乳腺癌前哨淋巴结活检术中不同染色淋巴结与肿瘤转移的关系

目的探讨乳腺癌前哨淋巴结活检术(SLNB)中不同染色情况的淋巴结与肿瘤转移的关系。方法选择我院2014年1月至2018年1月行前哨淋巴结活检的乳腺癌患者92例,以亚甲蓝为示踪剂,根据92例乳腺癌患者SLNB中淋巴结染色情况的不同分为无染色组、完全染色组和染色不均组,病理检测3组患者淋巴结的肿瘤转移情况并作比较。结果92例乳腺癌SLNB共取得淋巴结256枚,平均每例患者2.8枚,无染色组(80枚)肿瘤转移率为13.8%,完全染色组(112枚)肿瘤转移率为43.8%,染色不均组(64枚)肿瘤转移率为62.5%,3组间肿瘤转移率差异有统计学意义(P<0.05)。结论乳腺癌SLNB中染色不均的淋巴结最易出现肿瘤转移,其次为完全染色的淋巴结,染色淋巴结附近看到的未染色淋巴结也有肿瘤转移的可能,宜一并切除送检,有利于降低假阴性率。     

卵巢癌腹膜后淋巴结转移的特点及其临床意义

目的：研究卵巢上皮性癌淋巴结转移的解剖学和生物学特点及临床合理治疗。方法：40例Ⅰ期卵巢癌根据清除淋巴结与否分成A、B两组；40例Ⅲ-Ⅳ卵巢癌清除淋巴结20例为C组、不清除淋巴结20例为D组，C、D两组减瘤术后残余癌灶均2cm。化疗方法，药物及其剂量基本相同。结果：A组3例腹主动脉旁淋巴结转移者合并盆腔淋巴结转移2例，单纯盆腔淋巴结转移1者，共4例淋巴结转移，转移率20％。A、B两组5年生存率各为95％与80％。C组腹主动脉旁淋巴结转移10例中合并盆腔淋巴结转移9例，单独盆腔淋巴结转移2例，转移率为60％（12／20）。C、D两组5年生存率各为55％与15％。5年生存率A、B两组差异有显著意义（P〈0．05），C、D两组差异有极显著意义（P〈0．001）。结论：卵巢癌淋巴结转移率，随期别而升高，腹主动脉旁与盆腔淋巴结转移率几乎相等，但腹主动脉旁淋巴结转移是主要路线。恰当清除淋巴结可以提高生存率。     

TIMP-3基因甲基化与结直肠癌临床病理的关系

目的：探讨TIMP-3基因甲基化与结直肠癌临床病理指标和转移复发的关系。 方法： 采用巢式甲基化特异性PCR技术（nMSP法）检测100例结直肠癌组织和100例癌旁非癌组织TIMP-3基因甲基化；采用RT-PCR检测100例结直肠癌组织和100例癌旁非癌组织TIMP-3 mRNA的表达。 结果： 肿瘤组织TIMP-3 mRNA的表达阳性率为64%，肿瘤组织TIMP-3 mRNA的表达率明显低于癌旁非癌组织（P＜0.01）；TIMP-3 mRNA的表达率无淋巴结转移组（34/42）高于淋巴结转移组（30/58）（P＜0.01），甲基化阳性率Duke’s C+D期伴淋巴结转移组明显高于Duke’s A+B期不伴淋巴结转移组（P＜0.05）。结肠近端、分化程度差的结直肠癌组织甲基化阳性率明显高于远端直肠和分化程度高者（P＜0.05）。 结论： TIMP-3基因甲基化容易发生在结肠近端、Duke’s C、D期、伴淋巴结转移、细胞分化差和浸润型结直肠癌患者。     

影响乳腺癌前哨淋巴结数目的临床病理特征分析

目的分析影响乳腺癌前哨淋巴结数目的相关因素,探讨最佳的前哨淋巴结活检值。方法回顾性分析2007年1月-2011年12月中国医学科学院肿瘤医院乳腺癌前哨淋巴结活检病例578例。采用Logistic回归模型分析前哨淋巴结数目与临床病理特征的相关性。结果全组女性,平均年龄49.9(21～90)岁。总共获得2 222枚前哨淋巴结,平均每例3.8枚(1～15)。淋巴结转移率17.8%(103/578),转移组和无转移组淋巴结数目无差异。单因素分析显示,术式、显像方法和体质指数影响前哨淋巴结数目(P<0.05)。多因素分析中,单纯乳房切除、联合显像、BMI≤30者前哨淋巴结较多(P<0.05)。前哨淋巴结限于5枚时,转移病例检出率100%。18.7%(108/578)病例不必继续送检淋巴结,298枚淋巴结免于切除。结论乳腺癌前哨淋巴结活检数量受到显像方法、乳腺术式和体质指数的影响,5枚前哨淋巴结可能是一个比较合适的参考标准。     

