Proteome analysis of formalin-fixed paraffin-embedded colorectal adenomas reveals the heterogeneous nature of traditional serrated adenomas compared to other colorectal adenomas

Traditional serrated adenoma (TSA) remains the least understood of all the colorectal adenomas, although these lesions have been associated with a significant cancer risk, twice that of the conventional adenoma (CAD) and of the sessile serrated adenoma (SSA/P). This study was performed to investigate the proteomic profiles of the different colorectal adenomas to better understand the pathogenesis of TSA. We performed a global quantitative proteome analysis using the label-free quantification (LFQ) method on 44 colorectal adenoma (12 TSAs, 15 CADs, and 17 SSA/Ps) and 17 normal colonic mucosa samples, archived as formalin-fixed paraffin-embedded blocks. Unsupervised consensus hierarchical clustering applied to the whole proteomic profile of the 44 colorectal adenomas identified four subtypes: C1 and C2 were well-individualized clusters composed of all the CADs (15/15) and most of the SSA/Ps (13/17), respectively. This is consistent with the fact that CADs and SSA/Ps are homogeneous and distinct colorectal adenoma entities. In contrast, TSAs were subdivided into C3 and C4 clusters, consistent with the more heterogeneous entity of TSA at the morphologic and molecular levels. Comparison of the proteome expression profile between the adenoma subtypes and normal colonic mucosa further confirmed the heterogeneous nature of TSAs, which overlapped either on CADs or SSA/Ps, whereas CADs and SSAs formed homogeneous and distinct entities. Furthermore, we identified LEFTY1 a new potential marker for TSAs that may be relevant for the pathogenesis of TSA. LEFTY1 is an inhibitor of the Nodal/TGFβ pathway, which we found to be one of the most overexpressed proteins specifically in TSAs. This finding was confirmed by immunohistochemistry. Our study confirms that CADs and SSA/Ps form homogeneous and distinct colorectal adenoma entities, whereas TSAs are a heterogeneous entity and may arise from either SSA/Ps or from normal mucosa evolving through a process related to the CAD pathway. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cancer Proteomics: The State of the Art

Now that the human genome has been determined, the field of proteomics is ramping up to tackle the vast protein networks that both control and are controlled by the information encoded by the genome. The study of proteomics should yield an unparalleled understanding of cancer as well as an invaluable new target for therapeutic intervention and markers for early detection. This rapidly expanding field attempts to track the protein interactions responsible for all cellular processes. By careful analysis of these systems, a detailed understanding of the molecular causes and consequences of cancer should emerge. A brief overview of some of the cutting edge technologies employed by this rapidly expanding field is given, along with specific examples of how these technologies are employed. Soon cellular protein networks will be understood at a level that will permit a totally new paradigm of diagnosis and will allow therapy tailored to individual patients and situations.     

乳腺癌组织蛋白酶D和PSA表达与同侧腋淋巴结转移的关系

目的：探讨乳腺癌中组织蛋白酶Ｄ（ｃａｔｈｅｐｓｉｎＤ，ＣＤ）和前列腺特异性抗原（ＰＳＡ）的表达与同侧腋淋巴结转移的关系。方法：应用ＬＳＡＢ免疫组化法检测ＣＤ和ＰＳＡ在７８例乳腺癌中的表达，其中伴同侧腋淋巴结转移者３６例。结果：（１）７８例乳腺癌ＣＤ阳性表达率４１．０３％（３２／７８），ＰＳＡ阳性表达率４３．５％（３４／７８）；（２）同侧腋淋巴结转移组ＣＤ阳性表达率６９．４４％（２５／３６），无同侧腋淋巴结转移组ＣＤ阳性表达率１６．６７％（７／４２），差异有高度显著性（Ｐ＜０．０１）；（３）同侧腋淋巴结转移组ＰＳＡ阳性表达率２５．０％（９／３６），无同侧腋淋巴结转移组ＰＳＡ阳性表达率５９．５２％（２５／４２），差异有高度显著性（Ｐ＜０．０１）。结论：ＣＤ和ＰＳＡ的表达可作为乳腺癌预后检测的参考指标。ＣＤ表达阳性率与同侧腋淋巴结转移呈正相关，与预后负相关；而ＰＳＡ表达阳性率与同侧腋淋巴结转移呈负相关，与预后正相关     

