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cytokeratin 20 immunophenotype in colorectal adenocarcinomas with BRAF mutations
Pathology – Research and Practice 208 (2012) 163–166
Contents lists available at SciVerse ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.de/prp
Original article
Aberrant pattern of the cytokeratin 7/cytokeratin 20 immunophenotype in colorectal adenocarcinomas with BRAF mutations Simona Gurzu ∗ , Ioan Jung Department of Pathology, University of Medicine and Pharmacy of Targu-Mures, Romania
a r t i c l e
i n f o
Article history: Received 23 August 2011 Received in revised form 5 January 2012 Accepted 16 January 2012 Keywords: Colorectal carcinomas Cytokeratin 7 Cytokeratin 20 BRAF mutation Microsatellite instability
a b s t r a c t Cytokeratin 7 (CK7) and 20 (CK20) are used for the differential diagnosis of metastases from colorectal carcinomas (CRC), which are usually CK7−/CK20+, and other tumors. In our study, we performed immunohistochemical staining with CK7 and CK20 in 52 randomly selected cases of CRC and analyzed microsatellite instability status and BRAF mutations to identify those factors that may determine the changing pattern of CK7/CK20 immunophenotype in these tumors. CK7 was negative in all microsatellite stable tumors (MSS), but all carcinomas presenting microsatellite instability (MSI) and BRAF mutations were diffusely positive for this marker. CK20 was diffusely expressed in 79.06% of MSS tumors. Regarding MSI, in case with no BRAF mutations, a progressive decrease in CK20 expression was noted, and in BRAF-mutated adenocarcinomas, no expression of CK20 was observed. It seems that in case of MSI located on the proximal colon, which also presents BRAF mutations, CK20/CK7 may present a changing immunophenotype pattern, which may complicate the differential diagnosis of metastatic tumors. This is the first reported study of the relationship between CK20/CK7 immunophenotype, BRAF mutations and microsatellite status in CRC. © 2012 Elsevier GmbH. All rights reserved.
Introduction Bayrak et al. [1] recently suggested that 17.3% of colorectal adenocarcinomas (CRC) expressed cytokeratin 7 (CK7) and 18.9% of cases did not express cytokeratin 20 (CK20), although most authors admit that CK20 is of great importance in the differentiation of colonic metastases from other metastatic tumors like lung, bladder, breast carcinomas and cholangiocarcinomas [4,5,8]. On the one hand, CK7 is usually expressed in the normal epithelia of the lung, mesothelium, urinary bladder and female genital tract, and, rarely, it may be observed in gastric and intestinal normal glands, but most authors believe that it is not found in the normal colonic mucosa [9,10]. On the other hand, CK20 specifically stains the normal gland cells of the colonic mucosa and Merkel cells and, more rarely, its expression may be seen in the urothelium or other mucosas [10,14]. Based on these characteristics, the immunophenotype CK7/CK20 is used in daily diagnoses to differentiate CK20 expresssing metastases of colorectal adenocarcinomas (CRC) from lung, ovarian or bladder carcinomas, which are usually stained with CK7 [2,12].
∗ Corresponding author at: Department of Pathology, University of Medicine and Pharmacy, 38 Ghe Marinescu Street, 540139 Targu Mures, Romania. Tel.: +40 745673550; fax: +40 265210933. E-mail address: [email protected] (S. Gurzu). 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2012.01.003
In our study, we analyzed the immunohistochemical (IHC) expression of both CK7 and CK20 in CRC to emphasize the real importance of these antibodies in the differential diagnosis of metastatic adenocarcinomas and to identify those factors which may induce the changing pattern of CK7/CK20 immunophenotype in these tumors. We also tried to correlate the expression of these markers with the microsatellite status and BRAF mutations. These aspects have not been mentioned in the English literature yet.