乳腺癌非前哨淋巴结转移的预测

目的 :预测非前哨淋巴结 (non SLN)转移 ,以筛选出转移局限于前哨淋巴结 (SLN)的乳腺癌患者。方法 :采用99mTc SC作为示踪剂 ,对 95例乳腺癌患者行前哨淋巴结活检 ,对乳腺癌非前哨淋巴结转移进行单因素和多因素分析。结果 :95例患者中成功发现 91例患者有SLN (95 8% ) ,其中 85例患者SLN能准确反映腋窝淋巴结的病理状况 (93 4% )。临床肿块大小(P =0 0 2 8)、肿瘤分级 (P =0 0 40 )和原发灶cyclinD1蛋白 (P =0 0 17)的表达与non SLN转移显著相关。而Logistic多因素分析证实 ,临床肿块大小、肿瘤分级为独立的预测非前哨淋巴结转移的因子。结论 :可根据临床病理学特征 ,筛选出乳腺癌转移只局限于前哨淋巴结的患者 ,也存在免除腋窝淋巴结清扫的可能性     

uPA与NF-κB p65在乳腺癌组织表达的相关性研究及临床病理意义

目的：探讨尿激酶型纤溶酶原激活物（uPA ）基因与NF-κB p65在乳腺癌组织表达的相关性及临床病理意义。方法：采用实时荧光定量PCR法（FQ-PCR）检测了46例乳腺癌及其正常组织中的uPA mRNA（单位:2-△△Ct）及免疫组化检测NF-κB p65的表达。结果：乳腺癌 组织中均可检测到uPA mRNA表达，以2-△△Ct≥2为阳性标准，63％（29/46例）的乳 腺癌中uPA基因表达阳性；T/A 2-△△Ct值在不同NF-κB表达状态、肿瘤最大径及淋 巴结阳性数分组中，差异显著，P值分别为＜0.01和＜0.05；进一步经相关分析显示 ，uPA基因表达值与NF-κB表达状态、肿瘤最大径相关，r分别为0.451、0.512，均 P＜0.01；在患者年龄、肿瘤组织学类型及有无远处转移分组中，uPA基因表达未发现显 著性差异。结论：乳腺癌组织中uPA mRNA含量明显增高，并且与NF-κB 表达、肿瘤大小及淋巴结阳性数目有关。     

内源性E2,EGFR,PCNA在大肠癌中作用及其相互关系

目的：明确内源性雌激素（Ｅ２）和相生长因（ＥＧＦＲ）在大肠癌中的作用及相互关系，方法；免疫组化检测４８例大肠癌内源性Ｅ２ＥＧＦＲ和ＰＣＮＡ的表达。结果：内源性Ｅ２阳性２９（６０．４％）例，其阳性细胞平均百分率为４１．１％，与肿瘤淋巴结转移呈负相关（Ｐ〈０．０５），ＥＧＦＲ过表达３２（６６．７％）例，ＰＣＮＡ增殖指数高，且两者均与肿瘤淋巴结转移呈正相关（Ｐ〈０．０５），内源性Ｅ２阳性细胞百分率与ＥＧ     