Lymphatic vessel density in pulmonary adenocarcinoma immunohistochemically evaluated with anti-podoplanin or anti-D2-40 antibody is correlated with lymphatic invasion or lymph node metastases

In lung cancers, lymph node metastasis of cancer cells is one of the most important prognostic factors, and lymphatic vessel invasion (LVI) is very important in the stage preceding lymph node metastases. Recently, it has been reported that lymphatic vessel density (LVD) is associated with lymph node metastasis. The aim of the present study was to evaluate the relationship between LVD and LVI based on the immunohistochemical expression of podoplanin or D2-40, which are new specific markers for lymphatic endothelium. Using 76 cases of pulmonary adenocarcinoma, the relationship between LVD and LVI, lymph node metastases, vascular endothelial growth factor C (VEGF-C), VEGF-D or hepatocyte growth factor (HGF) expression was investigated. LVD was significantly associated with LVI, lymph node metastases and VEGF-D expression. LVI was also associated with lymph node metastases, histological subtype, VEGF-C or VEGF-D expression. High LVD, induced by VEGF-C or VEGF-D expression of cancer cells, is a good indicator of lymphatic metastases and LVI in pulmonary adenocarcinoma.     

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     

Duodenal gangliocytic paraganglioma with regional lymph node metastasis and a glandular component

Gangliocytic paraganglioma (GP) is generally considered to be a benign periampullary lesion, although it is unclear whether it should be classified as a hamartoma or as a neoplasm. Here, we present a GP case with lymph node metastasis. A 16-year-old boy complained of exertional dyspnea. Upper endoscopy and imaging studies revealed a polypoid ampullary tumor. Pancreaticoduodenectomy with lymph node dissection was performed due to swelling of peripancreatic lymph nodes. Histologically, the tumor consisted of three cell types: epithelioid; spindle; and ganglion cells. In addition to these typical components of GP, a distinct glandular component was also present. There was substantial invasion of tumor cells into the lymphovascular vessels, associated with lymph node metastases. These lymph node metastases were histologically similar to the primary tumor. To judge from these findings GP may be a true neoplasm with metastatic capacity. Pre- and intraoperative investigations for lymph node or distant metastases are required for adequate resection of this kind of tumor.     

Comparison of different tissue sampling methods for protein extraction from formalin-fixed and paraffin-embedded tissue specimens

Aims

Protein extracts from formalin-fixed and paraffin-embedded (FFPE) tissue for proteomic analysis has recently gained attention. In this study, we explored the possibility to standardize tissue sampling from paraffin blocks and compared the protein extracts with those obtained from fresh frozen material.

Materials and methods

Fresh frozen and FFPE material was obtained from five patients with pancreatic ductal adenocarcinoma either by cutting sections with a microtome or by stamping a cylinder with tissue micro-array technology. All samples were weighed, forwarded to protein extraction and analyzed by polyacrylamide gel electrophoresis and Western blotting. Immunohistochemistry allocated proteins in tissue sections.

Results

Sampling of tissue was highly reproducible, as assessed by sample weight. While protein concentrations were significantly higher in fresh frozen material compared to FFPE material, equal amounts of protein were extracted from FFPE using either paraffin sections or core cylinders in SDS-PAGE, all three procedures showed comparable protein patterns. In Western blotting, annexin I had the same molecular weight independent of the sample source and sampling procedure.

Conclusions

The sampling of FFPE specimens for protein extraction and analysis can be standardized, uncovering equal amounts of tissue and protein. In addition, the proteins extracted from FFPE tissue seem to be the same compared with those extracted from fresh frozen tissue.     