Material and methods In 52 randomly selected cases of colorectal adenocarcinoma (26 from proximal and 26 from distal colon and rectum), we performed immunohistochemical (IHC) staining with CK7 and CK20 and molecular examinations to determine BRAF mutations and microsatellite status. UltraVision system by D-Line, LabVision (Fremont, CA, USA), was used for immunohistochemical reactions in formalin-fixed, paraffin-embedded tissues. CK7 (clone OV-TL 12/30) and CK20 (clone Q6) antibodies were also provided by D-Line, LabVision. Sections were deparaffinized, incubated at 100 ◦ C with pepsin and washed with distilled water previous to hydrogen peroxide incubation. Subsequently, they were washed with Tris buffered saline (TBS) and incubated with primary antibodies for 60 min. Then, they were washed with TBS and covered with streptavidin peroxidase solution for 5 min. After this, they were washed with TBS
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Fig. 1. A microsatellite stable colon adenocarcinoma without BRAF mutations, with cytokeratin 7 immunoreactivity in normal mucosa (A). The tumor cells present diffuse expression of cytokeratin 20 (B) and no staining for cytokeratin 7 (A).
and covered with biotinylated goat anti-polyvalent solution for further 5 min. The development was performed with dyaminodihydrochlorid benzidine solution (DAB) for 3–5 min. Counterstaining was performed with Mayer’s hematoxylin. Both CK7 and CK20 expressions were evaluated in the cell membrane and also in the cytoplasm. We considered those cases negative which expressed these markers in less than 5% of the cells. Focal positivity was considered as the presence of antibody expression in 5–40% of the tumor cells, the other cases were considered to have diffuse positivity. To determine microsatellite instability, DNA was extracted from formalin-fixed, paraffin-embedded tissues. We used real-time PCR (Roche GmbH, Mannheim, Germany), the method of melting point analysis, as well as microsatellite mononucleotide markers BAT25 and BAT26 by Roche. Exon 15 BRAF mutations were determined using real-time PCR. Results Forty-three of the 52 cases were microsatellite stable carcinomas (MSS), and 9 presented microsatellite instability (MSI), all 9 being located on the proximal colon. Four of the 52 cases presented BRAF mutations. All four cases were also MSI carcinomas. Regarding the normal mucosa, independent of the microsatellite status, BRAF mutations or other factors, CK20 was positive in 41 of the 52 cases (78.84%), the number of stained cells being higher in the surface epithelium. CK7 was focally positive in the normal mucosa in 10 of the 52 carcinomas (19.23%), its intensity being higher in the crypt epithelium (Fig. 1). We found some differences between MSI and MSS cases regarding the CK7/CK20 immunophenotype in tumor areas. In 34 of the 43 MSS cases (79.06%), the tumor cells were diffusely positive for CK20, but no CK7 expression was observed in these tumors (Fig. 1).
In all 9 MSI cases, CK20 was either focally expressed or negative, independent of the other clinicopathological parameters (Table 1). No MSI cases with diffuse positivity were identified for CK20. The CK7 expression was dependent on BRAF mutations and lymph node status. It was diffusely positive in all MSI cases with BRAF mutations and negative in all carcinomas without BRAF mutations (Figs. 2 and 3), except one case. In this MSI case (case 5), which had no BRAF mutations but lymph node metastases, CK7 was focally expressed. Discussion According to other authors, the immunophenotype CK7−/CK20+ is characteristic of colorectal adenocarcinomas [2,12,17]. Few recent studies have revealed that this immunophenotype may present some changes. During carcinogenesis, some dysplastic glands were CK7-positive, but the normal mucosa was CK7−/CK20+ [9,14]. In colorectal adenocarcinomas, the superficial glands of the normal mucosa express CK20 and remain negative for CK7, but the crypts may present positivity for CK7 [1]. At the same time, both markers may have different patterns in the tumor cells. These cells may be either diffusely positive for CK20, or their reduced expression is obviously emphasized [6]. In some cases, CK7 may be expressed by the tumor cells, but the positivity rate ranges from 0 to 74% according to previously published studies [1,11,16]. Fewer than 10 published studies have analyzed the reasons for the changes in the immunophenotype pattern in CRC. In only one of these studies was microsatellite instability also considered [6]. These studies revealed that CK7 expression was more common in advanced high-grade carcinomas with lymph node metastases located on the proximal colon, but CK20 was expressed more frequently in carcinomas of the distal colon and rectum [1,3].