肿瘤引流淋巴结中免疫活性细胞分布的原位分析

目的:观察人乳腺癌和胃癌局部引流淋巴结(LDLN)从无转移、微转移到晚期转移过程中,免疫组织学变化及免疫活性细胞(ICC)的分布特征。方法:采用传统的病理学方法,对22例乳腺癌LDLN(71个)和7例进展期胃癌LDLN(28个)进行组织形态学分类,并以抗穿孔素、抗颗粒酶B、抗CD8、抗CD56、抗CD68、抗S-100、抗CD134及抗CD25单克隆抗体(mAb)进行催化信号放大(Catalyzedsignalamplification,CSA)免疫组化染色,检测肿瘤LNDN中ICC的分布。结果:肿瘤LDLN中以副皮质区增生和窦组织细胞增生为主,细胞毒性T淋巴细胞(CTL)及树突状细胞(DC)的数量,从无转移、微转移到晚期转移过程中有逐渐减少的趋势。在无和微转移的淋巴结内,穿孔素 、颗粒酶B 及S100 DC的数量高于晚期转移淋巴结(P<0.05);而S100 DC不仅数量减少,而且其形态也有变化,呈多角形、星形,并有胞质突起,与周围淋巴细胞接触呈活化状态的DC变为椭圆形,少有胞质突起或呈短突起的静止状态的DC。CD134 细胞及CD25 细胞的数量在晚期转移淋巴结中明显高于无和微转移淋巴结(P<0.01)。ICC在无和微转移的前哨和非前哨淋巴结中的分布无统计学意义(P>0.05)。结论:肿瘤LDLN中ICC分布的变化,提示随着肿瘤的进展,其免疫微环境向抑制机体抗肿瘤免疫的方向偏移。     

β-干扰素抑制裸鼠荷人肝癌切除术后复发和生长的实验研究

目的:研究β-干扰素(INF-β)对肝癌根治性切除后复发和生长的抑制作用。 方法:将人肝癌组织植入25只BALB/c裸鼠肝脏，建立人肝癌原位移植瘤模型。第10 d完整切除荷瘤后，随机分为3组：A组对照组（生理盐水皮下注射）；B组小剂量实验组（INF-β 3×105U/d皮下注射）;C组大剂量实验组（INF-β 6×105U/d皮下注射）。连续注射35 d后处死裸鼠，观察肿瘤复发情况，测量肿瘤大小和体积；免疫组织化学法检测肿瘤增殖核抗原(Ki-67)、诱导型一氧化氮合酶(iNOS)和半胱氨酸天冬氨酸蛋白酶-3(caspase-3)表达。 结果:A组肿瘤复发率为100%，B组为87.5%，C组为77.8%，3组比较无显著差异（P>0.05）。B组和C组抑瘤率分别为73.6%和94.3%，与A组比较差异显著（P<0.05）。B组和C组肿瘤组织iNOS、Ki-67的表达明显低于A组，而caspase3的表达明显高于A组（P<0.05）。 结论:β-干扰素可有效抑制肝癌切除术后复发肿瘤的生长。大剂量β-干扰素较小剂量对肝癌的生长抑制作用更强，其机制可能与肿瘤组织内iNOS、Ki-67和caspase3的表达有关。     

甲状腺乳头状癌 ALDH1A1表达与淋巴结转移的相关性

目的探讨甲状腺乳头状癌（ PTC）中ALDH1A1的表达情况及与淋巴结转移的相关性。方法收集首都医科大学附属北京同仁医院病理科2006年1月至2013年12月间PTC腺叶切除＋淋巴结清扫标本153例,在HE染色下观察其一般临床病理学特点（肿物直径、双侧、多灶、肿瘤边界、腺叶外浸润）,并采用免疫组织化学染色EnVision法,检测癌及癌旁组织中ALDH1A1的表达情况,分析ALDH1A1表达与淋巴结转移的关系。结果在153例PTC中,84例（54.9％）发生淋巴结内癌转移,126例癌组织高表达ALDH1A1,112例癌旁组织高表达ALDH1A1。通过单因素分析,发现年龄＜45岁、肿物直径＞10 mm、浸润性边缘及癌组织 ALDH1A1高表达与淋巴结转移明显相关（P＜0.05）。而性别、双侧、多灶、腺叶外浸润、癌旁组织ALDH1A1与淋巴结转移无关（P＞0.05）。进一步多因素分析发现浸润性边界和癌组织ALDH1A1高表达是PTC淋巴结转移的独立危险因素（P＜0.05）。在随访的82例PTC中,局部肿瘤复发4例,5年局部复发率为4.88％,包括淋巴结肿瘤复发3例和甲状腺肿瘤复发1例,无远处转移及疾病相关死亡病例。复发病例肿瘤组织ALDH1A1均为高表达。结论 ALDH1 A1在PTC癌组织内高表达与淋巴结转移明显相关,可作为PTC淋巴结转移的有效预测因子,有利于改善PTC患者治疗方法及随访方案。     

