adenomatous polyposis coli, mismatch repair, and ... · kanker kolorektal di rsup h. adam malik,...

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ORIGINAL ARTICLE

Acta Medica Indonesiana - The Indonesian Journal of Internal Medicine

Adenomatous Polyposis Coli, Mismatch Repair, and Microsatellite Instability in Colorectal Cancer Based on Different Locations

Rustam Effendi-YS1, Lukman H. Zain1, Gontar A. Siregar1, Harun R. Lubis2, Harun A. Damanik2, Lidya I. Laksmi3, Jessy Chrestella3

1 Department of Internal Medicine, Faculty of Medicine, University of Sumatera Utara, Adam Malik Hospital, Pirngadi Hospital, Medan, Indonesia.2 Department of Internal Medicine, Faculty of Medicine University of Sumatera Utara, Medan, Indonesia.3 Department of Anatomical Pathology, Faculty of Medicine University of Sumatera Utara, Medan, Indonesia.

Correspondence mail:Jalan Sutrisno, Gg. Sehati No. 754 Medan, Postal code 20216, Medan, Indonesia. email: [email protected].

ABstRAkTujuan: meneliti ekspresi protein negatif (EPN) Adenomatous Polyposis Coli (APC), Mismatch Repair

(MMR) dan Microsatellite Instability (MSI), serta membandingkannya dengan lokasi kanker pada pasien kanker kolorektal di RSUP H. Adam Malik, RSU Pirngadi, dan RSU lain yang mempunyai kerjasama dengan FK USU Medan, Indonesia. Metode: penelitian prospektif dikerjakan dari bulan April hingga Desember 2012. Obyek penelitian: jaringan yang baru diambil dari penderita tumor kolorektal. EPN-APC dan MMR (MLH1, MSH2, PMS2, MSH6) diperiksa dengan imunohistokimia, MSI dengan PCR mengunakan 5 penanda mikrosatelit (BAT25, BAT26, D2S123, D5S346, D17S250), sebagai variabel independen. Lokasi tumor sebagai variabel dependen: kolon proksimal (sekum, kolon asenden, kolon transversum), kolon distal (fleksura lienalis, kolon desenden, sigmoid), dan rektum. Analisis komparatif secara bivariat dan multivariat. Hasil: didapat 77 kasus adenokarsinoma kolorektal (KKR). EPN-MMR didapat 54 dari 77 (70,1%). Analisis bivariat: EPN-MLH1 berbeda antara kanker kolon distal dan rektum (p=0,008), EPN-MSH6 antara kolon proksimal dan rektum (p=0,020). Analisis multivariat: EPN-MLH1 berhubungan dengan lokasi kanker (p=0,006), OR 0,12 (95% CI 0,026-0,547). Probabilitasnya 0,12 kali di distal dibanding rektum. EPN-MLH1 10 kali lebih besar di proksimal dibanding di distal (p=0,037). EPN-MSH6 berhubungan dengan lokasi (p=0,026) dengan OR 0,165 (95% CI 0,034-0,803), dan mempunyai probabilitas 0,165 kali di proksimal dibanding rektum; juga 11 kali lebih besar di kolon distal dibanding proksimal (p=0,043). EPN-APC berhubungan dengan lokasi (p=0,020), dengan OR 6,897 (95% CI 1,359-34,995), probabilitasnya 6,89 kali lebih besar di kolon distal dibanding rektum, dengan mengendalikan variabel lain. MSI-H didapat pada 29 dari 77 (37,7%) dan MSI-L/MSS pada 48 (62,3%). Proporsi MSI-H mempunyai tendensi terjadi pada kolon proksimal daripada di distal atau rektum, secara statistik tidak bermakna. Kesimpulan: ada perbedaan jalur atau latar belakang molekuler karsinogenesis masing-masing lokasi kanker pada penderita KKR di daerah ini. EPN MLH1, karakteristik pada kanker kolon proksimal, EPN MSH6 pada kolon distal dan kanker rectum, dan EPN APC pada kolon distal.