Table 1 The correlation between clinico-pathological parameters, CK7 and CK20 expression and BRAF mutations in colorectal carcinomas with microsatellite instability (BRAFMUT = mutant; M = male; F = female). No. of case
Age (years)
Gender
Microscopy
Tumor stage
BRAF status
CK7
CK20
1 2 3 4 5 6 7 8 9
55 75 66 56 49 73 73 63 63
M M M F M F M F F
Mucinous carcinoma High-grade adenocarcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Mucinous carcinoma Low-grade adenocarcinoma
pT3N0 pT3N0 pT3N0 pT4N0 pT3N2 pT4N0 pT3N0 pT3N0 pT2N0
Wild type MUT MUT MUT Wild type Wild type Wild type Wild type MUT
Negative Diffuse positive Diffuse positive Diffuse positive Focally positive Negative Negative Negative Diffuse positive
Focally positive Negative Negative Negative Focally positive Focally positive Focally positive Negative Focally positive
S. Gurzu, I. Jung / Pathology – Research and Practice 208 (2012) 163–166
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Fig. 2. A colon adenocarcinoma with microsatellite instability without BRAF mutations. The tumor cells do not express cytokeratin 7 (A) but present focal positivity for cytokeratin 20 (B).
Fig. 3. A colon adenocarcinoma with microsatellite instability with BRAF mutations. The tumor cells strongly express cytokeratin 7 (A) but are negative for cytokeratin 20 (B).
Regarding the microsatellite status, McGregor et al. reported a decreasing expression of CK20 in MSI carcinomas, without data on the relationship between CK7 and molecular characteristics [6]. In our study, CK7 was negative in all MSS cases and diffusely expressed in MSI carcinomas of the proximal colon, which also presented BRAF mutations. Only one MSI case without BRAF mutations but with lymph node metastases was focally positive for CK7. This observation partially corresponds with Bayrak’s opinion, who mentioned that CK7 was strongly expressed in carcinomas with lymph node metastases, independently of other factors [1]. Regarding the expression of CK20, this was indeed decreased in cases of MSI, but all BRAF-mutated carcinomas were CK20-negative. To the best of our knowledge, this is the first report of the correlation between BRAF mutations, microsatellite status, CK20 and CK7 in colorectal cancer and the second report that reveals a decreasing expression of CK20 in MSI colorectal carcinomas, first published by McGregor et al. [6]. Based on an investigation of 11 cases, O’Brien et al. [7] recently suggested that MSI colorectal adenocarcinomas with BRAF mutations may originate from serrated polyps. Their findings were confirmed by Stefanius et al. [13], who investigated 42 serrated adenocarcinomas. They added that 42% of BRAF-mutated serrated adenocarcinomas were MSI cases. At the same time, they proved that 45% of serrated adenocarcinomas were also KRASmutated. Based on these aspects, they concluded that only a subset of sporadic MSI colorectal adenocarcinomas are indeed serrated, although in the earlier studies, all sporadic MSI adenocarcinomas have been attributed to the serrated pathway [15]. If our results are
confirmed by other studies, positivity for cytokeratin 7 and BRAF mutations in MSI cases could be used in the future as a base for the identification of serrated colorectal adenocarcinomas. Some further studies are required to elucidate the possible changing pattern of CK7/CK20 immunophenotype in MSI colorectal carcinomas with BRAF mutations. Analyses of this immunophenotype, in combination with examination of MSI status, BRAF and KRAS mutational status, could be clinically relevant since the treatment may differ in the future for this specific group of colorectal adenocarcinomas. If these results are confirmed by large-scale experiments, the CK7/CK20 is more useful for the differential diagnosis of colorectal cancer. Acknowledgments This work was partially supported by the Romanian National University Research Council (CNCSIS), Ministry of Education and Research, projects frame: PN II – PD, no. 504/2010 and by the Sectoral Operational Programme Human Resources Development, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/89/1.5/S/60782. References [1] R. Bayrak, S. Yenidunya, H. Haltas, Cytokeratin 7 and cytokeratin 20 expression in colorectal adenocarcinomas, Pathol. Res. Pract. 207 (2011) 156–160. [2] J. Chenevert, P. Besette, M. Plante, et al., Mixed ovarian large cell neuroendocrine carcinoma, mucinous adenocarcinoma, and teratoma: a report of two cases and review of literature, Pathol. Res. Pract. 205 (2009) 657–661.
