually choose to be treated in the nearest community hospital. It is questionable if any of the gynaecologists working in the community hospital will pass the learning curve. Results of a multicenter observational study on SN detection show low rate groin recurrence, excellent survival and minimal treatment-related morbidity in patients with negative SN. However, the authors suggest that the sentinel node dissection should be performed by a quality-controlled multidisciplinary team [35]. Papers describing the accuracy of sentinel lymph node procedure in squamous cell carcinoma of the
vulva were already published but negative data only in case reports. Our study points problems that may occur if this procedure would be wide implemented. Conclusion We conclude that the most accurate method of identification of the sentinel lymph node in vulvar carcinoma is preoperative lymphoscintigraphy (administration of Tc-99m) and intraoperative biopsy under gamma probe. The dye staining alone is insufficient for SLN identification. The RT-PCR detection of CA9 can be recommended for further systematic examination of its use for the detection of vulvar carcinoma cells in the lymph nodes. The RT-PCR test presents higher sensitivity than histopathologic examination with combined H–E and cytokeratin IHC stainings. In our settings, the SLN procedure cannot be recommended as a reliable procedure to avoid radical inguinofemoral lymphadenectomy in early stages of vulvar cancer because of high false-negative SLN rate. In summary, further investigation is needed, especially to assess indispensable conditions to gain the learning curve expertise. Conflict of interest statement The authors declare that there are no conflicts of interest.
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