78例腋淋巴结转移瘤活检的病理学分析

目的　分析腋淋巴结转移瘤的病理学特点 ,为判断腋淋巴结来源未明转移瘤 (MUO)的可能原发部位提供参考性诊断思路。方法　对近 2 0年收验的 78例腋淋巴结转移瘤患者的性别、年龄、转移瘤原发部位进行一般性分析 ,并对其中已知原发瘤部位 (MKO)的 6 2例 (男 16例、女 46例 )患者年龄、受累及淋巴结侧位、转移瘤基本组织学类型等与原发瘤部位进行相关性分析。结果　 (1)约 1/ 5 (2 0 5 %)病例未知原发部位 ,男性病例的MUO率 (30 4 3%)显著高于女性 (14 18%) (P <0 0 5 )。(2 )女性患者的年龄总体上显著低于男性 ,并且年龄分布范围较广泛。 (3)男性和女性的转移瘤都主要为腺癌 (分别为47 83%和 83 6 4%) ,但女性显著高于男性 (P <0 0 1) ;男、女各自的腺癌与鳞癌之比分别为 1∶0 36和 1∶0 0 7。 (3)女性原发瘤部位的数目 (6处 )多于男性 (3处 )。 (5 )女性的 6处中 ,来自乳腺和肺者分别居于左侧或右侧的首位和次位 ,其他为食管、子宫内膜、子宫颈和腕部皮肤。 (6 )男性的 3处中 ,来自肺者皆居于左、右侧之首 ,其他为胃和食管。结论　女性腋淋巴结转移瘤主要来自局部引流区域 ,以乳腺为主 ,占 82 6 6 %;男性转移瘤皆来自胸腔和腹腔等远处引流区域。提示腋淋巴结转移瘤 ,在男性主要考虑来自胸腔和腹     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Influence of formalin fixation duration on RNA quality and quantity from formalin-fixed paraffin-embedded hepatocellular carcinoma tissues

Analyzing RNA samples from formalin-fixed paraffin-embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next-generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1–240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi-CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over-fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6–72 h) for cancer patients.     

Expression and functional perspectives of miR-184 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumors, with its 5-year survival rate lower than 5%. MicroRNAs (miR) have been known as important regulators for the tumorigenesis, progression, invasion and metastasis of various cancers. MiR-184 was found to be abnormally expressed in various cancers including glioma and oral carcinoma. The expression and functional role of miR-184 in PDAC, however, remains unclear. PDAC cell line PANC-1 was transfected with miR-184 inhibitor. Real-time PCR was used to detect the expression of miR-184 in untreated PANC-1, miR-184 inhibitor transfected PANC-1 and controlled normal pancreatic ductal epithelial cell line HPDE6c7. MTT assay was used to detect the effect of miR-184 on the proliferation of PANC-1 cells, while invasion assay and Western blotting were employed to describe the effect on cell invasion ability and expression of caspase-3, respectively. In PANC-1 cells, miR-184 was abundantly expressed. The transfection of inhibitor effectively suppressed the expression of miR-184, and further inhibited both cell proliferation and invasion abilities, in addition to the up-regulation of pro-apoptotic protein caspase 3 expression. The up-regulation of miR-184 in PDAC may facilitate the proliferation and invasion ability, and inhibit apoptosis of tumor cells, thus potentiating the occurrence and development of PDAC. MiR-184, therefore, is a potential molecular target for therapy.     

A novel prognostic factor TIPE2 inhibits cell proliferation and promotes apoptosis in pancreatic ductal adenocarcinoma (PDAC)