Multiparameter flow cytometry as a tool for the detection of micrometastatic tumour cells in the sentinel lymph node procedure of patients with breast cancer

AIM: To investigate whether multiparameter flow cytometry (MP-FCM) can be used for the detection of micrometastasis in sentinel lymph nodes (SLNs) in breast cancer. METHODS: Formalin fixed, paraffin wax embedded sentinel lymph nodes (n = 238) from 98 patients were analysed. For each lymph node, sections for haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for cytokeratin (MNF116) were cut at three levels with a distance of 500 microm. The intervening material was used for MP-FCM. Cells were immunostained with MNF116, followed by an incubation with fluorescein isothiocyanate (FITC) labelled goat antimouse immunoglobulin. DNA was stained using propidium iodide. From each lymph node 100,000 cells were analysed on the flow cytometer. RESULTS: Thirty eight of the 98 patients with breast carcinoma showed evidence of metastatic disease in the SLN by one ore more of the three methods. In 37 of 38 cases where metastatic cells were seen in the routine H&E and/or IHC, more than 1% cytokeratin positive cells were detected by MP-FCM. In 24 patients, metastatic foci were more than 2 mm (macrometastasis) and in 14 these foci were smaller than 2 mm (micrometastasis). In three of these 14 cases, MP-FCM revealed positive SLNs, although this was not seen at first glance in the H&E or IHC sections. After revision of the slides, one of these three remained negative. However, MP-FCM analysis of the cytokeratin positive cells showed an aneuploid DNA peak, which was almost identical to that of the primary breast tumour. Duplicate measurements, done in 41 cases, showed a 99% reproducibility. In five of 14 patients with micrometastasis, one or two metastatic foci were found in the non-SLN. However, in 15 of 24 macrometastases multiple non-SLNs were found to have metastatic tumour. All micrometastases except for the remaining negative one mentioned above showed only diploid tumour cells, despite the fact that their primary tumours contained both diploid and aneuploid tumour cells. In primary tumours with more than 60% aneuploid cells, predominantly aneuploid macrometastasis were found, whereas diploid primary tumours only showed diploid micrometastases or macrometastases in their SLN. Aneuploid SLN macrometastases were associated with non-SLN metastases in five of seven patients, whereas diploid cases showed additional non-SLN metastases in only seven of 16 patients. CONCLUSION: In all cases, MP-FCM was sufficient to detect micrometastatic tumour cells in a large volume of lymph node tissue from SLNs. In some cases it was superior to H&E and IHC staining. Approximately 30% of SLN micrometastases are accompanied by additional non-SLN metastases. The size of the aneuploid fraction (> 60%) in the primary tumour may influence the risk of having both SLN and non-SLN metastases.     

Modes of benign mechanical transport of breast epithelial cells to axillary lymph nodes

The status of axillary lymph nodes is a key prognostic indicator available for the management of patients with breast cancer. Sentinel lymph node (SLN) evaluation as a predictor of lymph node status has led to increased use of ancillary methods, principally immunohistochemistry, to increase the sensitivity of the SLN biopsy. So-called "occult" micrometastases detected by such methods have led to speculation that some may have reached the SLNs by benign mechanical transport (BMT) rather than a metastatic process. We review evidence suggesting two potential modes of BMT: lymphatic transport of epithelial cells displaced by biopsy of the primary breast tumor and by breast massage-assisted SLN localization. The biopsy techniques under most scrutiny include fine needle aspiration and large-gauge core biopsy. The evidence implicating breast massage prior to SLN biopsy as a mode of BMT has been supported by statistical analysis; however, no method of distinguishing massage-associated cells in SLNs from true occult micrometastases is available. The significance of small epithelial clusters in SLNs is currently unknown. Thus, deviation from current biopsy and SLN-localizing practices is unwarranted.     