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S. Gurzu, I. Jung / Pathology – Research and Practice 208 (2012) 163–166
[3] B.Y. Hernandez, M.F. Frierson, C.A. Moskaluk, et al., CK20 and CK7 protein expression in colorectal carcinoma: demonstration of the utility of a population-based tissue microarray, Hum. Pathol. 36 (2005) 275–281. [4] T. Kidambi, A. Mahajan, D. DiBardino, Cholangiocarcinoma presenting as metastases to the cervical spine, Am. J. Med. 124 (2011) e1–e2. [5] M. Matsumoto, T. Nakajima, K. Kato, et al., Small invasive colon cancer with systemic metastasis: A case report, BMC Gastroenterol. 11 (2011) 59. [6] D.K. McGregor, T.T. Wu, A. Rashid, et al., Reduced expression of cytokeratin 20 in colorectal carcinomas with high levels of microsatellite instability, Am. J. Surg. Pathol. 28 (2004) 712–718. [7] M.J. O’Brien, S. Yang, C. Mack, H. Xu, C.S. Huang, E. Mulcahy, et al., Comparison of microsatellite instability, CpG island methylation phenotype BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma endpoints, Am. J. Surg. Pathol. 30 (2006) 1491–1501. [8] M. Okido, M. Seo, Y. Hamada, et al., Metastatic breast carcinoma simulating linitis plastica of the colon: report of a case, Surg. Today 41 (2011) 542–545. [9] S. Radovic, I. Selak, M. Babic, et al., Anti-cytokeratin 7: a positive marker for epithelial dysplasia in flat bowel mucosa, Bosn. J. Basci. Med. Sci. 4 (2004) 24–30.
[10] A. Righi, C.M. Betts, C. Marchetti, et al., Merckel cells in the oral mucosa, Int. J. Surg. Pathol. 14 (2006) 206–211. [11] R.S. Saad, J.F. Silverman, M.A. Khalifa, et al., CDX2, cytokeratin 7 and 20 immunoreactivity in rectal adenocarcinoma, Appl. Immunohistochem. Mol. Morphol. 17 (2009) 196–201. [12] K. Sato, Y. Ueda, K. Kawamura, et al., Plasmacytoid urothelial carcinoma of the urinary bladder: a case report and immunohistochemical study, Pathol. Res. Pract. 205 (2009) 189–194. [13] K. Stefanius, L. Ylitalo, A. Tuomiosto, R. Kuivila, T. Kantola, P. Sirniö, et al., Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma, Histopathology 58 (2011) 679–692. [14] R. Stenling, J. Lindberg, J. Rutegard, et al., Altered expression of CK7 and CK20 in preneoplastic and neoplastic lesions in ulcerative colitis, APMIS 115 (2007) 1219–1226. [15] K. Tuppurainen, J.M. Mäkinen, O. Juntilla, A. Liakka, A.P. Kyllönen, H. Tuominen, et al., Morphology and microsatellite instability in sporadic serrated and nonserrated colorectal cancer, J. Pathol. 207 (2005) 285–294. [16] P.J. Zhang, M. Shah, G.W. Spiegel, et al., Cytokeratin 7 immunoreactivity in rectal adenocarcinomas, Appl. Immunohistochem. Mol. Morphol. 11 (2003) 306–310. [17] X. Zhang, P.S. Zheng, Expression and significance of CK7 and CK19 in colon cancer, Xi. Bao. Yu. Fen. Zi. Mian. Yi. Xue. Za. Zhi. 26 (2010) 157–158.
