Identification of Cancer-Associated Fibroblasts in Metastatic Lymph Nodes of Colorectal Carcinoma.

Cancer-associated fibroblasts (CAF) are a heterogeneous group of tumor microenvironment cells that are barely studied in metastatic lymph nodes. The presence of CAF in regional metastases of colorectal cancer was assessed by using SMA, PDGFRb, and POD markers; the obtained subpopulations were compared with the primary tumor. A total of 26 cases of colon adenocarcinoma with metastases to regional lymph nodes were studied. Duplex immunohistochemical detection (POD+SMA and PDGFRb+SMA) was carried out by the immunohistochemical method. In most cases, POD was absent in metastases (65.4%) and PDGFRb was present (88.5%). The POD and PDGFRb staining in the invasive edge of the tumor did not correlate with metastasis. Attention was drawn to the absence of POD and PDGFRb reactions in a blood vessel embolus, as well as to negative PDGFRb in metastasis in the presence of pronounced PDGFRb in the primary tumor in patients after neoadjuvant therapy.

Detection, Role and Prognostic Value of Cancer-associated Fibroblasts in Colorectal Cancer.

Cancer-associated fibroblasts (CAFs) - mesenchymal cells in the tumor stroma, play one of the leading roles in tumor progression in many different tumors, including colorectal cancer. Scientists have described many markers for CAFs, but none of them is specific. We performed immunohistochemistry tests using five antibodies (αSMA, POD, FAP, PDGFRα, PDGFRß) to investigate CAFs in three zones of 49 colorectal adenocarcinomas: apical, central, and invasive edge. We revealed the reliable correlation between high PDGFRß and PDGFRα value in the apical zone and deeper invasion (T3-T4) (p = 0.0281 and p = 0.0137). High αSMA level in apical zone (p = 0.0001), αSMA level in central zone (p = 0.019), POD level in apical zone (p = 0.0222), POD level in central zone (p = 0.0206) and PDGFRß level in apical zone (p = 0.014) correlated reliably with the presence of metastasis in lymphatic nodules. For the first time, focused on the inner layer of CAF adjacent to tumor complexes. We observed that cases with inner αSMA expression were significantly more often (p = 0.023) characterized by the presence of regional lymph node metastasis compared with cases with mix of CAF markers (p = 0.007) and with cases with inner POD expression (p = 0.024). The found relationships between the level of markers and the presence of metastases indicate their clinical significance.

[Multiplex fluorescent imaging of cancer-associated fibroblasts in colorectal cancer]. / Mul'tipleksnaya flyuorestsentnaya vizualizatsiya opukholeassotsiirovannykh fibroblastov v kolorektal'nom rake.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a heterogeneous cell population in the tumor stroma and have important prognostic and clinical significance for solid tumors, including colorectal cancer. The identification of CAF presents difficulties due to the lack of a unique diagnostic marker. OBJECTIVE: Detection of CAF by multiplex immunohistochemical staining and assessment of their colocalization. MATERIAL AND METHODS: For multiplex IHC staining specimens of 10 colon adenocarcinomas without neoadjuvant treatment were selected. We used «OPAL 7-COLOR MANUAL IHC KIT¼ (Akoya Biosciences, USA) with five antibodies (FAP, PDGFRß, CD31, POD, PCK) for staining and Mantra 2 Quantitative Pathology Imaging System (Akoya Biosciences, USA) for evaluation of results. RESULTS: CD31 and CAF markers (FAP, PDGFRß, POD) are expressed fundamentally in different cells (p<0.0001) in all areas of the tumor (apical, central, invasive margin). Pairs FAP+PDGFRß in all zones demonstrated significantly higher (p<0.0001) square of tandem staining. It shows that these markers are expressed in the same stromal cells (probably CAF). In pair FAP+POD significant colocalization (p=0.011) was detected only in apical zone. We connect this finding rather with active proliferation of population of young fibroblasts in zones of ulceration and granulations than with CAF. CONCLUSION: We evaluated co-localization of CAF markers (FAP, PDGFRß, POD) and endothelial cells (CD31) in different zones of colorectal carcinomas. We showed colocalization of CAF markers for pairs FAP+PDGFRß in all tumor zones and for pair FAP+POD in apical zone.

[Podoplanin`s expression in cancer-associated fibroblasts close to tumor budding of invasive edge]. / Ekspressiya podoplanina v opukholeassotsiirovannykh fibroblastakh vblizi opukholevykh pochkovanii invazivnogo kraya.

