【作者】 张毅；

【导师】 姜军；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 乳腺癌是危害妇女健康的最严重的恶性肿瘤之一,并且逐年呈上升趋势,目前针对乳腺癌的治疗仍然是以手术治疗为首选的治疗方式。由于对乳腺癌的认识进一步的深入,手术方式也发生巨大变化,对大部分临床Ⅰ～Ⅱ期乳腺癌的手术已从以全乳切除向保留乳房手术过渡,在一些发达国家或地区,乳腺癌保留乳房手术占常规手术已超过50%。但是保留乳房手术依然存在一些问题,如术后局部复发、由于切除范围过大导致乳房形态不美观、因担心局部复发而所有保乳病人均术后进行全乳放疗而增加病人经济负担等等。这一系列问题均归根于对肿瘤在乳腺内空间结构缺乏深入研究,如何准确掌握保乳手术的切除范围目前临床研究尚没有很好解决,存在切除范围过广或不足等问题。新辅助化疗可对乳腺癌有降低临床病理分期的作用,可提高保乳手术率,但化疗后肿瘤及浸润特征变化特点尚缺乏研究。随着可视化人体计划实施并取得了一些列进展,数字化虚拟技术在现代医学中的应用越来越广泛,虚拟模拟外科手术、导航外科手术、遥控手术等虚拟现实技术逐渐应用于临床科研及实践,外科手术操作日渐精细。在乳腺外科方面,针对乳腺癌保留乳房手术存在的不足,有必要将三维可视化技术与乳腺癌研究结合起来,将数字化虚拟技术应用在乳腺癌术前评估、手术设计及术中导航等方面的研究,将有助于使保留乳房手术技术更加完善,切除范围更加准确。本研究中首次采用薄层断面三维重建技术,对人乳腺癌全乳标本进行三维重建,观察乳腺癌三维空间生长特点,将人体器官可视化应用在肿瘤形态学及生长、浸润研究中,期望通过对肿瘤空间结构特征的全面了解,使乳腺癌保留乳房手术切缘选择更加合理;导航外科的开展是建立在精确的影像学虚拟技术之上,我们通过与CT图像三维重建进行对比研究,为影像学检查进一步提高准确性提供依据;同时将全乳大切片技术应用在肿瘤的浸润特征研究方面,更全面地观察肿瘤在完整乳房中的浸润特征,以及针对新辅助化疗在保乳手术中的作用,通过全乳大切片全面观察化疗后肿瘤浸润特征的变化,为保留乳房手术切除范围的判定提供实验依据。研究方法与主要结果1.薄层断面数字化虚拟三维重建人乳腺癌的形态结构及生长特征取术前穿刺病理诊断明确的乳腺癌全乳切除标本,经修剪后10%福尔马林固定、明胶溶液包埋、冷冻,在相对密闭的-20～-25℃恒温低温实验室中,采用TK-T6350型数控卧式铣床进行铣切,铣切厚度为0.2mm,数码相机摄像。数码相机获取连续、清晰的薄层断面解剖图像,图像数据格式为.RAW,经格式转换为无数据压缩的TIFF图像格式后,再由.TIFF转换为.BMP格式。图像经对位后转换为JPG格式,导入3DDOCTOR1.0软件,经过对乳房边缘、皮肤、腺体、肿瘤及肿瘤周围血管等结构的边界勾画、分割后将边界数据集保存为.prj格式文件。运行3D Rendering simple surface,进行数据三维显示。共获取乳腺连续薄层断面数据集17套,首次利用薄层断面三维重建技术将乳腺癌全乳标本进行三维重建,通过对乳房解剖特点详细观察,主要有以下发现:首先通过对图像放大可以清晰观察到乳房的各个连续薄层断面,各个断面结构保留了乳房标本的基本色泽,三维重建可清楚显示各个结构的空间分布状况及结构特征;通过对肿瘤连续断面观察可清楚观察到肿瘤与周围正常组织的大致界限,在薄层断面中可以观察到肿瘤组织与正常腺体相比,色泽偏于灰暗,肿瘤组织与周围组织之间无明显包膜,表明乳腺癌向正常组织呈逐渐的浸润生长,与良性肿瘤呈压迫性或挤压性生长不同。在断面上还可以看到肿瘤实质中密度并不一致,肿瘤直径越大,密度差异表现越明显,可能是肿瘤迅速生长的同时由于肿块中央血供不足而发生中央区域性坏死。我们通过数字化虚拟技术首次清楚地展示了乳腺癌的三维空间形态特征;发现乳腺癌病灶在某些断面中表现为较为规则的形态,但其整体三维形态却呈现明显不规则生长,表明肿瘤在各个空间方向生长是无序的。通过对肿瘤周围血管的三维重建,发现围绕肿瘤周围有较丰富的血管网,在较大的肿瘤,肿瘤周围血流更丰富,而且肿瘤周边血供明显较肿瘤内部血运丰富,可以解释肿瘤向周围迅速浸润需要丰富的营养供应。因为解剖学是宏观观察研究,对于肿瘤对周围组织的微观浸润情况难以进行细致观察,但通过对肿瘤在乳房中的大体形态学研究,有助于了解乳腺癌的浸润性生长方式,对保留乳房手术肿块切除范围界定提供参照依据。2.人乳腺癌薄层断面三维重建与CT三维图像的对比研究将2004年至2006年术前穿刺明确病理诊断的乳腺癌病人15例,术前行16排螺旋CT扫描进行三维重建与术后离体标本进行薄层断面三维重建后进行对比研究。CT扫描层厚为1mm,扫描后数据传输到SYNGO工作站进行多平面重建和三维重建。手术切除全乳标本薄层铣切,层厚为0.2mm,铣切后数码摄像,获得连续薄层断面数据,在3DDOCTOR软件进行三维重建。经对比观察,薄层断面解剖特征较CT断层扫描更丰富,可清晰观察到更多的解剖结构。可以较清楚分辨肿瘤与正常腺体的大致界限,可观察到肿瘤内部结构特征,更充分了解肿瘤特征,还可观察到肿瘤血流分布,在肿瘤周围可见丰富血管分布。薄层断面三维重建后观察肿瘤形态特征及浸润情况与CT三维重建图像基本一致,CT增强扫描后可更充分显示肿瘤边缘形态特征。通过数字化虚拟技术三维重建后对肿瘤边缘的观察更加细致,CT图像上不能显示、无法辨认的肿瘤内部结构特征、边缘状况及血流分布等均可较清楚的显示。对比测量两种重建方法肿瘤大小,发现CT三维重建后肿瘤最长径均小于薄层断面三维重建肿瘤大小,说明CT图像有可能并不能完全显示肿瘤全貌,目前影像学对乳腺癌的肿瘤判定与真实状况仍存在偏差。两种重建方法的对比研究,反映目前影像学存在的不足,对今后进一步改进影像学对肿瘤精确性判定方面有促进作用。3.全乳大切片技术对肿瘤的浸润特征研究收集我院2004年6月至2006年8月乳腺癌手术全乳标本,术前均行空芯针穿刺活检获得准确病理结果,其中行新辅助化疗93例,未作化疗31例,制作次连续全乳大切片,进行全乳大切片观察,主要观察肿瘤的组织形态学特征、对大导管浸润特征、对周围皮肤、皮下浸润特征、对微血管的浸润等,并对新辅助化疗病例与未行化疗者进行浸润性特征对比。利用全乳大切片可以更清晰显示肿瘤在完整乳房的浸润特征,对两实验组进行对比观察发现,未化疗组肿瘤细胞分布密集,浸润性特征明显,肿瘤细胞浸润距离与肿瘤大小有关,肿瘤越大,浸润距离越远。而新辅助化疗后肿瘤浸润特征相应减弱,包括肿瘤细胞浸润距离明显减小,肿瘤中央出现大量坏死组织;化疗后肿瘤对皮肤皮下浸润减轻,主要表现为对皮肤皮下浸润阳性病例数减少;对乳头乳晕大导管浸润阳性例数明显减少。通过HE及CD34染色观察,未化疗时存在较多的微血管浸润,化疗后显著减少。肿瘤微血管密度也在化疗后显著减少。表明新辅助化疗可通过缩小肿瘤、缓解肿瘤对周围组织的浸润而增加保乳机会。主要结论:1.乳腺癌标本经固定、冷冻、铣切后能较好地保持乳腺大体结构特征,包括肿瘤在内的各个结构均能在薄层断面中连续清晰显示,适合进行三维重建。2.将薄层断面三维重建技术应用在乳腺癌形态结构及浸润生长研究中,可以良好再现肿瘤与周围结构的解剖特点,充分显示了乳腺癌生长的不规则性特点,表明肿瘤在各个空间方向生长的无序性,提示保留乳房手术时仅根据肿瘤表面估计切缘可能并不可靠;肿瘤周围丰富的血供特点,为肿瘤向周围浸润生长提供营养来源。3.针对CT下三维重建可能存在一定的偏差,薄层断面三维重建真实反映了乳房各结构特征,精细的乳腺癌薄层断面三维重建对CT下三维重建形态、结构特征判定有指导作用,为今后CT导航下保留乳房手术的开展提供了实验依据。4.全乳大切片能够更完整地观察乳腺癌组织学全貌,更直观地观察肿瘤的浸润特征,包括对组织形态学特征,对皮肤皮下、大导管及血管的浸润等,同时观察到不同大小的肿瘤向周围浸润距离不同,提示在保留乳房手术时应根据肿瘤大小适当选择切除范围。5.新辅助化疗可缓解肿瘤对周围组织的浸润,并能够增加保留乳房的机会及减少术后局部癌组织的残留。6.数字化虚拟三维重建在乳腺癌临床研究中是可行的,本研究结果提示如果具备精确的乳腺及肿瘤影像学虚拟技术,乳腺癌保留乳房导航外科手术有望实现。更多还原