Kata kunci: lokasi KKR, APC, MMR, MSI.

ABstRACtAim: to examine the protein expression negative (PEN) of Adenomatous Polyposis Coli (APC), Mismatch

Repair (MMR), and Microsatellite Instability (MSI) status of colorectal cancer (CRC), and establish a comparison

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of those molecular characteristics in CRC location among Indonesian patients in Adam Malik Hospital, Pirngadi Hospital, and other hospitals within the network of Faculty of Medicine University of Sumatera Utara Medan Indonesia. Methods: this prospective study was conducted from April to December 2012. Fresh tissues were obtained from colorectal tumor patients. The APC-PEN, MMR (MLH1, MSH2, PMS2, MSH6)-PEN, were assessed by immunohistochemistry, and MSI by PCR using 5 microsatellite markers (BAT25, BAT26, D2S123, D5S346, D17S250), as independent variables. The tumour locations as dependent variables were divided into proximal colon (caecum, ascending colon, transverse colon); distal colon (splenic flexure, descending colon, sigmoid) and rectum. The comparative study were done by bivariate and multivariate analysis. Results: there were 77 cases of colorectal adenocarsinoma. MMR-PEN was found in 54 of 77 (70.1%). MLH1-PEN was different between distal colon and rectal cancer (p=0.008); MSH6-PEN was different between proximal colon and rectal cancer (p= 0.020). Multivariate analysis showed: MLH1-PEN was related to cancer location (p=0.006) with OR 0.12 (95% CI 0.026-0.547). It had 0.12 times probability to be found in distal than rectum. MLH1-PEN had 10 times higher probability to be found in proximal than in distal (p=0.037). MSH6-PEN was related to the location (p=0.026) with OR 0.165 (95% CI 0.034-0.803), and had 0.165 times probability to be found in proximal than rectum; and 11 times higher probability in distal than proximal colon (p=0.043). APC-PEN was related to the location (p=0.020), with OR 6.897 (95% CI 1.359-34.995), and 6.89 times higher probability in distal than in rectum, with other variables controlled. MSI-H was found in 29 of 77 (37.7%) and MSI-L/MSS in 48 (62.3%). The proportion of MSI-H displayed a tendency to occur in proximal rather than in distal colon or rectal cancer. Conclusion: the underlying carcinogenic pathway or molecular background differs according to the cancer locations of CRC patients in this region. MLH1-PEN was prominent in proximal colon cancer, MSH6-PEN in distal colon and rectal cancer, and APC-PEN in distal colon respectively.

Key words: CRC Locations, APC, MMR, MSI.

IntRoDuCtIonThe molecular biological markers status

of colorectal cancer (CRC) patient is one of the important factors to determine either the possibility of successful treatment for CRC (predictive factor) or life-expectancy (prognostic factor).1 Most molecular studies have only compared between proximal and distal colon cancer, while we suggested that there are three different types of CRC in three locations. A research on the adenomatous polyposis coli (APC) protein expression negative (PEN), mismatch repair (MMR) PEN and microsatellite instability (MSI) status based on the three different locations of CRC (proximal, distal colon, and rectal cancer) had never been conducted.2

CRC is the third most common cancer in western countries, and is the second leading cause of cancer death in the United States.3,4-7 The incidence of this disease varies greatly. Developed countries have a higher incidence of CRC than developing countries,8 and approximately 70% of these cancer cases occur

in colon, while 30% in rectum.9 The ratio of colon to rectal cancer incidence is 2:1 or more common in the high-risk regions, and close to one in low-risk regions.10 Age is a significant risk factor for CRC, with more than 90% of cases developing after the age of 50. The median age at diagnosis is 73 years for colon cancer and 67 years for rectal cancer.3,11 The population in Indonesia is more than 235,000,000, and the age-standarized incidence rates per 100,000 for CRC by gender was 19.1 for male and 15.6 for female, and CRC displayed a tendency to occur in patients under the age of 40 with the proportion of 35.26%.12,13 The study of 760 patients who had been examined by colonoscopy at Pirngadi Hospital Medan revealed 197 (25.9%) of the patients had colorectal cancer, 16.8% of them were under 40 years old and most of CRC are located in the rectum (74.6%) than in colon (18.8% distal and 6.6% proximal colon).14,15