Background: Tumor necrosis factor (TNF)-alpha-induced protein 8-like 2 (TIPE2 or TNFAIP8L2) is a newly discovered negative immune regulator. Studies have shown that TIPE2 causes significant malignant biological effects and is differentially expressed in various malignant tumors. However, the expression and roles of TIPE2 in pancreatic ductal adenocarcinoma (PDAC) are largely unknown.Materials and Methods: The expression of TIPE2 in PDAC tissues was assessed by immunohistochemistry, qPCR and western blot analysis and related clinicopathological parameters including survival time were analyzed. After overexpression of TIPE2, cell proliferation and apoptosis analysis were conducted, and the associated underlying molecular mechanism was also explored.Results: In the present study, TIPE2 was upregulated in early PDAC tissues, and TIPE2 expression decreased as the tumor progressed (P<0.001). TIPE2 expression was negatively associated with tumor size, TNM stage and metastasis of lymph nodes. Furthermore, as an independent risk factor, TIPE2 could be used to predict the survival of patients with PDAC (P=0.035). TIPE2 overexpression significantly suppressed the viability, proliferation and induced apoptosis of PDAC cells by inhibiting survivin and increasing the activity of caspase3/7.Conclusions: For the first time, this study demonstrated that TIPE2 is an independent prognostic factor in PDAC. TIPE2 inhibited the proliferation and induced apoptosis via regulating survivin/caspase3/7 signaling pathway. These results indicated that TIPE2 is a potential biomarker for predicting the prognosis of PDAC patients and plays a pivotal role in the progression of PDAC.     

Quantitative RT-PCR assay of HER2 mRNA expression in formalin-fixed and paraffin-embedded breast cancer tissues

Detection of human epidermal growth factor receptor 2 gene (HER2, also known as erbB2) expression is a preparatory process to decide a treatment strategy for breast cancer patients. 20-30% of breast cancer patients have HER2 overexpression, and they usually show poor recovery rate. For detection of HER2 expression, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) methods are conventionally used. Although these methods are accurate and reliable, their time-consuming process and high cost need a concise method with high sensitivity and accuracy. As a complementary method to the current IHC/FISH standard techniques, PCR-based methods have been developed. Here we employed a quantitative PCR method to detect HER2 expression in one hundred ninety nine formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue samples from the patients treated over two years at the Yonsei University Severance Hospital, Republic of Korea. Relative expression of HER2 mRNA in the FFPE samples was analyzed using a quantitative RT-PCR (RT-qPCR) method and the obtained HER2 expression levels were compared with those from IHC/FISH methods. Our results show that the RT-qPCR method was highly concordant with IHC/FISH methods for detecting HER2 expression. Overall sensitivity and specificity of the BrightGen HER2 RT-qDx assay kit (Syantra, Calgary, Canada), which is a kit we used for RT-qPCR analyses, were 93.0% and 89.8% (P < 0.0001), respectively. The diagnostic cut-off value of HER2 RT-qDx for the clinical samples was determined by likelihood ratio, among which the highest likelihood ratio of relative HER2 mRNA levels was over 105.5 (AUC = 0.9466) with the highest sensitivity and specificity. Our study indicates that quantification of HER2 mRNA expression with the RT-qPCR could be an alternative method of conventional IHC/FISH methods.     

Sensitivity of endoscopic ultrasound-guided fine-needle aspiration cytology and biopsy for a diagnosis of pancreatic ductal adenocarcinoma: A comparative analysis

The utility of endoscopic ultrasound fine-needle aspiration cytology (EUS-FNAC) or endoscopic ultrasound fine-needle aspiration biopsy (EUS-FNAB) for diagnosis of small and large pancreatic ductal adenocarcinomas (PDACs) remains in question. We addressed this by analyzing 97 definitively diagnosed cases of PDAC, for which both EUS-FNAC and EUS-FNAB had been performed. We subclassified the 97 solid masses into small (n = 35) or large (n = 62) according to the maximum tumor diameter (<24 mm or ≥24 mm) and compared the diagnostic sensitivity (truly positive rate) of EUS-FNAC and of EUS-FNAB for small and large masses. Diagnostic sensitivity of EUS-FNAC did not differ between large and small masses (79.0% vs. 60.0%; p = 0.0763). However, the diagnostic sensitivity of EUS-FNAB was significantly higher for large masses (85.5% vs. 62.9%; p = 0.0213). Accurate EUS-FNAC-based diagnosis appeared to depend on the degree of cytological atypia of cancer cells, which was not associated with quantity of cancer cells. The accuracy of EUS-FNAB-based diagnosis appeared to depend on cancer cell viability in large masses and cancer volume in small masses. Based on the advantages or disadvantages in each modality, both modalities play an important role in the qualitative diagnosis of PDAC as a complementary procedure.     