【Abstract】 Breast cancer is one of the most common malignant tumors harming women’s health, with increasing trend year by year. Because of the anatomical characteristics, breast cancer is regarded as an emphasis in the study of malignant tumors. Surgery has still been the main treatment for breast cancer at present. Deeper recognition of the biological characteristics of breast cancer has led to a conception that breast cancer is a systemic disease at the beginning. And breast conserving therapy (BCT) has been regarded as the first choice for stage I-II invasive breast cancer recently. In developed countries, more than 50 percent of breast cancer patients are treated with BCT. But there still exist some problems in breast conserving surgery, like local recurrence, the postoperative shape of breasts, the economic problem from postoperative chemotherapy, etc. All these ascribe to the insufficient study of the spatial structure of the tumor. Adequate excision extent in breast conserving surgery has not been well solved clinically. There has been no good standard evaluation for excision extent now, and it is evaluated just according to doctors’ experiences, which lacks theoretical evidence. Neoadjuvant chemotherapy has the effect of reducing clinical pathological stage of breast cancer and can raise the rate of BCT. But there are few studies on the characteristics of tumor and its infiltration after chemotherapy. With the progress of visible human project (VHP), digital virtual technique has been used widely. Virtual modeling surgery, navigationg surgery and tele-control surgery have been applied to clinical research and practice gradually. In breast surgery, it is necessary to combine three-dimensional visualization with the study of breast cancer. Utilizing digital virtual technique to navigate surgery can help improve and perfect BCT and evaluate more accurately the excision margin. In this study we produced successfully computer-assisted thin sections anatomy and three dimensional (3D) reconstruction technique to reconstruct human breast cancer in whole breast specimens to observe the 3D growth characteristics of breast cancer and the contrast with 3D-CT reconstruction images, with an attempt of providing evidences for improving accuracy of imageology, and observing infiltrating features of breast cancer using whole breast sections technique, finally to provide theoretical evidences for evaluation of resection margin for BCT.Methods and results:1. Human breast cancer 3D reconstruction to observe the growth and morphological characteristics of the tumorAll whole breast specimens were pathologically diagnosed by core biopsy before surgery. First, every specimen was fixed by 10% formalin, embedded by gelatine solution and frozen at low temperature respectively. Then each specimen was cut into 0.2mm slices at -20～-25℃and imaging of each slice was acquired simultaneously by a 11 million pixels digital camera. The serial and clear images of thin sections anatomy were acquired, and many structures could be observed in detail. By means of