The differences in the characteristics of Chromosome Instability (CIN), MMR, and MSI of CRC found in the proximal and distal colon had been previously investigated; CIN more

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prominent in distal colon, while MMR and MSI in proximal. It has been suggested that those cancer locations differ in their associated genetic alterations in tumorigenesis or carcinogenesis based on studies in western countries, especially Australia and Europe.16 As a result, some researchers believed in the concept “ two colons two cancers”.17

In Indonesia, the study of individual characteristic or molecular profile of CRC patients specifically the APC-PEN, MMR-PEN (MLH1, MSH2, PMS2, MSH6) and MSI using 5 marker of microsatellite based on the three different locations had never been previously conducted. We aimed to investigate those molecular characteristic in CRC among Indonesian patients.

MethoDs

This prospective study was conducted from April to December 2012. Fresh tissues were obtained either by colonoscopic biopsy or surgery, from CRC patients at H. Adam Malik Hospital, Dr. Pirngadi Hospital and other related hospitals within the network of Faculty of Medicine University of Sumatera Utara. The minimum number of sample size patients had been statistically calculated and decided to be 24 patients for each location (24 proximal colon, 24 distal colon and 24 rectal cancer), as this was a comparative study. Inclusion criteria: Indonesian patients with primary colorectal adenocarcinoma confirmed histologically, no family history of adenoma or coloretal cancer, accepted and signed the informed consent. Exclusion criteria: histologically unconfirmed adenocarcinoma, or insufficient tissue specimen which cannot be examined for histology, immunohistochemistry and PCR examinations.

Tumor tissues immediately fixated by using 10% phosphate formaldehyde buffering solution and made into paraffin blocks or formalin-fixed and paraffin-embedded tissues (FFPET). It was used for examining histology of the tumor with Hematoxylin-Eosin staining. The tissues confirmed with adenocarcinoma were included in the study and then immunohistochemical test was done to examine the APC protein expression negative (APC-PEN), MMR-PEN

(MLH1, MSH2, MSH6, and PMS2); while the stability of microsatellite by polymerase chain reaction (PCR) based on 5 markers of BAT25, BAT26, D2S123, D5S346, D17S250.16 MSI-high (MSI-H) was considered if there were ≥2 of markers instable; MSI-Low (MSI-L) if one marker instable, and MS-Stable (MSS) if no instability was found. The tumours were classified according to anatomical subgroups of location: proximal (caecum, ascending colon, transverse colon), distal colon (splenic flexure, descending colon, sigmoid) and rectal cancer. The primary antibodies used for IHC examination were: MLH1: Flex monoclonal mouse antibody (Clone:ESO5, DAKO); MSH2: (3A2B8C); MSH6: Abcam Inc:Cambridge MA (ab92471); PMS2: (H-300): sc-11440.Inc, and APC: (F-3): sc-9998. Biotechnology. Dako Real Envision Rabbit/Mouse was used as Universal Secondary Antibody. Primers and Characteristics of Microsatellite Loci Analyzed: BAT-25 (Hex), Genbank no. 9834508, U63834; BAT-26 (Tet), Genbank no. 9834505, L47575; D2S123 (Tet), Genbank no. 187953; D5S346 (Fam), Genbank no. 181171, M73547; and D17S250 (Fam), Genbank no. 177030, X54562. Histopathological, immunohistochemical assessment and interobserver reliability (Kappa) were performed and reviewed by two pathologists. This study was approved by the Health Research Ethical Committee, Faculty of Medicine University of Sumatera Utara Medan, Indonesia.

statistical Analysis The analysis of the study (univariate,

bivariate and multivariate) were done. Bivariate analysis were performed with the Chi-square test, or Fisher’s exact test. It was used to test for an association between dependent variables (proximal, distal and rectum) and independent variables APC-PEN, MMR-PEN, and MSI.