Quantitative proteome analysis reveals annexin A3 as a novel biomarker in lung adenocarcinoma

Metastasis is a common phenomenon and the major lethal cause of lung adenocarcinoma (AdC). To discover novel potential biomarkers associated with lymph node metastasis and prognosis in lung AdC, we assessed differences in protein expression between primary lung AdC with (LNM AdC) and without lymph node metastasis (non-LNM AdC) using a quantitative proteomic approach. Laser capture microdissection was performed to purify the cancer cells from primary lung AdC tissues. The differential proteins between the pooled microdissected non-LNM AdC and LNM AdC tissues were identified by two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). In this study, twenty proteins were found to be differentially expressed in two types of lung AdC. ANXA3, significantly up-regulated in LNM AdC compared with non-LNM AdC, was validated by western blotting. Immunohistochemistry showed that ANXA3 over-expression was frequently observed in LNM AdCs and matched lymph node metastases compared with non-LNM AdCs. ANXA3 over-expression was significantly associated with advanced clinical stage (p < 0.001) and lymph node metastasis (p < 0.001) and increased relapse rate (p < 0.001) and decreased overall survival (p < 0.001) in lung AdCs. Cox regression analysis indicated ANXA3 over-expression was an independent prognostic factor. Our results indicate that ANXA3 might serve as a novel biomarker for lymph node metastasis and prognosis in lung AdC, and play an important role in lung AdC progression.     

Lymphatic vessel density in primary melanomas predicts sentinel lymph node status and risk of metastasis

Shayan R, Karnezis T, Murali R, Wilmott J S, Ashton M W, Taylor G I, Thompson J F, Hersey P, Achen M G, Scolyer R A & Stacker S A
(2012) Histopathology  61, 702–710 Lymphatic vessel density in primary melanomas predicts sentinel lymph node status and risk of metastasis Aims: Important prognostic factors in patients with cutaneous melanoma include primary tumour thickness/depth of invasion, ulceration and mitotic rate, and the presence of tumour cells in regional lymph nodes. More recently, features of stromal components, such as blood and lymphatic vessel density, have been suggested as additional indicators of metastatic potential. Our aim was to investigate the relationship between tumour lymphatic vessels and lymph node metastasis. Methods and results: Metastasizing (n = 11) and non‐metastasizing (n = 11) primary melanoma samples matched for depth/thickness, mitotic rate and ulceration were examined for lymphatic vessel density (LVD) in the primary tumour, using an antibody to podoplanin. Significant differences were found between LVD (vessels/unit area) in the peripheral (5.73 ± 0.67) versus central (1.72 ± 0.42) regions of the metastasizing tumour group (P < 0.001), and between LVD in the peripheral areas of metastasizing (5.73 ± 0.67) versus non‐metastasizing (4.21 ± 0.37) tumours (P < 0.01). No overall difference was found between total average LVD in the two tumour groups or between their vessel morphology. Conclusions: Our results show that LVD is associated with risk of lymph node metastasis. Furthermore, the ratio of peripheral LVD:central LVD is a useful marker of primary melanomas that are likely to metastasize to lymph nodes.     