Angiogenesis and mast cells in human breast cancer sentinel lymph nodes with and without micrometastases

AIMS: An increasing number of mast cells have been reported in angiogenesis associated with solid and haematopoietic tumours. Data concerning the number of mast cells in neoplastic lymph nodes and their relationship with microvessel density are controversial. The aim was to correlate the extent of angiogenesis with the number of mast cells reactive with tryptase in biopsy specimens of sentinel lymph nodes with and without micrometastases obtained from patients with breast cancer. METHODS AND RESULTS: Specimens from sentinel lymph nodes obtained from 80 patients (40 with and 40 without micrometastases) were investigated immunohistochemically by using anti-CD31 and anti-tryptase antibodies. Angiogenesis, measured as microvessel counts, increased in parallel with the number of tryptase-positive mast cells and their values were significantly higher in lymph nodes with micrometastases compared with those without. CONCLUSIONS: Tryptase-positive mast cells may contribute, at least in part, to angiogenesis occurring in sentinel lymph nodes with micrometastases from patients with breast cancer.     

Plasmacytoid dendritic cells represent a major dendritic cell subset in sentinel lymph nodes of melanoma patients and accumulate in metastatic nodes

Plasmacytoid dendritic cells (pDC) represent the main source of interferon-alpha, a cytokine with antitumor activity. However, in vitro studies point to pDC as a key subset for induction of tolerance. Herein, we investigated pDC in sentinel lymph nodes (SLN) of melanoma patients. We report that pDC were constantly found in SLN and represented, with Langerhans cells, the most frequent dendritic cell subset. Their frequency in positive (with metastasis) SLN was significantly higher than in negative (without metastasis) SLN. PDC were observed in the T cell-rich areas of lymph nodes, particularly around high endothelial venules and, in metastatic nodes, they accumulated in close vicinity with melanoma nests. Finally, pDC capability to produce interferon-alpha in situ was impaired. Consistently, pDC expressed CD86, but neither CD80 nor CD83, suggesting a not complete activation in melanoma-draining lymph nodes. These results are consistent with the hypothesis of a tolerogenic role played by pDC in tumor immunology.     

Intraoperative cytologic evaluation of sentinel lymph nodes in patients with breast carcinoma by scrape preparation

Intraoperative evaluation of sentinel lymph nodes (SLNs) in patients with breast carcinoma allows surgeons to complete axillary lymph node dissection in one procedure if any SLN shows metastasis. The accuracy of intraoperative pathological diagnosis is critical for decision-making. The purpose of this study was to evaluate our rapid intraoperative cytologic diagnosis of SLN through comparing with the final surgical pathologic diagnosis of the corresponding lymph nodes. A total of 454 SLNs from 159 consecutive female patients with a preoperative diagnosis of breast carcinoma over 3-year period were included in this study. After gross examination of each bisected lymph node, a scrape preparation was prepared for each submitted lymph node and was stained by the rapid Papanicolaou method. The intraoperative cytologic diagnosis was compared with the final surgical pathologic diagnoses. The overall sensitivity of intraoperative cytology was 52.5% with specificity of 100%. There were 17 false-negative cases. Of them, six nodes had isolated tumor cells, seven nodes had micrometastasis (0.2-2 mm), and four nodes had macrometastasis (>2 mm). There were no interpretive errors identified. The size of metastasis and tumor grade appeared to be significant factors in detecting metastasis by cytology. In addition, subsequent non-SLN involvement was 9% in patients with micrometastasis versus 50% in patients with macrometastasis (P < 0.05). Our study shows that the intraoperative cytologic evaluation of SLNs in breast carcinoma is a reasonably accurate method. The majority of false-negative cases were due to micrometastasis and isolated tumor cells.     

Detection of micrometastases in bone marrow and sentinel lymph nodes of breast cancer patients

Objective: To study the sensitivity and clinical significance of HE-staining,IHC and RT-PCR in detecting breast cancer micrometastases in bone marrow and sentinel lymph nodes (SLNs). Methods:After general anesthesia, all patients underwent bone marrow puncture and sentinel lymph node biopsy (SLNB) by 1% isosulfan blue, and then HE-staining,IHC and RT-PCR were used to detect micrometastases. Results:Of 62 patients with breast cancer whose axillary lymph nodes showed negative HE-staining results, 15 cases presented with positive RT-PCR and 9 cases showed positive IHC results positive in bone marrow micrometastases detection. PT-PCR and IHC showed good uniformity(kappa=0.6945)and there was significant difference in detective rate between these two methods (χ2=4.1667,P=0.0412). In SLN samples, 13 showed positive RT-PCR results, while 7 showed positive IHC results. PT-PCR and IHC showed good uniformity (kappa=0.6483)and significant difference was also found in detective rate between these two methods (χ2=4.1667,P=0.0412). Both bone marrow and SLN samples were RT-PCR positive in 3 cases,which indicated that bone marrow micrometastases did not always accompany SLN micrometastases(χ2=0.067,P=0.796). Conclusion: Even if no axillary lymph node involvement or distant metastases are present in routine preoperative examination, micrometastases can still be detected in bone marrow or SLNs. Because the bone marrow micrometastases and axillary node micrometastses are not present simultaneously, combination test of multiple indicators will detect micrometastases more accurately.     

