摘要
目的:
宫颈癌是女性最常见的妇科恶性肿瘤,淋巴转移是导致宫颈癌治疗失败和死亡的主要原因。本课题研究早期宫颈癌淋巴管新生与淋巴结转移的相关性,寻找预测宫颈癌淋巴结转移的关键指标,为抑制宫颈癌淋巴结转移、提高宫颈癌疗效提供治疗新靶点。
方法:
(1)采用透射电镜和光学镜观测宫颈癌内、癌旁、正常区域以及淋巴结周围微小淋巴管的分布和形态特征,分析不同部位淋巴管新生、淋巴管面积、淋巴管侵犯以及淋巴内皮细胞间的开放连接与宫颈癌前哨淋巴结转移的关系。
(2)采用免疫组织化学(IHC)和逆转录聚合酶连反应(RT-PCR)技术检测宫颈癌内、癌旁、正常区、前哨淋巴结及非前哨无转移淋巴结组织中VEGF-C/D的表达,分析VEGF-C/D的表达与宫颈癌淋巴管新生、淋巴管侵犯以及与前哨淋巴结转移的关系,探讨癌旁区淋巴管新生、淋巴管侵犯以及前哨淋巴结转移机制。
(3)分析癌旁区LVI、SLN检测以及二者联合应用对宫颈癌淋巴结转移的预测价值。
结果:
(1)宫颈癌组织淋巴管的分布与形态特征:宫颈癌组织内可见极少数塌陷的淋巴管,无法计数;癌旁区淋巴管密度显著高于正常区淋巴管密度(p=0.000);癌旁区淋巴管总面积显著低于正常区淋巴管总面积(p=0.002)。淋巴结转移组癌旁区淋巴管密度和总面积均显著高于无淋巴结转移组(p=0.000,p=0.037)。癌旁区可见肿瘤细胞侵入淋巴管内,正常区无淋巴管侵犯。
(2)宫颈癌旁区淋巴管侵犯与前哨淋巴结转移的关系:宫颈癌癌旁区淋巴管侵犯35.29%(18/51),其中前哨淋巴结转移阳性者16例,HE、IHC分别检出前哨淋巴结阳性例数为13例、14例。前哨淋巴结采用HE、IHC检测方法预测宫颈癌前哨淋巴结转移的准确率分别为94%(47/50)、96%(48/50),假阴性率分别为18.75%(3/16)、12.50%(2/16)。宫颈癌癌旁区淋巴管侵犯与宫颈癌淋巴结转移具有一致性(McNemar Test p=0.500)。阳性预测值为83.33%(15/18),阴性预测值为93.93%(31/33),准确率为90.19%(46/51)。
(3) VEGF-C蛋白在宫颈癌组织中的表达及临床意义:VEGF-C蛋白在宫颈癌组织中的阳性表达率70.59%(36/51),癌旁区阳性表达率68.63%(35/51);17例转移淋巴结(其中前哨转移淋巴结16例)阳性表达率94.12%(16/17)。宫颈正常组织和非前哨无转移淋巴结均无表达,差异有统计学意义p=0.000)。VEGF-C阳性表达组淋巴结转移率44.44%(16/36)高于VEGF-C阴性表达组6.67%(1/15); VEGF-C阳性表达组宫颈癌浸润深度>1/2者77.78%(28/36),高于VEGF-C阴性表达组13.33%(2/15);VEGF-C阳性表达组宫颈癌淋巴管侵犯44.44%(16/36),高于VEGF-C阴性表达组6.67%(1/15); VEGF-C阳性表达组宫颈癌II期患者47.22%(17/36),高于VEGF-C阴性表达组II期宫颈癌患者13.33%(2/15)(p均<0.05);VEGF-C阳性组宫颈癌癌旁区淋巴管密度和总面积均高于VEGF-C阴性组(p<0.05)。
(4) VEGF-D蛋白在宫颈癌组织中的表达及临床意义:宫颈癌组织中VEGF-D蛋白阳性表达率60.78%(31/51),癌旁区阳性表达率56.86%(29/51);17例转移淋巴结(其中前哨转移淋巴结16例)VEGF-D蛋白阳性表达率82.35%(14/17)。宫颈癌正常组织和非前哨无转移淋巴结均无表达,VEGF-D蛋白在宫颈癌组织中、癌旁、前哨转移淋巴结的阳性表达高于宫颈癌正常组织和非前哨无转移淋巴结的表达(p=0.000;p=0.000;p=0.000)。VEGF-D阳性表达组淋巴结转移率51.61%(16/31),高于VEGF-D阴性表达组3.33%(1/30);VEGF-D阳性表达组LVI的发生率48.39%(15/31),高于VEGF-D阴性表达组10%(3/30)(p<0.05)。