To investigate the independent variables in relation to CRC locations, stastical analysis using multinomial (polytomous) logistic regression (also called nominal regression) was used. Likelihood ratio test evaluates the comparison of odds ratio (OR). Three steps of analysis was done. Step 1. To exclude each marker of the independent variables which had no significant


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difference based on CRC locations. Step 2. To compare the marker of each MMR-PEN and APC-PEN between proximal colon and rectal cancer, and between distal and rectal cancer. Step 3. To compare each marker of MMR-PEN between proximal and distal cancer.

Statistical test were 2-sided, with p<0.05 considered significant. Data were analyzed by using computer with statistical software (SPSS).

ResuLts A total of 77 cases of CRC patients were

enrolled, consisted of 24 proximal colon, 24 distal, and 29 rectal cancer. Male patients had a larger proportion 42 (54.5%) compared to female 35 (45.5%). Mean age of 77 CRC patients was 53.99 and median age was 54. From the histopathology results, 49.4% well differentiated adenocarcinoma was found, followed by moderately differentiated adenocarcinoma (27.2%), poorly differentiated adenocarcinoma (13.0%), Signet ring cell (5.2%), mucinuous moderately adenocarcinoma (2.6%), and papillary adenocarcinoma (2.6%) respectively (Table 1).

APC-PEN was found 79.2% in proximal, 87.5% in distal colon and 69.0% in rectum. The proportion of MMR-PEN based on locations was 62.5% in proximal, 66.7% in distal and 79.3% in rectum. MSI-H was found in 29 of 77 (37.7%), with a proportion 11 (45.8%) in proximal, 8 (33.3%) in distal, 10 (34.5%) in rectal cancer; and MSI-L/MSS in 48 cases (62.3%) (Table 2).

Table 3 and 4 shows that there was significant difference of MLH1-PEN between distal versus rectal cancer (p=0.008), and MSH6-PEN between proximal vs rectal cancer (p=0.020). There was no significant differences of MSH2-PEN, PMS2-PEN and MSI-H found among the three locations of cancer.

The proportion of MSI status based on sex, age, locations, MMR and MLH1-PE were 29 cases with MSI-H, 17 (31.5%) with MMR-PEN and 12 (52.2%) with MMR-PE positive (Table 5).

Table 6 shows the analysis of each MMR-PEN in relation to the CRC locations. Multivariate analysis showed that MSH6-PEN was significantly related to the locations (p=0.026) with OR 0.165 (95% CI 0.034-0.803).

table 1. Characteristics of study subjects

Characteristics n (%)

Tumor Location

- Proximal Colon 24 (31.2)

- Distal Colon 24 (31.2)

- Rectum 29 (37.6)

- Total 77 (100.0)

Sex

- Male 42 (54.5)

- Female 35 (45.5)

Age (Years):

- 20 – 29 6 (7.8)

- 30 – 39 7 (9.1)

- 40 – 49 17 (22.1)

- 50 – 59 16 (20.7)

- 60 – 69 17 (22.1)

- 70 – 79 14 (18.2)

Ethnicity

- Toba Bataknese 25 (32.5)

- Mandailing Bataknese 6 (7.8)

- Karo Bataknese 7 (9.1)

- Simalungun Bataknese 5 (6.5)

- Pakpak Bataknese 1 (1.3)

- Javanese 15 (19.5)

- Banten 1 (1.3)

- Minang 2 (2.6)

- Malay 5 (6.5)

- Chinese 5 (6.5)

- Nias 1 (1.3)

- Aceh 4 (5.2)

Histopathology

- Well Differentiated Adenocarcinoma 38 (49.4)

- Moderately Differentiated Adenocarcinoma

21 (27.2)

- Poorly Differentiated Adenocarcinoma

10 (13.0)

- Papilary Adenocarcinoma 2 (2.6)

- Mucinous Adenocarcinoma 2 (2.6)

- Signet Ring Cell Carcinoma 4 (5.2)

MLH1-PEN was significantly related to the location of the cancer (p=0.006) with OR 0.12 (95% CI 0.026-0.547). APC-PEN was also significantly related to the locations (p=0.020), with OR 6.897 (95% CI 1.359-34.995).