Contents lists available at SciVerse ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.de/prp
Original article
Aberrant pattern of the cytokeratin 7/cytokeratin 20 immunophenotype in colorectal adenocarcinomas with BRAF mutations Simona Gurzu ∗ , Ioan Jung Department of Pathology, University of Medicine and Pharmacy of Targu-Mures, Romania
a r t i c l e
i n f o
Article history: Received 23 August 2011 Received in revised form 5 January 2012 Accepted 16 January 2012 Keywords: Colorectal carcinomas Cytokeratin 7 Cytokeratin 20 BRAF mutation Microsatellite instability
a b s t r a c t Cytokeratin 7 (CK7) and 20 (CK20) are used for the differential diagnosis of metastases from colorectal carcinomas (CRC), which are usually CK7−/CK20+, and other tumors. In our study, we performed immunohistochemical staining with CK7 and CK20 in 52 randomly selected cases of CRC and analyzed microsatellite instability status and BRAF mutations to identify those factors that may determine the changing pattern of CK7/CK20 immunophenotype in these tumors. CK7 was negative in all microsatellite stable tumors (MSS), but all carcinomas presenting microsatellite instability (MSI) and BRAF mutations were diffusely positive for this marker. CK20 was diffusely expressed in 79.06% of MSS tumors. Regarding MSI, in case with no BRAF mutations, a progressive decrease in CK20 expression was noted, and in BRAF-mutated adenocarcinomas, no expression of CK20 was observed. It seems that in case of MSI located on the proximal colon, which also presents BRAF mutations, CK20/CK7 may present a changing immunophenotype pattern, which may complicate the differential diagnosis of metastatic tumors. This is the first reported study of the relationship between CK20/CK7 immunophenotype, BRAF mutations and microsatellite status in CRC. © 2012 Elsevier GmbH. All rights reserved.
Introduction Bayrak et al. [1] recently suggested that 17.3% of colorectal adenocarcinomas (CRC) expressed cytokeratin 7 (CK7) and 18.9% of cases did not express cytokeratin 20 (CK20), although most authors admit that CK20 is of great importance in the differentiation of colonic metastases from other metastatic tumors like lung, bladder, breast carcinomas and cholangiocarcinomas [4,5,8]. On the one hand, CK7 is usually expressed in the normal epithelia of the lung, mesothelium, urinary bladder and female genital tract, and, rarely, it may be observed in gastric and intestinal normal glands, but most authors believe that it is not found in the normal colonic mucosa [9,10]. On the other hand, CK20 specifically stains the normal gland cells of the colonic mucosa and Merkel cells and, more rarely, its expression may be seen in the urothelium or other mucosas [10,14]. Based on these characteristics, the immunophenotype CK7/CK20 is used in daily diagnoses to differentiate CK20 expresssing metastases of colorectal adenocarcinomas (CRC) from lung, ovarian or bladder carcinomas, which are usually stained with CK7 [2,12].
∗ Corresponding author at: Department of Pathology, University of Medicine and Pharmacy, 38 Ghe Marinescu Street, 540139 Targu Mures, Romania. Tel.: +40 745673550; fax: +40 265210933. E-mail address: [email protected] (S. Gurzu). 0344-0338/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2012.01.003
In our study, we analyzed the immunohistochemical (IHC) expression of both CK7 and CK20 in CRC to emphasize the real importance of these antibodies in the differential diagnosis of metastatic adenocarcinomas and to identify those factors which may induce the changing pattern of CK7/CK20 immunophenotype in these tumors. We also tried to correlate the expression of these markers with the microsatellite status and BRAF mutations. These aspects have not been mentioned in the English literature yet.