(5) VEGF-C/DmRNA的表达:荧光定量RT-PCR检测VEGF-C mRNA在宫颈癌内、癌旁、前哨转移淋巴结中的表达显著高于宫颈正常组织和非前哨无转移淋巴结(P<0.01)。VEGF-C mRNA在前哨转移淋巴结中的表达显著高于宫颈癌内和癌旁组织(P<0.05)。VEGF-D mRNA在宫颈癌内、癌旁、前哨转移淋巴结中的表达显著高于宫颈正常组织(P<0.01)和非前哨无转移淋巴结(P<0.05)。VEGF-D mRNA在前哨转移淋巴结中的表达显著高于宫颈癌内(P<0.05),与宫颈癌旁组织比较无显著差异(P>0.05)。VEGF-C/DmRNA在宫颈癌组织中的表达与淋巴结转移、淋巴管侵犯、肿瘤浸润深度方面均有显著性差异(p均<0.05)。
(6)核素+染料法检测SLN成功率98.04%(50/51);全部检测SLN141个,均经HE、IHC检测,HE染色检测阳性淋巴结35个(24.82%),IHC检出阳性淋巴结42个(29.79%),HE与IHC之间检出率的差异无统计学意义(p=0.283)。
(7)癌旁区淋巴管侵犯阳性18例,其中15例前哨淋巴结转移阳性。淋巴管侵犯-IHC预测宫颈癌前哨淋巴结转移状态的敏感度为93.75%(15/16) ,特异度为90.32%(28/31),准确率为98%(49/50),假阴性率为6.25%(1/16)。联合淋巴管侵犯-IHC和SLN检测预测宫颈癌淋巴结转移敏感度100%(16/16),准确率100%(50/50),假阴性率0%(0/16)。
(8)宫颈癌不同临床分期对SLN的影响:全组SLN检测失败1例。Ib1期诊断准确率100%(25/25),Ib2期诊断准确率100%(7/7),IIa期诊断准确率88.89%(16/18),假阴性率16.67%(2/12)。
结论
(1)宫颈癌淋巴管新生发生于癌旁区,新生淋巴管管腔小,肿瘤细胞通过淋巴管壁的内皮细胞开放连接和破坏癌旁区淋巴管进入淋巴引流,可能是宫颈癌形成淋巴结转移的两种主要方式;癌旁区LVI-IHC有可能成为准确预测淋巴结转移的敏感指标。
(2) VEGF-C、D在宫颈癌组织中都表达上调,且有一定的协同性,它们可能参与了宫颈癌发生、发展的过程,在宫颈癌的淋巴结转移中起重要作用;VEGF-C、D既可以诱导宫颈癌癌旁区淋巴管新生又可以促使淋巴管侵犯、淋巴结转移增加。VEGF-C、D有可能成为预测宫颈癌淋巴结转移的关键指标,并可能成为抑制宫颈癌淋巴结转移、提高宫颈癌疗效的治疗靶点。
(3)对Ib期宫颈癌联合应用LVI-IHC和前哨淋巴结检测,可以提高诊断的敏感度,降低假阴性率。
Objection: Cervical cancer is the most common gynecological malignant tumor. Lymphatic metastasis is the most important cause for treatment failure and death of cervical cancer. This research studied the relationship between neoplasm of lymphatic vessel and metastasis of lymph node in early stage of cervical cancer, in order to find the critic index for prediction of lymphatic metastasis, and to provide the new therapy target for suppression lymphatic metastasis and improvement the treatment effect.
