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Promoter methylation of tumor suppressor genes in pre-neoplastic lesions; potential marker of disease recurrence.

Rengucci C, De Maio G, Casadei Gardini A, Zucca M, Scarpi E, Zingaretti C, Foschi G, Tumedei MM, Molinari C, Saragoni L, Puccetti M, Amadori D, Zoli W, Calistri D - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry.Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions.Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence.

Methods: A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry.

Results: Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively.

Conclusions: Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

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IHC staining of FHIT protein in adenoma samples. A) High cytoplasmic staining in 85% of colonic glands (grade 3+), a small fraction of glands (15%-20%) showing low intensity staining (grade 2+). Magnification 2.5 ×. B) High cytoplasmic staining in 85% of colonic glands (grade 3+). Magnification 20×. C) Medium cytoplasm staining in 80% of colonic glands (grade 2+). Magnification 20×. D) Low cytoplasmic staining in 60% of colonic glands; 40%, grade 1+ and 20%, grade 2+. Magnification 2.5×. E) Negative cytoplasmic staining of colonic glands. Magnification 2.5 x.
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Figure 3: IHC staining of FHIT protein in adenoma samples. A) High cytoplasmic staining in 85% of colonic glands (grade 3+), a small fraction of glands (15%-20%) showing low intensity staining (grade 2+). Magnification 2.5 ×. B) High cytoplasmic staining in 85% of colonic glands (grade 3+). Magnification 20×. C) Medium cytoplasm staining in 80% of colonic glands (grade 2+). Magnification 20×. D) Low cytoplasmic staining in 60% of colonic glands; 40%, grade 1+ and 20%, grade 2+. Magnification 2.5×. E) Negative cytoplasmic staining of colonic glands. Magnification 2.5 x.

Mentions: Good concordance was observed between MS-MLPA and the other two methods used (Table 1). In particular, a comparison between the MS-MLPA and pyrosequencing methods showed a 79% (57/72 cases) agreement in samples for MLH1 and a 73% (56/77cases) agreement for ATM, respectively. The concordance between MS-MLPA results and IHC was 84% for FHIT (48/57 cases) (Figure 3). This validation was not performed on samples for which there was insufficient biological material.


Promoter methylation of tumor suppressor genes in pre-neoplastic lesions; potential marker of disease recurrence.

Rengucci C, De Maio G, Casadei Gardini A, Zucca M, Scarpi E, Zingaretti C, Foschi G, Tumedei MM, Molinari C, Saragoni L, Puccetti M, Amadori D, Zoli W, Calistri D - J. Exp. Clin. Cancer Res. (2014)

IHC staining of FHIT protein in adenoma samples. A) High cytoplasmic staining in 85% of colonic glands (grade 3+), a small fraction of glands (15%-20%) showing low intensity staining (grade 2+). Magnification 2.5 ×. B) High cytoplasmic staining in 85% of colonic glands (grade 3+). Magnification 20×. C) Medium cytoplasm staining in 80% of colonic glands (grade 2+). Magnification 20×. D) Low cytoplasmic staining in 60% of colonic glands; 40%, grade 1+ and 20%, grade 2+. Magnification 2.5×. E) Negative cytoplasmic staining of colonic glands. Magnification 2.5 x.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4274757&req=5

Figure 3: IHC staining of FHIT protein in adenoma samples. A) High cytoplasmic staining in 85% of colonic glands (grade 3+), a small fraction of glands (15%-20%) showing low intensity staining (grade 2+). Magnification 2.5 ×. B) High cytoplasmic staining in 85% of colonic glands (grade 3+). Magnification 20×. C) Medium cytoplasm staining in 80% of colonic glands (grade 2+). Magnification 20×. D) Low cytoplasmic staining in 60% of colonic glands; 40%, grade 1+ and 20%, grade 2+. Magnification 2.5×. E) Negative cytoplasmic staining of colonic glands. Magnification 2.5 x.
Mentions: Good concordance was observed between MS-MLPA and the other two methods used (Table 1). In particular, a comparison between the MS-MLPA and pyrosequencing methods showed a 79% (57/72 cases) agreement in samples for MLH1 and a 73% (56/77cases) agreement for ATM, respectively. The concordance between MS-MLPA results and IHC was 84% for FHIT (48/57 cases) (Figure 3). This validation was not performed on samples for which there was insufficient biological material.

Bottom Line: MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry.Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions.Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence.

Methods: A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry.

Results: Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively.

Conclusions: Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

Show MeSH
Related in: MedlinePlus