Multivariate analysis also showed that MLH1-PEN was significantly related to the cancer location (p=0.037) with OR 0.10 (95% CI 0.011-0.874). MSH6-PEN was also related


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table 2. APC-PE, PE of each MMR, and MSI status based on cancer locations

Protein expressionCRC location

total n (%)Proximal n (%) Distal n (%) Rectum n (%)

APC-PE

- Negative 19 (79.2) 21 (87.5) 20 (69.0) 60 (77.9)

- Positive 5 (20.8) 3 (12.5) 9 (31.0) 17 (22.1)

MLH1-PE

- Negative 9 (37.5) 4 (16.7) 15 (51.7) 28 (36.4)

- Positive 15 (62.5) 20 (83.3) 14 (48.3) 49 (63.6)

MSH2-PE

- Negative 11 (45.8) 11 (45.9) 12 (41.4) 34 (44.2)

- Positive 13 (54.2) 13 (54.2) 17 (58.6) 43 (55.8)

PMS2-PE

- Negative 4 (16.7) 4 (16.7) 4 (13.8) 12 (15.6)

- Positive 20 (83.3) 20 (83.3) 25 (86.2) 65 (84.4)

MSH6-PE

- Negative 3 (12.5) 7 (29.2) 12 (41.4) 22 (28.6)

- Positive 21 (87.5) 17 (70.8) 17 (58.6) 55 (71.4)

MSI status

- MSI-high 11 (45.8) 8 (33.3) 10 (34.5) 29 (37.7)

- Low+MSS 13 (54.2) 16 (66.7) 19 (65.5) 48 (62.3)

table 3. MLH1-PE between distal v.s. rectum

Protein expression

Cancer locationP valueDistal colon

n (%)Rectum

n (%)MLH1-PE

Negative 4 (16.7) 15 (51.7) 0.008

Positive 20 (83.3) 14 (48.3)

table 4. MSH6-PE between proximal v.s. rectum

Protein expression

Cancer locationP valueProximal colon

n (%)Rectum

n (%)MSH6-PE

Negative 3 (12.5) 12 (41.4) 0.020

Positive 21 (87.5) 17 (58.6)

to the locations (p=0.043) with OR 11.046 (95% CI 1.081-112.911) (Table 7).

In step 1, Likelihood ratio test showed p value 0.016 with pseudo R square or estimated R2 value (Cox and Snell) 0.248 (24.8%). It indicates that MMR and APC determined 24.8% molecular characteristics associated to cancer locations. After using the reduced model likelihood ratio test, p values of MSH2 and PMS2 were >0.05. In step 2, both MSH2 and PMS2 were exluded, pseudo R-square (Cox and Snell) was 0.231 (23.1%) and p value was found to be more significant (p=0.002). In step 3, we found estimated R2 (Cox and Snell) to be 0.166 (16.6%); this indicates that MLH1 and MSH6 determined cancer locations weighting by 16.6 %.

DIsCussIon

Gender, Age and ethnicity

There were more male patients compare to female. Similar result was discovered in the studies done in Sweden, United States, and Europe, especially in older patients.4,18 Interestingly, a different result was found in the data from endoscopic unit at Dr. Pringadi Hospital where female patients (51.3%) were more predominant than male patients (48.7%), however the difference was not significant.14 These variation need to be further studied. Older persons are more at risk for proximal cancer and the aging and growing population has contributed to this increase.19,20


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The greatest proportion of CRC patients in this study was found in the age group of 40s and 60s. There were 13 out of 77 CRC (16.9%) under 40 years (Table 1). This number was lower than the data from Department of Pathology Medical Faculty University of Indonesia which was 35.26%.12 In this study proximal colon cancer appeared to be more common in female patients in the age group 70-79, and male patients in the age group 40-49.

