Mucosal Lymphatic Vessels of the Esophagus Distant From
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Okajimas Folia Anat. Jpn., 88(2): 43–47, August, 2011 Mucosal lymphatic vessels of the esophagus distant from the cancer margin: morphometrical analysis using 27 surgically removed specimens of squamous cell carcinoma located in the upper or middle thoracic esophagus By Hidefumi NIsHIMorI1, shogo HayasHI2*, Munekazu NaIto3, Gen MurakaMI4, Masahiro FujIta5, Masao Hosokawa1 1Division of Surgery, Keiyu-kai Sapporo Hospital, Sapporo, Hokkaido, Japan 2Medical Education Center, Aichi Medical University, Nagakute, Aichi, Japan 3Department of Anatomy, Tokyo Medical University, Shinjuku, Tokyo, Japan 4Iwamizawa Kojin-kai Hospital, Iwamizawa, Hokkaido, Japan 5Keiyu-kai Institute of Clinical and Surgical Pathology, Sapporo, Hokkaido, Japan – Received for Publication, March 7, 2011 – key words: lymphatic vessel, esophageal cancer, morphometric study, immnohistochemistry abstract: Purpose: to clarify the configuration of the esophageal mucosal lymphatics distant from cancer using D2-40 immuno histochemistry. Methods: D2-40 immunohistochemistry for human lymphatic epithelium was performed at sites about 10 cm anal from the pathologically examined margin of upper or mid- thoracic squamous cell carcinoma (27 patients). we measured the entire length of mucosal lymphatic vessels within a ×10 objective field (1.2 mm along the muscularis mucosae). Results: the present morphometrical study demonstrated significant individual differences in the amount of mucosal lym- phatic vessels, within a range of more than 10-fold (8.4 mm– 0.8 mm within an objective field). However, the difference in length of the mucosal lymphatic epithelium did not correlate with either N-factor, t-factor including cancer depth or prognosis. Conclusion: a higher density of pre-existing mucosal lymphatic vessels may not always be correlated with larger numbers of nodal metastases. Lymphatic proliferation or dilation induced by cancer seems to occur irrespective of whether pre-existing vessels are rich or sparse. Introduction lymphatic vessel morphology are present in the esophagus distant from the cancer, although systemic changes in Lymphatic vessel invasion has been shown to be a lymphatics other than those of the esophagus have been strong and independent prognostic factor in cases of suggested in cancer patients.8, 9) esophageal squamous cell cancer.1,2,3) In contrast to the Recently, using donated cadavers without cancer, Yajin identification of lymphatic cancer invasion using conven- et al.10) demonstrated individual differences of more than tional staining, recently developed antibodies such as D2- 2-fold in the “length or number of the mucosal lymphatics 40 (anti-human podoplanin antibody) have allowed more per 1-mm length of the esophageal epithelium”. This led acurate identification of gastrointestinal mucosal lym- them to suggest that, before the onset of cancer pathology, phatic vessels including those of the esophagus.4) In this the “pre-existing” mucosal lymphatic vessels exhibit sig- context, many research groups have focused on individ- nificant individual differences. On the basis of their s tudies, ual differences in lymphatic vessel density among pa- we hypothesized that, even in cancer patients, the mor- tients with esophageal squamous cell carcinoma.5,6,7) These phology of lymphatics distant from the cancer site is likely studies have revealed that lymphatic density is significantly to reflect any “pre-existing” morphology. How much of a correlated with nodal involvement, and thus possibly with distance from the cancer site is necessary? In the present prognosis. However, to our knowledge, no previous study study, we hypothesized that a distance of 10 cm anal from has investigated whether certain individual differences in the pathologically estimated cancer margin would be free * Correspondence to Shogo Hayashi, MD, PhD, Medical Education Center, Aichi University School of Medicine, Nagakute, Aichi 480-1195 Japan. Phone: +81 561 62 3311 ext. 1447. Fax: +81 561 63 1037. E-mail: [email protected] 44 H. Nishimori et al. of cancer invasion because of the dominant upward longi- “u pper or mid” thoracic esophagus. The sites examined by tudinal flow along the esophagus.10,11,12,13) Consequently, to D2-40 immunohistochemistry were located 10 cm anal to assess the anatomical background of cancer prognosis, we the pathologically examined cancer margin. This distance conducted the present morphometrical study to analyze was easily estimated because, in the hospital that had mucosal lymphatic vessels in the esophagus around 10 cm treated the present patients, pathological sections had been anal from squamous cell carcinoma occupying the upper routinely prepared along the “longitudinal axis” of the or middle thoracic esophagus. esophagus (Fig. 1A). Conversely, for the present aim, cross-sectional histology was considered better because, in previous studies, 5,6,7,10) the morphometry had been Materials and Methods p erformed on cross-sections. Therefore, the longitudinal paraffin blocks were melted and the specimens were