INTRODUCTION: Tumor budding was declared as independent prognostic factor for early cancer in 2016. Tumor-associated fibroblasts (CAFs) are one of the main components of tumor microenvironment. Plenty of different markers are used for detection of CAFs, including podoplanin (POD). OBJECTIVE: The aim of this study is to identify correlation between tumor budding, that indicates tumor invasive potential and is considered to be a negative prognostic factor, and CAFs near tumor buds using POD. MATERIAL AND METHODS: 43 cases of colon adenocarcinoma are included in the study. Double staining immunohistochemical technology with PCK and POD was used for detection of tumor budding and evaluation of POD expression near tumor buds. RESULTS: Significant correlations are revealed between tumor budding and depth of tumor invasion (p=0.023)/regional lymph node metastasis (p=0.068), but between POD expression and depth of tumor invasion (p=0.088) only a tendency of correlation is identified. These facts demonstrate critical prognostic value of tumor budding instead POD expression near tumor buds. It was found that intensity of POD expression near tumor buds is statistically analogous to POD expression in the invasive margin (p=0.0016). That means it is not necessary to evaluate POD expression exactly near tumor buds. For the first time stronger POD expression near mucin complexes is reported. That, probably, will allow to use mucin complexes as alternative prognostic factor instead tumor budding, as tumor buds are absent in mucinous adenocarcinoma.

[Coexpression of CD44 and Ki-67 in colon`s neoplast]. / Koékspressiia CD44 i Ki-67 v novoobrazovaniiakh tolstoi kishki.

AIM: the evaluation of Ki-67 and CD44 expression in the 'serrated' polyps of the colon and comparison them with adenocarcinomas and tubular and tubule-villous adenomas of the colon. MATERIAL AND METHODS: The study is including 49 'serrated' polyps, 34 tubular (AT) and tubulo-villous (ATV) adenomas and 32 adenocarcinomas of the colon. Antibodies CD44 and Ki-67 were used as immunohistochemical markers in this study. RESULTS: A statistically significant difference (p<0.01) was observed between traditional serrated adenomas (TSA) from hyperplastic polyps (HP) and sessile serrated adenomas (SSA) in the Ki-67 level and the localization of the Ki-67 and CD44 reaction: surface areas of the crypts (upper third) in TSA and base of crypts (lower third) in HP and SSA. There was no difference between HP and SSA (p>0.05), neither by marker localization, nor by their level. In all 'serrated' polyps of the colon, the Ki-67 reaction was nuclear; CD44 - membrane (except for 1 TSA). CONCLUSION: we are the first ones who suggested to evaluate not the overall level of reactions of CD44 and Ki-67, but particular level for each third part of crypts. The similarities of TSA, AT and ATV and between HP and SSA are shown as well as the principal statistical difference between these two groups. The cytoplasmic reaction of CD44 in adenocarcinomas and the membrane reaction of CD44 in 98% of the 'serrated' polyps of the colon are described. For the first time coexpression of CD44 and Ki-67 on particulate thirds of crypts in neoplasms of the colon is shown and the potential reasons for this phenomenon are discussed.

[Gastric adenocarcinoma arising in giant hyperplastic polyp]. / Adenokartsinoma v gigantskom giperplasticheskom polipe zheludka.

Gastric hyperplastic polyps are usually solitary, their development is supposed to be associated with excessive proliferation of foveolar cells. It is essential to differentiate hyperplastic polyps from other sporadic polyps (adenomatous and fundic gland polyps) and lesions, included in familial polyposis syndromes. The frequency of adenocarcinoma in large gastric hyperplastic polyps (more than 1-2 cm in size) is about 2,1%. This article includes case report of gastric adenocarcinoma arised in large hyperplastic polyp in a 56-year-old patient. On histological examination a well-differentiated adenocarcinoma without invasion in the peduncle was identified. Immunohistochemically cells of adenocarcinoma showed elevated expression of claudin-3, CDX2, p53 and Ki67 compared to hyperplastic glands and dysplastic areas of the polyp. Also focal expression of MUC2 was revealed in adenocarcinoma. Expression of MUC5AC, CD44 and cyclin D1 was less prominent in cancer areas compared to hyperplastic and dysplastic glands. Levels of expression of claudin-1, claudin-4 and ß-catenin were equal in adenocarcinoma and hyperplastic structures. Control endoscopic examination with following morphologic examination was performed three months after surgical operation. No signs of tumor growth were identified.

[The expression of claudins in colonic neoplasms]. / Osobennosti ekspressii klaudinov v novoobrazovaniyakh tolstoi kishki.

AIM: the evaluation of localization claudin-1, -3 and -4 types of cancer and colon polyps. MATERIAL AND METHODS: The study included 32 colon adenocarcinoma and 86 polyps. Antibody claudin-1; -3 and -4 were used as immunohistochemical markers in this study. RESULTS: 84/118, 64/118, 52/118 reaction with claudin-1, claudin-3 and claudin-4 in cancer and colon polyps had a membrane localization, respectively. In 33 (27.9%) cases was found paradoxical reaction claudin-1; in 50 (42.4%) - a paradoxical reaction claudin-3 in 66 (55.9%) - a paradoxical reaction claudin-4. Among the paradoxical claudin reaction nuclear localization of marker was observed relatively rarely: claudin-3 in 2.5% cases of colon cancer; claudin-4 in 8.5% of colon polyps. CONCLUSION: Mislocalization claudin-3 to nucleus in colon cancer and mislocalization claudin-4 to nucleus in adenomas of the colon were detected for the first time. The potential reasons for the paradoxical expression are discussed and a review of the literature, related all the alleged mechanisms of this mislocalization is provided.