Most of CRC found were among Bataknese, followed by Javanese and other ethnic groups. A study performed from 1998 to 2001 revealed that the black population was more likely to develop distal cancer than the white population.20 However, we could not determine that ethnicity played a role in the carcinogenesis of CRC solely based on those results.

MMR DefectMLH1-PEN was more frequent in rectum

than in distal colon cancer (p=0.008). MSH6-PEN was more frequent in rectal cancer than proximal colon cancer (p=0.020). Our study

table 5. The MSI status based on sex, age, location, MMR and MLH1-PE

MsI-h n (%)

MsI-L+Mssn (%) p

Sex

- male 13 (44.8) 29 (60.4) 0.18

- female 16 (55.2) 19 (39.6)

Age (years)

- <50 11 (37.9) 19 (39.6) 0.88

- >50 18 (62.1) 29 (60.4)

Location

- Proximal vs Distal11 (57.9) 13 (44.8) 0.38

8 (42.1) 16 (55.2)

- Distal vs. rectum8 (44.4) 16 (45.7) 0.93

10 (55.6) 19 (54.3)

- Proximal vs. rectum

11 (52.4) 13 (40.6) 0.40

10 (47.6) 19 (59.4)

Protein expression

- MMR PE positive 12 (41.4) 11 (22.9) 0.09

- MMR PE negative 17 (58.6) 37 (77.1)

Protein expression

- MLH1 PE positive 18 (62.1) 31 (64.6) 0.82

- MLH1 PE negative 11 (37.9) 17 (35.4)

table 6. Odds ratio for MLH1-PEN, MSH6-PEN, and APC-PEN related to CRC locations with rectum as base

Coefficient se p oR (95% CI)Proximal ColonIntercept -0.472 0.568 0.406

MLH1-PEN -0.332 0.693 0.631 0.717 (0.185-2.787)

Msh6-Pen -1.800 0.806 0.026 0.165 (0.034- 0.803)APC-PEN 1.208 0.740 0.102 3.348 (0.785-14.280)

Distal ColonIntercept -0.930 0.678 0.170

MLh1-Pen -2.119 0.773 0.006 0.120 (0.026-0.547)MSH6-PEN -0.166 0.755 0.826 0.847 (0.193-3.724)

APC-Pen 1.931 0.829 0.020 6.897 (1.359-34.995)

table 7. Odds ratio for MLH1-PEN and MSH6-PEN between proximal and distal colon cancer

Coefficient se p oR (95% CI)

MLh1- Pen -2.304 1.107 0.037 0.100 (0.011-0.874)Msh6- Pen 2.402 1.186 0.043 11.046 (1.081-112.911)Constant 0.104 0.356 0.771 1.109


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also showed MSH6, MLH1 and APC-PEN were significantly related to the cancer locations. MLH1 and MSH6-PEN were significantly related to cancer location.

Multivariate analysis showed that MLH1-PEN was related to cancer location (p=0.006). It had 0.12 times probability to be found in the distal colon than in the rectal cancer, or 8.33 times higher probability to be found in rectal than in distal colon cancer, and it had 10 times higher probability to be found in proximal than in distal colon cancer (p=0.037). This indicates that proximal colon cancer was characterized by MLH1-PEN.

MSH6-PEN was related to the location of cancer (p=0.026). It had 0.165 times probability to be found in proximal than in rectal cancer or 6.06 times higher probability to be found in rectal cancer than in proximal colon cancer (Table 6), and 11.046 times higher probability in distal colon than in proximal colon cancer (p=0.043) (Table 7). It indicates that rectal cancer as well as distal colon cancer were characterized by MSH6-PEN.