Factors affecting metastases to non-sentinel lymph nodes in breast cancer

AIMS: Because sentinel lymph node (SLN) biopsy for breast cancer has become well established, one of the challenges now is to determine which patients require a completion axillary dissection following a positive SLN biopsy. METHODS: A prospective database of patients who underwent SLN biopsy for invasive breast cancer from July 1999 to November 2002 (n = 180) was analysed. Fifty four patients (30%) had one or more positive SLN, and all underwent a completion axillary dissection. This subgroup was further analysed to delineate which factors predicted non-SLN metastasis. RESULTS: Twenty six of the 54 patients with a positive SLN had additional metastases in non-SLNs. Significant variables that predicted non-SLN metastasis included extranodal extension (odds ratio (OR), 17.399; 95% confidence interval (CI), 1.69 to 178.96) and macrometastasis within the SLN (OR, 6.985; 95% CI, 1.291 to 37.785). CONCLUSIONS: In patients with invasive breast cancer and a positive SLN, extranodal extension or macrometastasis within the SLN were both independent predictors of non-SLN involvement.     

Metastasis to sentinel lymph nodes in breast cancer is associated with maturation arrest of dendritic cells and poor co-localization of dendritic cells and CD8+ T cells

The regional immune systems of patients with breast cancer are immunosuppressed. Dendritic cells are professional antigen-presenting cells and present cancer-associated antigens to the adaptive immune system in sentinel lymph nodes. Dendritic cells may promote, or inhibit, an adaptive immune response to specific antigens. Our aim was to assess whether dendritic cells were associated with nodal metastasis in patients with breast cancer. Sentinel lymph nodes of 47 patients with breast cancer with varying degrees of nodal disease and ten controls were evaluated using immunohistochemistry for the accumulation of dendritic cells in general (CD1a⁺), mature dendritic cells (CD208⁺), and plasmacytoid dendritic cells (CD123⁺). Cytotoxic T cell and regulatory T cell accumulation were also evaluated. Sentinel lymph nodes with macrometastases demonstrated fewer mature dendritic cells than sentinel lymph nodes without metastasis (p = 0.028), but not controls. There were fewer mature dendritic cells to cytotoxic T cells in sentinel lymph nodes with metastasis than those without (p = 0.033). Also, there were more regulatory T cells to mature dendritic cells in sentinel lymph nodes with metastasis than those without (p = 0.02). In conclusion, our study suggests that sentinel lymph nodes with metastasis have arrest of maturation of dendritic cells, fewer mature dendritic cell interactions with cytotoxic T cells, and more regulatory T cells than sentinel lymph nodes without metastasis in patients with breast cancer. These findings extend our understanding of regional immunosuppression and suggest that most regional immunosuppressive changes are associated with nodal metastasis in breast cancer.     

Metastases in axillary sentinel lymph nodes in breast cancer as detected by intensive histopathological work up

AIM: To assess the value of the intensive histological work up of axillary sentinel lymph nodes (SLN) to demonstrate regional metastatic disease. METHODS: From a series of 58 successful lymphatic mapping procedures, 78 SLN were analysed by serial sections (mean of 49 levels/SLN) and by immunostaining to cytokeratin and epithelial membrane antigen, and the results compared with those obtained by assessing the central cross section. RESULTS: The central cross section would have failed to detect metastases in eight of 26 lymph nodes (31%) in patients with breast cancer metastasising to the SLN only. This would have led to a false negative node status in six of 21 patients (29%). Two micrometastases were detected with the aid of immunostains. CONCLUSIONS: The results suggest the need to examine SLN at multiple levels and to use immunohistochemistry in negative cases. Serial sections are also useful in the case of micrometastases, as some of these may convert to macrometastases at deeper levels. Multiple level investigation of SLN and immunohistochemistry in the event of the negativity of standard stains would result in improved staging and an increase in the proportion of node positive disease detected.