Methods:
(1) Using Transmission electronic microscopy and Optical microscopy to observe the distribution and morphologic character of micro lymphatic vessels in the region of intra-cancer, para-cancer, normal cancer and sentinel lymph nodes (SLN) of cervical cancer. Analysis the relationship between metastasis of sentinel lymph nodes and neoplasm of lymphatic vessels, lymphatic vessels total area (LVTA), lymphatic vessels invasion (LVI) and opening connection of lymphatic endothelia cells.
(2) Using Immunohistochemistry (IHC) and Real time Reverse transcription polymerase chain reaction (RT-PCR) to detect the expression of VEGF-C/D in the tissue of intra-cancer, para-cancer, normal, SLN and non-SLN. And analysis the relationship of the expression of VEGF-C/D and neoplasm of lymphatic vessels, invasion of lymphatic vessels and metastasis of SLN, exploration the mechanism of neoplasm of lymphatic vessels, invasion of lymphatic vessels and metastasis of SLN. (3) Analyzing the detection of LVI-IHC and SLN biopsy, and the value of combining LVI-IHC and SLN for predicting the metastasis of lymph nodes in cervical cancer.
Results:
(1) Distribution and morphologic characters of lymphatic vessels of cervical cancer: There were few collapsed lymphatic vessels in the region of cervical cancer tissue and could hardly be counted. The density of lymphatic vessels (LVD) in para-cancer region was significantly higher than that in normal region (p=0.000). LVTA in para-cancer region was significantly lower than that in normal region (p=0.002). The LVD and LVTA of metastasis lymph nodes were significantly higher than that of lymph nodes without metastasis. There were cancer cells invasion into lymphatic vessels in para-cancer region, but no cancer cells invasion into lymphatic vessels in normal region.
(2) Relationship between invasion of lymphatic vessel in para-cancer region and metastasis of SLN: There were 35.92% (18/51) para-cancer regions of cervical cancer having lymphatic vessel invasion, among which there were 16 cases having positive SLN. HE detected 13 cases of positive SLN, its correct rate for predicting SLN was 94%(47/50), false negative was 18.75%(3/16). While IHC detected 14 cases of positive SLN, its correct rate for predicting SLN was 96%(48/50), false negative was 12.50%(2/16). There were uniformity between invasion of lymphatic vessel in para-cancer region and metastasis of SLN (McNemar Test p=0.500). Using invasion of lymphatic vessel in para-cancer region to predict metastasis of SLN, the positive predictive value was 83.33%(15/18),negative predictive value was 93.93%(31/33),correct rate was 90.19%(46/51).
(3) The expression of VEGF-C in cervical cancer tissue and its clinical significance: The positive rate of VEGF-C in cervical cancer was 70.59%(36/51),in para-cancer region was 68.63%(35/51), in 17 cases of metastasis lymph node(among them 16 was SLN) was 94.12%(16/17). There was no VEGF-C expression in normal cervical tissue and lymph node without metastasis. The expression of VEGF-C in different tissues has significant difference( p=0.000). The metastasis rate was 44.44%(16/36) in VEGF-C positive group , which was significant higher than VEGF-C negative group 13.33%(2/15). The invasion of lymphatic vessel was 44.44%(16/36)in VEGF-C positive group, which was significant higher than VEGF-C negative group 6.67%(1/15). The positive expression of VEGF-C 47.22%(17/36)was significantly higher than negative expression of VEGF-C 13.33%(2/15)in Stage II of cervical cancer(p<0.05). Both LVD and LTA of VEGF-C positive group were significantly higher VEGF-C negative group(p<0.05).