Material and methods In 52 randomly selected cases of colorectal adenocarcinoma (26 from proximal and 26 from distal colon and rectum), we performed immunohistochemical (IHC) staining with CK7 and CK20 and molecular examinations to determine BRAF mutations and microsatellite status. UltraVision system by D-Line, LabVision (Fremont, CA, USA), was used for immunohistochemical reactions in formalin-fixed, paraffin-embedded tissues. CK7 (clone OV-TL 12/30) and CK20 (clone Q6) antibodies were also provided by D-Line, LabVision. Sections were deparaffinized, incubated at 100 ◦ C with pepsin and washed with distilled water previous to hydrogen peroxide incubation. Subsequently, they were washed with Tris buffered saline (TBS) and incubated with primary antibodies for 60 min. Then, they were washed with TBS and covered with streptavidin peroxidase solution for 5 min. After this, they were washed with TBS
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Fig. 1. A microsatellite stable colon adenocarcinoma without BRAF mutations, with cytokeratin 7 immunoreactivity in normal mucosa (A). The tumor cells present diffuse expression of cytokeratin 20 (B) and no staining for cytokeratin 7 (A).
and covered with biotinylated goat anti-polyvalent solution for further 5 min. The development was performed with dyaminodihydrochlorid benzidine solution (DAB) for 3–5 min. Counterstaining was performed with Mayer’s hematoxylin. Both CK7 and CK20 expressions were evaluated in the cell membrane and also in the cytoplasm. We considered those cases negative which expressed these markers in less than 5% of the cells. Focal positivity was considered as the presence of antibody expression in 5–40% of the tumor cells, the other cases were considered to have diffuse positivity. To determine microsatellite instability, DNA was extracted from formalin-fixed, paraffin-embedded tissues. We used real-time PCR (Roche GmbH, Mannheim, Germany), the method of melting point analysis, as well as microsatellite mononucleotide markers BAT25 and BAT26 by Roche. Exon 15 BRAF mutations were determined using real-time PCR. Results Forty-three of the 52 cases were microsatellite stable carcinomas (MSS), and 9 presented microsatellite instability (MSI), all 9 being located on the proximal colon. Four of the 52 cases presented BRAF mutations. All four cases were also MSI carcinomas. Regarding the normal mucosa, independent of the microsatellite status, BRAF mutations or other factors, CK20 was positive in 41 of the 52 cases (78.84%), the number of stained cells being higher in the surface epithelium. CK7 was focally positive in the normal mucosa in 10 of the 52 carcinomas (19.23%), its intensity being higher in the crypt epithelium (Fig. 1). We found some differences between MSI and MSS cases regarding the CK7/CK20 immunophenotype in tumor areas. In 34 of the 43 MSS cases (79.06%), the tumor cells were diffusely positive for CK20, but no CK7 expression was observed in these tumors (Fig. 1).
In all 9 MSI cases, CK20 was either focally expressed or negative, independent of the other clinicopathological parameters (Table 1). No MSI cases with diffuse positivity were identified for CK20. The CK7 expression was dependent on BRAF mutations and lymph node status. It was diffusely positive in all MSI cases with BRAF mutations and negative in all carcinomas without BRAF mutations (Figs. 2 and 3), except one case. In this MSI case (case 5), which had no BRAF mutations but lymph node metastases, CK7 was focally expressed. Discussion According to other authors, the immunophenotype CK7−/CK20+ is characteristic of colorectal adenocarcinomas [2,12,17]. Few recent studies have revealed that this immunophenotype may present some changes. During carcinogenesis, some dysplastic glands were CK7-positive, but the normal mucosa was CK7−/CK20+ [9,14]. In colorectal adenocarcinomas, the superficial glands of the normal mucosa express CK20 and remain negative for CK7, but the crypts may present positivity for CK7 [1]. At the same time, both markers may have different patterns in the tumor cells. These cells may be either diffusely positive for CK20, or their reduced expression is obviously emphasized [6]. In some cases, CK7 may be expressed by the tumor cells, but the positivity rate ranges from 0 to 74% according to previously published studies [1,11,16]. Fewer than 10 published studies have analyzed the reasons for the changes in the immunophenotype pattern in CRC. In only one of these studies was microsatellite instability also considered [6]. These studies revealed that CK7 expression was more common in advanced high-grade carcinomas with lymph node metastases located on the proximal colon, but CK20 was expressed more frequently in carcinomas of the distal colon and rectum [1,3].