MsI statusThe primers proposed by National Cancer

Institute workshop on MSI (D2S123, D5S346, D17 S250, BAT25 and BAT 26) were used in this study.16 MSI-H was found in 29 of 77 (37.7%) CRC, while MSI-L+MSS 48 (62.3%). The result was lower than Ashktorab’s 43%,21 however it was higher than Chang’s 31%,18 and Boardman’s 14%.22

In this study MSI-H had a higher proportion in proximal colon 45.8%. Cho, found 105 of 164 (64%) MSI-H CRC were located in proximal and 59 (36%) in distal colon, with proximal CRC predominantly found in female (p=0.014).17 We also discovered each cancer location had different MSI-H proportion with no statistically significant difference (p=0.341). MSI-H was found in 55.2% female and 44.8% male, no statistical difference. From 47 patients aged ≥50 years there were 18 (38.3%) MSI-H, and 29 (61.7%) MSI-L+MSS cases. In the age group <50 years, 11 of 30 (36.7%) MSI-H and 19 of 30 (63.3%) MSI-L+MSS. MSI-H seemed to occur more frequently in female patients and those over 50; with no statistical difference.

A previous molecular studies indicated that 10-15% CRC was due to a defect in DNA MMR which causes MSI-H, and CRC patients with MSI-H showed a better prognosis than CRC with MSS or CIN.7,21,23

In this study defect in DNA MMR with MSI-H were 17 of 54 (31.5%) cases. It was higher than what was found by Funkhouser (15%).23 Non MSI-H CRC (MSS) showed that CIN, imbalanced in chromosome translocation, gain or loss of entire chromosome, cause a change in gene expression.23,24 Gupta discovered that all of the MSI-H CRC patients (hispanic origin) exhibited loss of MMR expression, and 3.5% of MSS CRC also showed a defect of MMR.25

APC-PeThe result of APC-PE negative in this study

was 60 of 77 (77.9%), nearly equivalent to the result of Seidler,26 and it was distributed with the higher probability in distal colon. APC-PEN was related to CRC location (p=0.020). The probability of APC-PEN in distal colon was 6.897 times higher than in rectum, independently (Table 6). This results revealed that distal colon cancer is a cancer type with a predominant APC-PEN.

Oncogen activation, tumour suppressor gene (TSG) inactivation and genetic instability (DNA methylation), CIN, MSI and CIMP play a fundamental role in CRC carcinogenesis.27,28 As previously stated, there was a concept of two colons two cancers. However, we suspected three locations of CRC and three cancers, for each location has specific carcinogenesis pathways.16,29

ConCLusIon

The MLH1-PEN was prominent in proximal colon cancer, MSH6-PEN in distal colon and rectal cancer, and APC-PEN in distal colon respectively. The proportion of MSI-H displayed a tendency to occur in proximal rather than in distal or rectal cancer. Nevertheless, these findings suggest that the underlying carcinogenic pathway or molecular backgrounds differ according to the cancer locations among CRC patients in this region. The cancer in each


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colorectal location has its specific molecular characteristics.

ACknowLeDGMentsWe thank Prof. Dr. dr. Syahril Pasaribu,

DTM&H, MSc (CTM), Sp.A (K), Rector of University of Sumatera Utara ; Prof. dr. Gontar A Siregar, Sp.PD-KGEH, Dean of Faculty of Medicine University of Sumatera Utara; Prof. dr. Chairuddin P Lubis, DTM&H, Sp.A(K); Prof. Dr. dr. Delfitri Munir, Sp.THT-KL(K); Prof. dr. Lukman Hakim Zain, Sp.PD-KGEH; Prof. dr. Harun Rasyid Lubis, Sp.PD-KGH; Prof. Dr. dr. Harun Alrasyid, Sp.PD, Sp.GK; dr. Gino Tann, PhD, Sp.PK; dr. Adang Bachtiar, MPH, DSc; Prof. dr. Sorimuda Sarumpaet, MPH; Prof. dr. Marcellus Simadibrata, PhD, Sp.PD-KGEH; and Dr. dr. Aru W. Sudoyo, SpPD-KHOM; Director and Hospital team of Adam Malik Hospital, Pirngadi Hospital, and other Associated Hospitals; dr. T. Ibnu A, Sp.PA, Head of Pathology Department of Faculty of Medicine, Head and staff of Pathology Department at Adam Malik and Pirngadi Hospital, PT. Prodia Widya Husada, BPPT Jakarta, Dr. Miswar Fattah, MSi; who supported this study, and patients whom participated in this study.

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