[Markers of angiogenesis in tumor growth].

Angiogenesis is a process of new blood vessels formation. The role of angiogenesis in growth, invasion and metastasis of malignant tumours is nowdays universally recognized. Though, investigation of mechanisms of blood vessels formation and elaboration methods for assessment of tumour angiogenesis are still up-dated. Another important concern are different aspects of usage of immunohistochemical markers of blood vessels endothelium (CD31 and CD34) for assessment of tumour aggressiveness and prognosis. The problems of malignant lymphangiogenesis are also up-to-date. The focus is on methods of immunohistochemical visualization of forming lymphatic vessels, role of podoplanin, the most reliable marker of lymphatic vessels, in their identification, and formulization of the main criteria for lymphangiogenesis estimation, its correlation with metastatic activity and prognostic potential. Studying of angiogenesis and lymph angiogenesis in malignant tumors is important and challenging direction for researching tumour progression and invention of antiangiogenic therapy.

[Serrated lesions of the large intestine: Prevalence analysis and clinical and morphologic characteristics].

OBJECTIVE: to retrospectively estimate the prevalence of serrated lesions of the large intestine and to reveal the features of dis- tribution of serrated lesions in the previously diagnosed classical adenomas and hyperplastic polyps. MATERIAL AND METHODS: A continuous sampling method was used to take biopsy specimens from the archive material of 440 polypoid lesions of the large intestine, which had entered in 2012-2013, and which were be morphologically reexamined by three experts. The mor- phological criteria for diagnosing different types of serrated lesions were taken according to the 2010 WHO classification of premalignant lesions of the large intestine and to the recommendations from an expert panel (Am J of Gastroenterology 2012). RESULTS: A group of serrated lesions included tubular (13.7%), tubulovillous adenomas (12.5%), and villous (50%) adenomas. The reexamination revealed 121 serrated lesions, including hyperplastic polyps (n = 55 (45.5%)), sessile serrated adenomas/ polyps (SSA/P) (n = 24 (19.8%)), out of which there were those with dysplasia (n=6 (5%)), traditional serrated adenomas (TSAs) (n = 20 (16.5%)), out of which there were those with dysplasia (n = 1 4 (11.6%)), and unclassified serrated polyps (n =22 (18.2%)). Most hyperplastic polyps (89%) and SSA/P (71%) were located distal to the splenic flexure of the colon; TSAs were equally distributed between the proximal and distal portions of the large intestine. The proportion of serrated lesions with dysplasia was 16.5%; that among SSA/P and TSAs was 25 and 70%, respectively. Serrated lesions with dysplasia were more often located proximally and were larger in size than those without dysplasia. CONCLUSION: Serrated lesions of the large intestine are its more common abnormalities than previously thought. The reexamination has revealed that serrated lesions occur in both the preexisting group of hyperplastic polyps and all groups of classical adenomas, suggesting the need for widespread introduction of the current classification intO everyday diagnostic practice.

[Serrated lesions of the large bowel].

The 2010 WHO classification identifies a new group of pretumor lesions of the large bowel--serrated masses, which includes hyperplastic polyps (HPs), sessile serrated adenomas/polyps (SSA/P), and traditional serrated adenomas (TSA). Serrated masses are a heterogeneous group characterized by serrated gland lumens and, in most cases, without dysplasia. An enlarged proliferative zone, elongated crypts, and no cytological atypia in addition to a serrated lumen are typical of HPs. SSA/P is characterized by the migration of the proliferative zone to the crypt walls, giving rise to specific architectural disorders, such as expanded and horizontally growing basal gland segments along the lamina muscularis mucosae. TSA is typified by short ectopic crypts that cannot reach the lamina muscularis and by epithelial eosinophilic changes. SSA/P and TSA have peculiar molecular genetic profiles and proven malignant potential.

[The alternative way of colorectal cancer developing. The histogenetic and molecular features of serrated lesions (review, continued)].

The occurrence of colorectal cancer can be traced in two ways: from conventional adenomas with the APC-gene mutation (model Fearon-Vogelstein) and the "serrated way", that has a unique genetic profile and morphological characteristics at the early stages. These neoplasms are determined from 7 to 9%. The risk of developing cancer of them is 7.5-15%. Precursors of epithelial neoplasia are aberrant crypts foci. About 20% of colorectal cancer demonstrated the common defects in DNA methylation (CIMP-positive profile), mutations BRAF (KRAS)--oncogenes, microsatellite instability (MSI). The serrated lesions may have these mutations. Serrated polyposis syndrome has specific genetic changes associated with biallelic mutation MUTYH also. Risk of colorectal cancer is very high in this syndrome and is more than 50%. Often the synchronous or metachronous cancers presence. They are usually accompanied by MSI-H and represented serrated morphology too. Understanding epigenetic ways and molecular features of serrated lesions gives an knowledge of their clinical significance and provides the evidence for the treatment and monitoring of patients with this disease. This review is devoted to these issues.