Marjun Philip Duldulao

Prognostic and Predictive Roles of KRAS Mutation in Colorectal Cancer

The RAS gene family is among the most studied and best characterized of the known cancer-related genes. Of the three human ras isoforms, KRAS is the most frequently altered gene, with mutations occurring in 17%–25% of all cancers. In particular, approximately 30%–40% of colon cancers harbor a KRAS mutation. KRAS mutations in colon cancers have been associated with poorer survival and increased tumor aggressiveness. Additionally, KRAS mutations in colorectal cancer lead to resistance to select treatment strategies. In this review we examine the history of KRAS, its prognostic value in patients with colorectal cancer, and evidence supporting its predictive value in determining appropriate therapies for patients with colorectal cancer.

Distribution of residual cancer cells in the bowel wall after neoadjuvant chemoradiation in patients with rectal cancer.

BACKGROUND: The standard treatment for locally advanced rectal cancer is preoperative cheimoradiation and total mesorectal excision. After surgery, tumors are classified according to the depth of tumor invasion, nodal involvement, and tumor regression grade. However, these staging systems do not provide information about the distribution of residual cancer cells within the bowel wall. OBJECTIVE: This study aimed to determine the distribution of residual cancer cells in each layer of the bowel wall in rectal cancer specimens. DESIGN: This was a secondary analysis of data from a prospective phase II study. SETTING: This study was performed in a multi-institutional setting. PATIENTS: Included were 153 patients with stage II or stage III rectal cancer. INTERVENTIONS: Patients were treated with chemoradiation and surgery. The surgical specimen tumor tissue was analyzed, and the distribution of residual cancer cells in each layer of the bowel wall was determined. MAIN OUTCOME MEASURES: Statistical analysis was used to examine the correlation of residual cancer cells in each layer of the bowel wall with the clinical/pathologic stage and tumor regression grade. RESULTS: Forty-two of 153 (27%) patients had complete response in the bowel wall (ypT0). Of the remaining 111 patients who had residual cancer cells, 5 (3%) were ypTis, 12 (8%) were ypT1, 41 (27%) were ypT2, 50 (33%) were ypT3, and 3 (2%) were ypT4. Of the 94 patients with ypT2-4 tumors, 12 (13%) had cancer cells in the mucosa, and 53 (56%) had cancer cells in the submucosa; 92 (98%) had cancer cells in the muscularis propria. Pretreatment cT correlated with the distribution of residual cancer cells. Tumor regression grade was not associated with the distribution of residual cancer cells after chemoradiation. LIMITATIONS: Patients received different chemotherapy regimens. CONCLUSIONS: Residual cancer cells in rectal cancer specimens after chemoradiation are preferentially located close to the invasive front. This should be considered when designing strategies to diagnose complete pathologic response and when investigating the mechanisms of tumor resistance to chemoradiation.

Socioeconomic Factors Impact Colon Cancer Outcomes in Diverse Patient Populations

PurposeCancer disparities among racial and ethnic groups are major public health concerns. Our objective was to examine the impact of socioeconomic status (SES) on survival of colon cancer patients within major racial and ethnic groups.MethodsPatients with colon adenocarcinoma from Los Angeles County (LAC) were assessed. SES was utilized as an indicator of healthcare access and categorized by tertiles (high, middle, and low). Patient characteristics were compared and survival analyses were performed.ResultsIn our heterogeneous LAC cohort, we confirmed survival disparities. Asians had the best survival followed by Hispanics, whites, and blacks. For each stage of disease, Asians and Hispanics had better outcomes than whites and blacks. Then, we evaluated the impact of SES on survival within each racial and ethnic group. We observed significantly longer survival for high SES patients compared to middle and low SES patients for all racial/ethnic groups.ConclusionsWhile disparities across racial/ethnic groups are well-documented, our study is the first to identify socioeconomic disparities in survival for patients within the same group. These novel findings demonstrate the complex role of SES on race and ethnicity and identify the need to improve healthcare access even within select populations.