Sineoculis homeobox homolog 1 protein overexpression as an independent biomarker for pancreatic ductal adenocarcinoma

Sineoculis homeobox homolog 1 (SIX1) is a member of the SIX gene family. It is highly expressed in cancers derived from tissues that play a fundamental role during embryogenesis. Recent studies suggest that inappropriate expression of SIX1 can both initiate tumorigenesis and promote metastasis. To investigate the clinicopathological significance of SIX1 expression in pancreatic ductal adenocarcinoma (PDAC), and to further identify its role as a potential biomarker and therapeutic target in PDAC, 103 PDAC tissue samples and 45 normal pancreatic tissue samples were immunohistochemically stained for SIX1 protein. The localization of SIX1 protein was detected in Panc-1 cancer cells using immunofluorescence staining. Correlations between SIX1 overexpression and the clinicopathological features of pancreatic cancer were evaluated using Chi-square (χ²) tests, differences in survival curves were analyzed using log-rank tests, and multivariate survival analysis was performed using the Cox proportional hazard regression model. In results, SIX1 protein showed mainly cytoplasmic/perinuclear staining pattern in PDAC with immunohistochemistry. The strongly positive rate of SIX1 protein was 60.2% (62/103) in PDAC, which was significantly higher than normal pancreatic tissue (6.7%, 3/45). SIX1 overexpression was positively correlated with tumor size, TNM stage, lymph node metastasis, and grade of PDAC (P < 0.001). SIX1 high expression levels influenced overall survival rates in G1, G2, stage I–II and stage III–IV groups of PDAC; and high expression levels had significantly lower overall survival rates than SIX1 low expression levels. In conclusion, SIX1 emerged as a significant independent prognostic factor in PDAC. SIX1 overexpression appears to be associated with PDAC, and may be a potential biomarker for early diagnosis and prognostic evaluation of PDAC.     

Histochemical localization of caldesmon isoforms in colon adenocarcinoma and lymph node metastases

Alternative splicing of the caldesmon gene results in high (h-caldesmon) and low (l-caldesmon) molecular weight isoforms of the cytoskeleton-associated protein caldesmon. h-Caldesmon is predominantly expressed not only in smooth-muscle cells but also in pericryptal fibroblasts in colon. l-Caldesmon is widely expressed and localized in podosomes/invadopodia. Studies with transformed and cancer cell lines suggest that a reduction in l-caldesmon facilitates podosome/invadopodia formation and metastasis. We investigated caldesmon isoforms in colon adenocarcinoma and their lymph node metastases using immunohistochemistry. Caldesmon immunoreactivity of colon adenocarcinoma primary tumors and lymph node metastases was very similar. l-Caldesmon immunoreactivity of cancer epithelial cells in primary tumors and lymph node metastases varied ranging from reduced to stronger as compared to immunoreactivity of normal areas. The variation did not show a consistent relation to the tumor center or invasive margin. While h-caldesmon immunoreactivity of pericryptal fibroblasts and blood vessels in the stroma of primary tumors and lymph node metastases was markedly reduced, cancer-associated fibroblasts and blood vessels in the stroma were strongly immunoreactive for l-caldesmon. Our results show a differential behavior of h- and l-caldesmon isoforms in epithelium and stroma of colon adenocarcinoma and lymph node metastases.     

Upstaging of early colorectal cancers following improved lymph node yield after methylene blue injection

Jepsen R K, Ingeholm P & Lund E L
(2012) Histopathology  61, 788–794 Upstaging of early colorectal cancers following improved lymph node yield after methylene blue injection Aims: To evaluate whether the use of intra‐arterial methylene blue injection improves lymph node yield, and to determine whether a higher lymph node count results in upstaging in colorectal cancer. Method and results: We performed a retrospective study of colorectal cancer specimens (n = 234) 1 year after implementation of the method. All colorectal cancer specimens from the previous year served as our control group. Data concerning tumour characteristics, lymph node count, number of positive lymph nodes and success of methylene injection had been prospectively collected in accordance with the department’s ongoing registration. The method was easy to implement and perform with a high rate of success (86%). The number of identified lymph nodes was highly significantly improved in the study group (P < 0.0001). In resections with pT1/T2 tumours, we demonstrated a significant increase in the number of resection specimens containing positive lymph nodes, with an increase in pN1 resections from 9.4% in the control group to 26.7% in the study group (P = 0.04). Conclusions: The methylene blue technique significantly improves lymph node identification in colorectal cancer specimens, and the improved lymph node identification leads to upstaging of International Union Against Cancer (UICC) pT1/pT2 cancers.