(4) The expression of VEGF-D in cervical cancer tissue and its clinical significance: The positive rate of VEGF-D in cervical cancer was 60.78%(31/51),in para-cancer region was 56.86%(29/51), in 17 cases of metastasis lymph node(among them 16 was SLN) was82.35%(14/17). There was no VEGF-D expression in normal cervical tissue and lymph node without metastasis. The expression of VEGF-D in different tissues has significant difference( p=0.000). The metastasis rate was 51.61%(16/31) in VEGF-D positive group , which was significant higher than VEGF-D negative group 3.33%(1/30). The invasion of lymphatic vessel was 48.39%(15/31)in VEGF-D positive group, which was significant higher than VEGF-D negative group 10%(3/30)(p<0.05).
(5) The expression of VEGF-C/DmRNA: Using Real time quantity of RT-PCR to detect the expression of VEGF-C mRNA. The expression of VEGF-C mRNA was significantly higher in intra-cancer, para-cancer, and metastasis SLN than that in normal cervical cancer (P<0.01)and non-SLN without metastasis(P<0.01). The expression of VEGF-C mRNA was significantly higher in metastasis SLN than that in intra-cancer and para-cancer region(P<0.05). The expression of VEGF-D mRNA was significantly higher in intra-cancer, para-cancer and, metastasis SLN than that in normal region(P<0.01)and non-SLN without metastasis(P<0.05). The expression of VEGF-D mRNA was significantly higher in metastasis SLN than that in intra-cancer, but had no significantly different with para-cancer region(P>0.05). There were significantly difference in expression of VEGF- C/D mRNA in LN metastasis, LVI and depth of cancer invasion(P<0.05).
(6) The success rate of combining 99mTc and dye to detect SLN was 98.04%(50/51). There were 141 SLN that were detected. HE detected 35(24.82%) positive LN, IHC detected 42(29.79%)positive LN. There were no significant difference between them(p=0.283).
(7) There were 18 cases having LVI, among them there were 15 cases of metastasis of SLN. Using LVI-IHC to predict metastasis of SLN, the sensitive rate was 93.75% (15/16), specific rate was 90.32% (28/31), correct rate was 98% (49/50), false negative rate was 6.25%(1/16). Combining LVI-IHC and SLN detection to predict metastasis of lymph node, the sensitive rate was 100%(16/16), specific rate was 90.32% (28/31), correct rate was 100%(50/50), false negative was 0%(0/16).
(8) The influence of clinical stages on SLN: There was only 1 case which was failed to detect SLN. The diagnosis correct rate was 100%(25/25)in stage Ib1, 100%(7/7) in stage Ib2, 88.89%(16/18)in stage IIa, false negative rate was 16.67%(2/12).
Conclusion:
(1) The neoplasm of lymph vessels of cervical cancer happened in para-cancer region. The lumen of neo-lymphatic vessel was small. There maybe existed two main methods for lymph node metastasis: tumor cell pass through the open connection of lymphatic vessel wall, or tumor cell distroy lymphatic vessel of para-cancer region. LVI-IHC of para-cancer region may become a sensitive index of predicting metastasis of lymph nodes.
(2) Both VEGF-C and VEGF-D were up regulated in cervical cancer tissue, and they have synergistic effect. Both VEGF-C and VEGF-D could induce neoplasm of lymphatic vessel in para-cancer region, and improve invasion of lymphatic vessel, and increase metastasis of lymph node. Maybe they take part in the process of happen and development of cervical cancer, and act important effect in lymph node metastasis. They may become a significant index for lymph node metastasis and treatment target for suppression lymph node metastasis and improvement treatment effect.
(3) The combining detection of LVI-IHC and SLN for Ib cervical cancer, may improve the sensitivity of diagnosis for lymph node metastasis and decrease the false negative.
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53. Nieweg OE,Bartelink H.Implication of lymphatic mapping for staging and adjuvant treatment of patients with breast cancer.Eur J Cancer,2004,40(2):179-181.
54. Hauspy J,Beiner M,Harley I,et al.Sentinel lymph node in vulvar.Cancer,2007,110(5):1015-1023.
55. Gipponi M. Clinical applications of sentinel lymph-node biopsy for the staging and treatment of solid neoplasms. Minerva Chir, 2005, 60(4): 217-233.