Table 1 The correlation between clinico-pathological parameters, CK7 and CK20 expression and BRAF mutations in colorectal carcinomas with microsatellite instability (BRAFMUT = mutant; M = male; F = female). No. of case
Age (years)
Gender
Microscopy
Tumor stage
BRAF status
CK7
CK20
1 2 3 4 5 6 7 8 9
55 75 66 56 49 73 73 63 63
M M M F M F M F F
Mucinous carcinoma High-grade adenocarcinoma Mucinous carcinoma Mucinous carcinoma Mucinous carcinoma Signet ring cell carcinoma Signet ring cell carcinoma Mucinous carcinoma Low-grade adenocarcinoma
pT3N0 pT3N0 pT3N0 pT4N0 pT3N2 pT4N0 pT3N0 pT3N0 pT2N0
Wild type MUT MUT MUT Wild type Wild type Wild type Wild type MUT
Negative Diffuse positive Diffuse positive Diffuse positive Focally positive Negative Negative Negative Diffuse positive
Focally positive Negative Negative Negative Focally positive Focally positive Focally positive Negative Focally positive
S. Gurzu, I. Jung / Pathology – Research and Practice 208 (2012) 163–166
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Fig. 2. A colon adenocarcinoma with microsatellite instability without BRAF mutations. The tumor cells do not express cytokeratin 7 (A) but present focal positivity for cytokeratin 20 (B).
Fig. 3. A colon adenocarcinoma with microsatellite instability with BRAF mutations. The tumor cells strongly express cytokeratin 7 (A) but are negative for cytokeratin 20 (B).
Regarding the microsatellite status, McGregor et al. reported a decreasing expression of CK20 in MSI carcinomas, without data on the relationship between CK7 and molecular characteristics [6]. In our study, CK7 was negative in all MSS cases and diffusely expressed in MSI carcinomas of the proximal colon, which also presented BRAF mutations. Only one MSI case without BRAF mutations but with lymph node metastases was focally positive for CK7. This observation partially corresponds with Bayrak’s opinion, who mentioned that CK7 was strongly expressed in carcinomas with lymph node metastases, independently of other factors [1]. Regarding the expression of CK20, this was indeed decreased in cases of MSI, but all BRAF-mutated carcinomas were CK20-negative. To the best of our knowledge, this is the first report of the correlation between BRAF mutations, microsatellite status, CK20 and CK7 in colorectal cancer and the second report that reveals a decreasing expression of CK20 in MSI colorectal carcinomas, first published by McGregor et al. [6]. Based on an investigation of 11 cases, O’Brien et al. [7] recently suggested that MSI colorectal adenocarcinomas with BRAF mutations may originate from serrated polyps. Their findings were confirmed by Stefanius et al. [13], who investigated 42 serrated adenocarcinomas. They added that 42% of BRAF-mutated serrated adenocarcinomas were MSI cases. At the same time, they proved that 45% of serrated adenocarcinomas were also KRASmutated. Based on these aspects, they concluded that only a subset of sporadic MSI colorectal adenocarcinomas are indeed serrated, although in the earlier studies, all sporadic MSI adenocarcinomas have been attributed to the serrated pathway [15]. If our results are
confirmed by other studies, positivity for cytokeratin 7 and BRAF mutations in MSI cases could be used in the future as a base for the identification of serrated colorectal adenocarcinomas. Some further studies are required to elucidate the possible changing pattern of CK7/CK20 immunophenotype in MSI colorectal carcinomas with BRAF mutations. Analyses of this immunophenotype, in combination with examination of MSI status, BRAF and KRAS mutational status, could be clinically relevant since the treatment may differ in the future for this specific group of colorectal adenocarcinomas. If these results are confirmed by large-scale experiments, the CK7/CK20 is more useful for the differential diagnosis of colorectal cancer. Acknowledgments This work was partially supported by the Romanian National University Research Council (CNCSIS), Ministry of Education and Research, projects frame: PN II – PD, no. 504/2010 and by the Sectoral Operational Programme Human Resources Development, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/89/1.5/S/60782. References [1] R. Bayrak, S. Yenidunya, H. Haltas, Cytokeratin 7 and cytokeratin 20 expression in colorectal adenocarcinomas, Pathol. Res. Pract. 207 (2011) 156–160. [2] J. Chenevert, P. Besette, M. Plante, et al., Mixed ovarian large cell neuroendocrine carcinoma, mucinous adenocarcinoma, and teratoma: a report of two cases and review of literature, Pathol. Res. Pract. 205 (2009) 657–661.