several transformations of image data format, pinpointed by Photoshop 7.0, the breast structures were dissected. We drew the outline of the breast margin, skin, glandular tissue, tumor, blood vessel and so on, and then the 3D reconstruction of the dissected breast structures were made including the volume and surface measurement of some structures by 3D DOCTOR1.0 to show 3D manifestation.A total of 17 data sets of serial thin section anatomy were acquired. By the observation of thin sections anatomy and 3D reconstruction’s characteristics, each slice could show the structures clearly, such as skin, glandular tissue, subcutaneous tissue, tumor and so on. We could distinguish the border between tumor and normal glandular tissue in the whole-breast sections. In different sections, the shapes of the tumors were different, round or irregular. The observation of the interior of the tumor showed the shade was also different with glandular tissue, which was gloomy than normal glandular shade. Parenchymatous tissue of tumor was also not well-distributed. Some areas were pyknotic, and other areas were porous. The 3D images were acquired after reconstruction, including skin, cutaneous tissue, tumor, glandular tissue and blood vessels around the tumor. Every structure of breast in thin sectionss was clear stereo image of breast soild and could be shown satisfactorily. Every shape of stereo image and the structure of breast could be shown by revolving the three dimensional image to different directions, and the tumor’s dimensional characteristics were irregular even if it was regular in certain direction. By means of transparent presentation of different structures, the dimensional distance of every structure could be measured. Meanwhile, we could find the blood stream which was rich around the tumor.2. Comparative study between thin sections anatomic 3D reconstruction and 3D CT reconstructionFrom 2004 to 2006, 15 breast cancer patients pathologically diagnosed by preoperative core biopsy were examined with 16 slices helical CT and 3D reconstruction of CT images. 3D reconstruction of the postoperative specimens was also performed with 3D DOCTOR software. The thickness of CT scanning was 1mm, and the data was transmitted to SYNGO workstation to perform 2D and 3D reconstructions of CT images. The specimens were sliced into 0.2mm continuous sections on transversal plane with the computerized miller, and photographed with digital camera. 3DDOCTOR software was used to make three dimensional block diagram.It was found that the characteristics of thin sections anatomy were more plentiful than CT scanning images, and more anatomic structures could be observed. The border between tumor and normal glandular tissue in CT images could be distinguished more clearly. The different shades of tumor interior and blood stream could also be observed in thin sections anatomy, but they could not be seen in CT images. The tumor shapes and infiltrating status observed after 3D reconstruction were basically consistent with CT images. But the tumor margins of thin sections anatomic 3D reconstructions were more accurate. The measurement of the tumor sizes from the two reconstruction methods found the tumor diameter of CT 3-D reconstruction was smaller than