56. Fader AN,Edwards RP,Cost M,et al.Sentinel lymph node biopsy in erly cervical cancer:utility of intraoperative versus postoperative assessment.Gynecol Oncol,2008,111(1):13-17.
57. Veronesi U,Paganelli G,Galimberti V,et al.Sentinel node biopsy to avoid axillary dissection in breast cancer with clinically negative lymph node.Lancet,1997, 349(9069):1864-1867.
58. EI-Ghodashv AE,Saidi SA.Sentinel lymph node sampling in gynaecological cancers:Techiques and clinical applications.Eur J Surg Oncol,2008,11,1.
59. McMasters KM,Giuliano AE ,Ross MI ,et al. Sentinel2lymph node biopsy for breast cancer : not yet the standard of care . N Engl J Med ,1998 ,399 (14);990-995.
60. Alex JC ,Krag DN. The gamma2probe2guided resection of radiolabeled primary lymph nodes. Surg Oncol N Am ,1996 ,5(1):33-41.
61. Albeyrtini JJ ,Lyman GH ,Cox C , et al. Lymphatic mapping and sentinel biopsy in the patients with breast cancer[J ] . JAMA ,1996 ,276 (5):1818-1822.
62. Martin RC 2nd,Edwards MJ,Wong SL,et al.Practical guidelines for optimal gammaprobe detection of sentinel lymph nodes in breast cancer:results of a multi-institutional study.For the University of Louisville Breast cancer Study Group.Surgery,2000,128(2):139-144.
63. Rob L,Strnad P,Robova H,et al.Study of lymphatic mapping and sentinel node identification in early stage cervical cancer.GynecolOncol,2005,98(2):281-288.
64. Niikura H,Okamura C,Akahira J,et al.Sentinel lymph node detection in early cervical cancer with combination 99mTc phytate and patent blue.Gynecol Oncol,2004,94(2):528-532.
65. Plentl AA,Friedman EA.Lymphatic system of the female genitalia.The morphologic basis of oncologic diagnosis and therapy.Major Probl Obstet Gynecol,1971,2:1-223.
66. Angioli R, Palaia I, Cip riani C, et al. Role of sentinel lymph node biop sy p rocedure in cervical cancer: a critical point of view. Gynecol Oncol, 2005, 96 (2) : 5042-5091.
67. Silva LB, Silva Filho AL, Traiman P, et al. Sentinel node detection in cervical cancer with ( 99m Tc) phytate . Gynecol Oncol, 2005, 97 (2) : 5882-5951.
68. Malus S, Krasuse N,Kohler C,et al.Sentinel lymph node detection in patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257.
69. 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(3):688-693.
70. Plante M,Renaud MC,Tetu B,ey al.Laparoscopic sentinel node mapping in early-stage cervical cancer.Gynecol Oncol,2003,91(3):493-503.
71.李斌,吴令英,李晓光,等.早期子宫颈癌宫旁淋巴结的识别及其临床意义.中华妇产科杂志,2006,41(9):608-611.
72. Sevcik L,Klat ,Graf P,et al.Lymphatic mapping in cervical cancer.Klin Onkol,2008,21(1):26-30.
73. Giuliano AE ,Jones RC ,Brennan M, et al. Sentinel lymphadenectomy in breast cancer . J Clin Oncol ,1997 ,15(6):2345-2350.
74. Paramo JC,Summeral J,Poppiti R,et al.Validation of sentinel node mapping in patients with colon cancer.Ann Surg,2002;9(6):550-554.
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56. Fader AN,Edwards RP,Cost M,et al.Sentinel lymph node biopsy in erly cervical cancer:utility of intraoperative versus postoperative assessment.Gynecol Oncol,2008,111(1):13-17.