Molecular Diagnosis of Response to Neoadjuvant Chemoradiation Therapy in Patients with Locally Advanced Rectal Cancer

BACKGROUND Pathologic complete response (pCR) to neoadjuvant chemoradiation (CRT) is an important prognostic factor in locally advanced rectal cancer. However, it is uncertain if histopathological techniques accurately detect pCR. We tested a novel molecular approach for detecting pCR and compared it with current histopathological approaches. STUDY DESIGN Pretreatment tumor biopsies and surgical specimens were collected from 96 patients with locally advanced rectal cancer treated with neoadjuvant CRT and surgery. Tumor response was categorized by tumor regression grade. Tumor DNA from pre-CRT tumor biopsies was screened for K-ras and p53 mutations. DNA from paired surgical specimens was then screened for the same mutations using highly sensitive polymerase chain reaction-based techniques. RESULTS Sixty-eight of 96 (71%) pretreatment biopsies harbored K-ras and/or p53 mutation; 36 (38%) had K-ras mutations, 52 (54%) had p53 mutations, and 20 (21%) carried both mutations. Of 70 patients with tumor regression grades 1 to 3, sixty-six (94%) had a concordant K-ras and p53 mutation profile in pre- and post-treatment tissues. Of 26 patients with tumor regression grade 0 (pCR), 12 had K-ras or p53 mutations in pretreatment biopsies. Of these, 2 (17%) patients had the same K-ras (n = 1) or p53 (n = 1) mutation detected in post-treatment tissue. CONCLUSIONS Sensitive molecular techniques detect K-ras and p53 mutations in post-CRT surgical specimens in some patients with a pCR. This suggests histopathological techniques might not be completely accurate, and some patients diagnosed with a pCR to CRT might have occult cancers cells in their surgical specimens.

TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRASmut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53−/−, TP53+/−, TP53mut/+, and TP53mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRASmut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (−/− or −/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer.

Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

Cancer stem cells (CSCs) have key roles in treatment resistance, tumour metastasis and relapse. Using colorectal cancer (CC) cell lines, patient-derived xenograft (PDX) tissues and patient tissues, here we report that CC CSCs, which resist chemoradiation, have higher SUMO activating enzyme (E1) and global SUMOylation levels than non-CSCs. Knockdown of SUMO E1 or SUMO conjugating enzyme (E2) inhibits CC CSC maintenance and self-renewal, while overexpression of SUMO E1 or E2 increases CC cell stemness. We found that SUMOylation regulates CSCs through Oct-1, a transcription factor for aldehyde dehydrogenases (ALDHs). ALDH activity is not only a marker for CSCs but also important in CSC biology. SUMO does not modify Oct-1 directly, but regulates the expression of TRIM21 that enhances Oct-1 ubiquitination and, consequently, reducing Oct-1 stability. In summary, our findings suggest that SUMOylation could be a target to inhibit CSCs and ultimately to reduce treatment resistance, tumour metastasis and relapse.

Gene polymorphisms predict toxicity to neoadjuvant therapy in patients with rectal cancer.

Toxicity from neoadjuvant chemoradiation therapy (NT) increases morbidity and limits therapeutic efficacy in patients with rectal cancer. The objective of this study was to determine whether specific polymorphisms in genes associated with rectal cancer response to NT were correlated with NT‐related toxicity.

Gene Expression Variations in Microsatellite Stable and Unstable Colon Cancer Cells

BACKGROUND Microsatellite instability (MSI) is a marker of chemoresistance, but it is associated with improved survival compared with microsatellite-stable (MSS) colon cancers. We hypothesized that MSI tumors overexpress chemoresistance-associated genes and underexpress DNA damage/repair genes. We used ultra high-throughput sequencing (UHTS) to assess the expression of representative genes in MSI and MSS colon cancer cell lines. METHODS Solexa UHTS was used to examine gene expression in HCT116 (MSI) and HT29 (MSS) cells, and normal colonic mucosa (NCM). We compared expression of 40 genes involved in chemoresistance, DNA repair, DNA damage, and drug metabolism pathways. RESULTS We observed gene expression differences between MSI and MSS cell lines in 8 out of 40 genes involved in mismatch repair (MMR), DNA repair, drug metabolism, and chemoresistance. MMR gene expression was lower in MSI cells, which is consistent with the MSI phenotype, whereas DNA repair genes were highly expressed in these cells. Genes associated with chemoresistance and drug metabolism also had increased expression in MSI cells. No difference in expression of DNA damage genes was observed between MSI and MSS cell lines. CONCLUSION Using UHTS gene expression analysis, we identified differential expression of genes between MSI and MSS cell lines which may account for resistance to chemotherapy in MSI tumors. UHTS expression analysis has the potential to identify genome-wide predictors of response or resistance to chemotherapy.