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[3] B.Y. Hernandez, M.F. Frierson, C.A. Moskaluk, et al., CK20 and CK7 protein expression in colorectal carcinoma: demonstration of the utility of a population-based tissue microarray, Hum. Pathol. 36 (2005) 275–281. [4] T. Kidambi, A. Mahajan, D. DiBardino, Cholangiocarcinoma presenting as metastases to the cervical spine, Am. J. Med. 124 (2011) e1–e2. [5] M. Matsumoto, T. Nakajima, K. Kato, et al., Small invasive colon cancer with systemic metastasis: A case report, BMC Gastroenterol. 11 (2011) 59. [6] D.K. McGregor, T.T. Wu, A. Rashid, et al., Reduced expression of cytokeratin 20 in colorectal carcinomas with high levels of microsatellite instability, Am. J. Surg. Pathol. 28 (2004) 712–718. [7] M.J. O’Brien, S. Yang, C. Mack, H. Xu, C.S. Huang, E. Mulcahy, et al., Comparison of microsatellite instability, CpG island methylation phenotype BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma endpoints, Am. J. Surg. Pathol. 30 (2006) 1491–1501. [8] M. Okido, M. Seo, Y. Hamada, et al., Metastatic breast carcinoma simulating linitis plastica of the colon: report of a case, Surg. Today 41 (2011) 542–545. [9] S. Radovic, I. Selak, M. Babic, et al., Anti-cytokeratin 7: a positive marker for epithelial dysplasia in flat bowel mucosa, Bosn. J. Basci. Med. Sci. 4 (2004) 24–30.
[10] A. Righi, C.M. Betts, C. Marchetti, et al., Merckel cells in the oral mucosa, Int. J. Surg. Pathol. 14 (2006) 206–211. [11] R.S. Saad, J.F. Silverman, M.A. Khalifa, et al., CDX2, cytokeratin 7 and 20 immunoreactivity in rectal adenocarcinoma, Appl. Immunohistochem. Mol. Morphol. 17 (2009) 196–201. [12] K. Sato, Y. Ueda, K. Kawamura, et al., Plasmacytoid urothelial carcinoma of the urinary bladder: a case report and immunohistochemical study, Pathol. Res. Pract. 205 (2009) 189–194. [13] K. Stefanius, L. Ylitalo, A. Tuomiosto, R. Kuivila, T. Kantola, P. Sirniö, et al., Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma, Histopathology 58 (2011) 679–692. [14] R. Stenling, J. Lindberg, J. Rutegard, et al., Altered expression of CK7 and CK20 in preneoplastic and neoplastic lesions in ulcerative colitis, APMIS 115 (2007) 1219–1226. [15] K. Tuppurainen, J.M. Mäkinen, O. Juntilla, A. Liakka, A.P. Kyllönen, H. Tuominen, et al., Morphology and microsatellite instability in sporadic serrated and nonserrated colorectal cancer, J. Pathol. 207 (2005) 285–294. [16] P.J. Zhang, M. Shah, G.W. Spiegel, et al., Cytokeratin 7 immunoreactivity in rectal adenocarcinomas, Appl. Immunohistochem. Mol. Morphol. 11 (2003) 306–310. [17] X. Zhang, P.S. Zheng, Expression and significance of CK7 and CK19 in colon cancer, Xi. Bao. Yu. Fen. Zi. Mian. Yi. Xue. Za. Zhi. 26 (2010) 157–158.