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62. Martin RC 2nd,Edwards MJ,Wong SL,et al.Practical guidelines for optimal gammaprobe detection of sentinel lymph nodes in breast cancer:results of a multi-institutional study.For the University of Louisville Breast cancer Study Group.Surgery,2000, 128(2):139-144.
63. Rob L,Strnad P,Robova H,et al.Study of lymphatic mapping and sentinel node identification in early stage cervical cancer.GynecolOncol,2005,98(2):281-288.
64. Niikura H,Okamura C,Akahira J,et al.Sentinel lymph node detection in early cervical cancer with combination 99mTc phytate and patent blue.Gynecol Oncol,2004,94(2):5 28-532.
65. Plentl AA,Friedman EA.Lymphatic system of the female genitalia.The morphologic basis of oncologic diagnosis and therapy.Major Probl Obstet Gynecol,1971,2:1-223.
66. Angioli R, Palaia I, Cip riani C, et al. Role of sentinel lymph node biop sy p rocedure in cervical cancer: a critical point of view. Gynecol Oncol, 2005, 96 (2) : 5042-5091.
67. Silva LB, Silva Filho AL, Traiman P, et al. Sentinel node detection in cervical cancer with ( 99m Tc) phytate . Gynecol Oncol, 2005, 97 (2) : 5882-5951.
68. Malus S, Krasuse N,Kohler C,et al.Sentinel lymph node detection in patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257.
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70. Plante M,Renaud MC,Tetu B,ey al.Laparoscopic sentinel node mapping in early-stage cervical cancer.Gynecol Oncol,2003,91(3):493-503.
71.李斌,吴令英,李晓光,等.早期子宫颈癌宫旁淋巴结的识别及其临床意义.中华妇产科杂志,2006,41(9):608-611.
72. Sevcik L,Klat ,Graf P,et al.Lymphatic mapping in cervical cancer.Klin Onkol,2008,21(1):26-30.
73. Giuliano AE ,Jones RC ,Brennan M, et al. Sentinel lymphadenectomy in breast cancer . J Clin Oncol ,1997 ,15(6):2345-2350.
74. Paramo JC,Summeral J,Poppiti R,et al.Validation of sentinel node mapping in patients with colon cancer.Ann Surg,2002;9(6):550-554.
1. Malus S, Krasuse N, Kohler C, et al.Sentinel lymph node detection in patients with cervical cancer.Gynecol Oncol,2001,80:254-257.
2. Gipponi M. Clinical applications of sentinel lymph-node biopsy for the staging and treatment of solid neoplasms. Minerva Chir, 2005, 60: 217-233.
3. Cabanas RM. An approach for the treatment of penile carcinoma. Cancer, 1977, 39: 456-466.
4. Rob L, Strnad P, Robova H, et al.Study of lymphatic mapping and sentinel node detection in early stage cervical cancer.Gynecol Oncol,2005,98:281-288.
5. Niikura H, Okamura C, Akahira J, et al.Sentinel lymph node detection in early cervical cancer with combination 99mTc phytate and patent blue.Gynecol Oncol,2004, 94:528-532.
6. Martin RC 2nd, Edwards MJ,Wong SL,et al.Practical guidelines for optimal gamma probe detection of sentinel lymph nodes in breast cancer:results of a multi-institutional study.For the University of Louisville Breast cancer Study Group.Surgery, 2000,128:139-144.
7. Plentl AA, Friedman EA. Lymphatic system of the female genitalia.The morphologic basis of oncologic diagnosis and therapy.Major Probl Obstet Gynecol,1971,2:1-223.
8. Paramo JC, Summeral J, Poppiti R,et al.Validation of sentinel node mapping in patientswith colon cancer.Ann Surg,2002;9:550-554.
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10. Plante M, Renaud MC, Tetu B, et al.Laparoscopic sentinel node mapping in early-stage cervical cancer. Gynecol Oncol,2003,91:493-503.
11. Li B, Wu LY, Li XG, et al. Lymphatic mapping and parametrial lymph nodes detection in patients with early stage cervical cancer. Chinese Journal of Obstetrics and Gynecology 2006; 41: 608-611.
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