that of thin sections anatomic 3-D reconstruction, explaining that CT images could not give a full view of breast cancer. The comparative study of the two reconstruction methods demonstrated shortcomings of imaging, which may facilitate the improvement of imaging for accurate evaluation of tumors.3. The study of tumor infiltrating characteristics with whole breast section techniqueWhole breast section technique was used to study infiltrating characteristics of breast cancer. Ninety-three cases of breast cancer with neoadjuvant chemotherapy and 31 cases without neoadjuvant chemotherapy (the control group) were studied from June, 2004 to August, 2006. All patients acquired preoperative pathological diagnosis. After surgery, all specimens were made into subserial sections to observe the morphological features, tumor infiltrating to skin, cutaneous tissue, big duct of breast and microvessels, and the different infiltrating features between the neoadjuvant chemotherapy specimens and the control group were compared.In the control group, tumor cells distributed intensively, the infiltrating features were very obvious. The infiltrating distance was relevant with tumor size. The greater size of the tumor, the farther the infiltrating distance. After neoadjuvant chemotherapy , the infiltrating features would be weakened, presenting the infiltrating distance was shortened, and necrotic tissue appeared in tumor center. In the neoadjuvant chemotherapy group, infiltrating to skin and subcutaneous tissue was lessened, showing positive cases and infiltrating to nipple-areolar ducts decreased obviously. The observation of HE staining and CD34 immuohistological staining showed infiltrating to microvessels in more positive cases in the control group than in the neoadjuvantt chemotherapy group. Tumor microvessel density (MVD) also declined after neoadjuvant chemotherapy.Conclusion:1. The specimens of breast cancer can keep their basic characteristics after fixation, freezing and slicing, and every structure is clear. It is adapted to 3D reconstruction.2. Thin sections anatomic 3D reconstruction of breast cancer can show irregular features of tumor and structural characteristics around tumor, indicating that tumor growing at every space direction is random. It is fallible to evaluate resection margin according to tumor surface only.3. There are some differences between thin sections anatomic 3D reconstruction and 3D CT reconstruction. Accurate thin sections anatomic 3D reconstruction can help to guide to 3D reconstruction of CT images, and provide theoretical evidence for CT navigation BCT in the future.4. Whole breast sections can observe the full view of breast cancer and the infiltrating characteristics more intuitively. Meanwhile, the infiltrating distance is relevant with tumor size. Surgeon should choose proper excision extent according to different tumor size while using BCT.5. Neoadjuvant chemotherapy can prevent carcinoma from infiltrating to peripheral tissues and increase the chance of BCT and lessen local recurrence rates.6. Digital virtual 3D reconstrustion is feasible in the study of breast cancer. If accurate imagelogy virtual technique is provided with breast cancer, navigation surgery in BCT will be hoped to come true.更多还原