Correlation of Chromosomal Instability, Telomere Length and Telomere Maintenance in Microsatellite Stable Rectal Cancer: A Molecular Subclass of Rectal Cancer

<div><p>Introduction</p><p>Colorectal cancer (CRC) tumor DNA is characterized by chromosomal damage termed chromosomal instability (CIN) and excessively shortened telomeres. Up to 80% of CRC is microsatellite stable (MSS) and is historically considered to be chromosomally unstable (CIN+). However, tumor phenotyping depicts some MSS CRC with little or no genetic changes, thus being chromosomally stable (CIN-). MSS CIN- tumors have not been assessed for telomere attrition. </p> <p>Experimental Design</p><p>MSS rectal cancers from patients ≤50 years old with Stage II (B2 or higher) or Stage III disease were assessed for CIN, telomere length and telomere maintenance mechanism (telomerase activation [TA]; alternative lengthening of telomeres [ALT]). Relative telomere length was measured by qPCR in somatic epithelial and cancer DNA. TA was measured with the TRAPeze assay, and tumors were evaluated for the presence of C-circles indicative of ALT. p53 mutation status was assessed in all available samples. DNA copy number changes were evaluated with Spectral Genomics aCGH. </p> <p>Results</p><p>Tumors were classified as chromosomally stable (CIN-) and chromosomally instable (CIN+) by degree of DNA copy number changes. CIN- tumors (35%; n=6) had fewer copy number changes (<17% of their clones with DNA copy number changes) than CIN+ tumors (65%; n=13) which had high levels of copy number changes in 20% to 49% of clones. Telomere lengths were longer in CIN- compared to CIN+ tumors (p=0.0066) and in those in which telomerase was not activated (p=0.004). Tumors exhibiting activation of telomerase had shorter tumor telomeres (p=0.0040); and tended to be CIN+ (p=0.0949).</p> <p>Conclusions</p><p>MSS rectal cancer appears to represent a heterogeneous group of tumors that may be categorized both on the basis of CIN status and telomere maintenance mechanism. MSS CIN- rectal cancers appear to have longer telomeres than those of MSS CIN+ rectal cancers and to utilize ALT rather than activation of telomerase. </p> </div

Histology, C-circle dot blot and aCGH summary for a MSS CIN- ALT + rectal cancer without activation of telomerase and MSS CIN+ ALT - rectal cancer with activation of telomerase.

<div><p>Panel A. Hematoxylin and Eosin tissue sections from an MSS CIN- , ALT+,Telomerase- rectal cancer (left) and from MSS CIN+, ALT-, Telomerase + rectal cancer. Both are moderately differentiated adenocarcinomas. The gland-to-stroma ratio is higher in the ALT+/tel- case, and it has less desmoplastic stroma.</p> <p>Panel B. Dot/blot showing presence of C-circles. C circles, extrachromosomal telomeric DNA, are strongly associated with ALT. Assessed in tumor DNA with isothermic amplification of C-circle complementary strand and hybridization with ³²P-(CCCTAA)₃ probe by Capital Biosciences (Capital Biosciences, Maryland, U. S. A. ), a sample was called ALT+ if C-circles were detected. The presence of C-circles are illustrated by the presence of radioactive tracer in the image on the left, and the absence of radioactivity in the blot on the right indicates absence of C-circles in the ALT- tumor. </p> <p>Panel C. Ideograms summarizing chromosomal gains and losses across all chromosomes evaluated by aCGH. The ALT+, telomerase negative tumor on the left had <10% of BAC clones showing aberrant hybridization and is classified as a CIN- tumor. The ALT-,,telomerase positive tumor on the right had 40% of clones with aberrant hybridization and is classified as a CIN+ tumor. </p> <p>Panel D. aCGH results of raw data for chromosome 17 for each tumor corresponding to the ideograms in Panel C. </p></div

Clone gains/losses by ALT activity.

<p>Tumors that were ALT- exhibited significantly more chromosomal gains/losses than ALT+ rectal cancer (p=0.0091).</p

Telomere length in tumor DNA based on CIN status and telomerase activation.

<div><p>Panel A: The telomere length of tumor DNA in chromosomally stable (CIN-) rectal cancer is significantly longer than that of chromosomally unstable (CIN+) rectal cancer (p=0.0066). CIN status is determined as CIN- if from <20% of clones show DNA copy number gains or losses. Rectal cancer is CIN+ if more than 20% of clones have gains or losses. </p> <p>Panel B: Activation of telomerase (Telomerase +) in rectal cancer correlates with shorter tumor telomere length than in tumors that do not utilize telomerase (Telomerase -) as a telomere maintenance mechanism (p=0.0040).</p></div