re- D2-40 immunohistochemistry for human lymphatic embedded to allow transverse sections (6 μm thick) to be epithelium was performed on surgically removed speci- cut. After taking photos using a ×10 objective (at 3 or 4 mens of the “mid or lower” thoracic esophagus from 27 sites along the entire circumferential wall of the esopha- patients with squamous cell carcinoma occupying the gus at the 10 cm anal level; e.g., Fig. 1B), we measured Figure 1. Materials and methods of the present study. This is an example with a cancer in the mid-thoracic esophagus: the oral part of the removed esophagus is omitted in this figure. Panel A displays the measured site almost 10 cm anal to the cancer (star. In the hospital in which the surgery was performed, the removed esophagus was cut longitudinally, as indicated by dotted lines, to show the longitudinal extension of the cancer. From the long belt-like paraffin embedded specimens, we prepared cross section histology with D2-40 immunohistochemistry ( panel B) and traced the vessels ( panel C). Lymph and carcinoma of esophagus 45 the entire length of mucosal lymphatic vessels within an Using cross-section histology with D2-40 immuno- objective field (1.2 mm along the muscularis mucosae). histochemistry, the fourth author (G.M.) took photos at On the basis of 164 pathological reports (April 2003– hot spots of lymphatic vessel density (Weidner et al., September 2004) of curative surgeries of esophageal squa- 1991) in each of 3 sections at the 10-cm anal level mous cell carcinoma at Keiyu-kai Sapporo Hospital, we (Fig. 1B). After tracing of the D2-40-positive vessels chose patients with upper or mid-thoracic squamous carci- (Fig. 1C), the third author (M.N.) measured them using noma from whom the esophagus had been surgically the ImageJ program (developed at the U.S. National Insti- r emoved more than 10 cm anal to the cancer margin. We tutes of Health and available on the internet at http://rsb. tried to eliminate any bias related to T-factor, N-factor and info.nih.gov/ij/ ). The third author also calculated 1) the prognosis. The T and N factors were classified according total number of mucosal lymphatic vessels in an objective to the criteria of the Japanese Society for Esophageal Dis- field (1.2 mm along the muscularis mucosae) and 2) total eases.14) The population examined (Table 1) comprised 23 circumferential length of vessels in an objective field. The males and 4 females ranging in age from 79 to 51 years second author (S.H.) evaluated the measured data using (70 –79 years, 14 patients; 60 – 69, 10; 51–59, 3; other fac- t-analysis, chi-squared analysis and one-way analysis of tors, see footnote of Table 2). None of the 27 patients had variance. The present study was conducted in accordance Barrett’s esophagus. with the provisions of the Declaration of Helsinki, 1995 For D2-40 immunohistochemistry, the primary anti- (as revised in Edinburgh in 2000). The protocol was ap- body (monoclonal anti-human podoplanin; Nichirei D2- proved by the ethics committees of the hospital of the first, 40 (code No. 413451), Tokyo, Japan; 1 : 100 dilution) was fifth and sixth authors (H.N., M.F., M.H.) who performed used after immersion in a ligand activator (Histofine SAB- the surgery or pathological examination. PO Kit, code No. 415211, Nichirei, Tokyo, Japan) with autoclave treatment (105 °C, 10 min). The second anti- body (Dako Chem Mate Envision Kit, Dako, Glostrup, results Denmark) was labeled with horseradish peroxidase, and antigen-antibody reactions were detected using HRP- In all 27 specimens examined, D2-40 successfully catalysis with diaminobenzidine. Counterstaining with stained vessels in any layer of the esophagus. However, hematoxylin was performed on the same samples. the measured lymphatic structures were limited to D2-40- positive vessels on the mucosal side of the muscularis mu- cosae (Fig. 1B). There was a difference in the density and amount of vessels among 3 or 4 sections at a level almost Table 1. Indivisual patient data analyzed in this study 10 cm anal from the cancer margin. However, the differ- age sex LV length LV number length/number T N outcome ence was restricted to within 80% of the mean in the spec- imen. In this study, we averaged the data obtained from 60 F 8.4 5.7 1.48 3 0 dead the 3– 4 quadrants of the circumferential esophageal wall 77 M 4.7 8.3 0.56 1 2 alive irrespective of the quadrants occupied by the cancer. We 75 M 4.1 17.0 0.24 1 2 dead 72 M 3.2 14.0 0.23 2 0 dead did not measure lymphatic vessels in and around the can- 57 M 3.1 17.0 0.19 3 2 dead cer because cross-sections were not obtained from this 73 M 2.9 10.7 0.27 2 0 dead area. 72 M 2.9 9.7 0.30 3 2 alive The amount of mucosal lymphatic vessels (the total cir- 60 M 2.8 17.3 0.16 2 2 alive cumferential length of vessels in an objective field) ranged 65 M 2.8 22.8 0.12 3 4 dead 71 F 2.5 15.3 0.17 2 1 alive from 8.4 mm to 0.8 mm (mean, 1.22 mm), while the nu- 57 M 2.5 10.3 0.24 3 4 dead merical density (the total number of mucosal lymphatic 73 M 2.3 10.3 0.22 1 1 alive vessels in an objective field) ranged from 22.8 to 5.7 73 M 2.3 14.0 0.16 2 0 alive (mean, 6.50).