Genetic polymorphisms by race/ethnicity among colon cancer patients in Los Angeles County.

492 Background: Though disparate outcomes in colon cancer may be multifactorial, racial/ethnic disparities persist despite adjusting for these factors. Genetic alterations, including single-nucleotide polymorphisms (SNPs), have been shown to predict treatment response in colorectal cancer. Here, we evaluate the variability of genetic alterations in colon cancer patients by examining SNPs in major racial/ethnic groups. METHODS We obtained 76 normal tissue specimens from colon cancer patients of different race/ethnicity at our institution. DNA was extracted and analyzed for 6 SNPs (MTHFR 677C>T, XRCC1 685C>T, XRCC1 1301G>A, XPD 2282A>C, TP53 215G>C, and CCND1 870G>A) by polymerase chain reaction and direct sequencing. Prevalence of each allelic combination was compared between groups by the chi-square test. RESULTS Archived colon cancer tissues were obtained from whites, blacks, Hispanics and Asians (n=19 per group). From genotyping, we identified statistically significant differences in prevalence of CCND1 870G>A between groups ( Table , p=0.0428). Specifically, whites and Asians had significantly higher prevalence of CCND1 870G>A than blacks and Hispanics (100% and 94%, vs. 68% and 79%, respectively). Furthermore, whites and Asians were more frequently heterozygous (G/A) for the SNP (89% in both), whereas Hispanics comprised the largest proportion (16%) of patients homozygous for CCND1 870G>A. Overall, the allelic frequency of CCND1 870G>A varied among the racial/ethnic groups. Other SNPs in treatment-related pathways were evaluated, but there was no difference in prevalence of the other 5 SNPs between groups. CONCLUSIONS Our findings demonstrate that detection of specific genetic polymorphisms differs between the major racial/ethnic groups with colon cancer. Differences in the prevalence of CCND1 870G>A, which affects cell-cycle progression, suggests a potential mechanism for disparities in cancer susceptibility and progression. Therefore, identification of such genetic variability may support a genetic basis for racial disparities in cancer outcomes. [Table: see text].

The extent of lymphadenectomy and overall survival depend on the timing of radiotherapy for rectal cancer.

540 Background: Accurate staging and local disease control depend on the extent of lymphadenectomy (LAD) in rectal cancer. Previous studies suggest that lymph node (LN) number varies with neoadjuvant therapies. Our objectives were to measure the impact of timing of radiotherapy on extent of LAD and to determine the prognostic role of LN number in rectal cancer. METHODS Patients undergoing curative-intent surgery for rectal adenocarcinoma (1988-2006) in Los Angeles County were identified from the Cancer Surveillance Program. Patients were grouped according to radiotherapy timing (neoadjuvant, adjuvant, or none). To measure prognostic significance, an optimal cutoff was assessed for patients with N0 disease by dichotomizing LN numbers from 3-7. RESULTS Query of the registry identified 6,358 patients. Of these, 20% (n = 1,280), 25% (n = 1,573), and 55% (n = 3,545) received neoadjuvant, adjuvant, and no radiotherapy, respectively. There was no difference in LN number in patients with and without radiotherapy (7 vs. 8 LNs, p = NS). However, within the radiotherapy cohort, there was significantly lower LNs in the neoadjuvant group (5 vs. 9 LNs, respectively; p < 0.001). Survival differences favored the groups with higher LN number. The optimal LN cutoff with no survival difference was 7 in the adjuvant radiotherapy group; there was no optimal cutoff for neoadjuvant therapy patients. CONCLUSIONS From our population-based cohort, we observed that patients receiving neoadjuvant radiotherapy had decreased LN retrieval and that LN number was non-prognostic. In contrast, the extent of LAD is a prognostic factor for overall survival in patients receiving adjuvant radiotherapy. [Table: see text] No significant financial relationships to disclose.