Interaction of molecular markers and physical activity on mortality in patients with colon cancer

Abstract

PURPOSE

Physical activity in colon cancer survivors has been associated with lower cancer recurrences and improved survival. Whether molecular features of the tumor portend more or less likelihood for benefit from exercise is unknown.

EXPERIMENTAL DESIGN

Utilizing two large prospective cohort studies with physical activity assessments after colon cancer diagnosis, we examined expression of fatty acid synthase (FASN), p53, p21, and p27 and mutational status of K-ras and phosphatidylinositol 3-kinase(PI3KCA). We calculated hazard ratios (HRs) of colon cancer-specific mortality, adjusted for tumor and patient characteristics, and tested for molecular interactions with exercise.

RESULTS

In a cohort of 484 men and women with stage I-III colon cancer, patients who engaged in at least 18 metabolic equivalent task (MET)-hours per week after diagnosis had an adjusted HR for colon cancer-specific mortality of 0.64 (95% CI, 0.33-1.23) and for overall mortality of 0.60 (95% CI, 0.41-0.86). A statistically significant interaction was detected based on p27 expression (p=0.03). For tumors with loss of p27 (n=195), physical activity ≥18 MET-hours/week led to a HR for colon cancer mortality of 1.40 (95% CI, 0.41-4.72), compared to those with <18 MET-hours/week. However, for tumors with expression of p27 (n=251), the adjusted HR was 0.33 (95% CI, 0.12-0.85). Molecular status of FASN, K-ras, p53, p21, PI3KCA did not influence the association between exercise and colon cancer-specific or overall mortality.

CONCLUSION

The benefit of physical activity on outcomes in patients with stage I—III colon cancer may be influenced by p27 status. Further studies are warranted to confirm these findings.

INTRODUCTION

Physically active people have a reduced risk of developing colon cancer (1-11). A meta-analysis of 19 cohort studies showed a statistically significant 22% reduction in the risk of colon cancer in active males and 29% reduction in active females (12). The International Agency for Research on Cancer concluded that the evidence supports a causal relation between inactivity and colon cancer risk (13). In two large prospective observational studies of colon cance patients, physical activity after colon cancer diagnosis was associated with significant improvements in colon cancer recurrences (14) or colon cancer-specific mortality (15) and overall mortality (14, 15). Colon cancer survivors who engaged in higher levels of physical activity experienced a 50-60% improvement in long-term outcomes compared to inactive patients.

Studies have suggested that energy balance and physical activity influence certain molecular features of tumors. Obesity and / or reduced physical activity have been associated with colon cancers with p53 overexpression (16) and K-ras mutations (17, 18). Fatty acid synthase (FASN) is physiologically regulated by energy balance, and high carbohydrate / low fat diets up-regulate FASN (19). On the contrary, exercise and energy restriction down-regulate FASN through AMP-activated kinase (20).

Obesity, insulin and insulin-like growth factor-1 influence growth and inhibit apoptosis through phosphatidylinositol 3-kinase (PIK3CA), which in turn activates Akt/protein kinase B (PKB) via phosphorylation (21). Phosphorylation of Akt (phospho-Akt) results in cell proliferation and escape from apoptosis (22). Approximately 45% of colon cancers overexpress phospho-Akt (23), which inhibits transcription and promotes degradation of the cyclin-dependent kinase inhibitor p27 (24), and in vitro studies demonstrate that insulin and IGF-1 similarly result in down-regulation of p27 (25). In animals, p27 expression increased in a dose-dependent manner in response to energy restriction and/or physical activity (26-28).

Given the association between post diagnosis physical activity and improved survival outcomes in patients with surgically resected colon cancer (14, 15) and the influence of physical activity on molecular features of tumors, we sought to determine whether the influence of physical activity on patient survival differs depending on molecular features of the tumor. Using two large, prospective cohorts with self reported post-diagnosis physical activity data and tumor blocks, we tested the interaction between exercise and molecular markers on colon cancer-specific and overall mortality in patients with stage I, II and III colon cancer.

MATERIALS AND METHODS

Study Population

We utilized the databases of two large prospective cohort studies, the Nurses’ Health Study (N=121,700 women followed since 1976), and the Health Professional Follow-up Study (N=51,500 men followed since 1986). Every 2 years, participants have been sent follow-up questionnaires to update information on potential risk factors and to identify newly diagnosed cancer and other diseases. This study was approved by the Human Subjects Committees at Brigham and Women’s Hospital and the Harvard School of Public Health, both in Boston, Massachusetts.

Measurement of Colon Cancer and Mortality

On each biennial follow-up questionnaire, participants were asked whether they had a diagnosis of colon cancer during the previous 2 years. When a participant (or next of kin for decedents) reported colon cancer, we sought permission to obtain medical records. Study physicians, while blinded to exposure data, reviewed all records related to colon cancer, and recorded AJCC (American Joint Committee on Cancer) tumor stage and tumor location. For nonresponders, we searched the National Death Index to discover deaths and ascertain any diagnosis of colon cancer that contributed to death or was a secondary diagnosis. The ascertainment of cases of colon cancer has been described in detail (29). The response rate for participants who had nonfatal outcomes was 96% of the possible number of person-years. We collected paraffin-embedded tissue blocks from hospitals where colon cancer patients underwent resections of primary tumors. Tissue sections from all colon cancer cases were reviewed by a pathologist (S.O.). Tumor grade was categorized as high (≤50% glandular area) or low (>50% glandular area). Based on availability of tissue samples, the current analyses have up to 487 tumor samples with physical activity assessments.

Patients were observed until death or June 2006, whichever came first. Ascertainment of deaths included reporting by the family or postal authorities. In addition, the names of persistent nonresponders were searched in the National Death Index (30). The cause of death was assigned by physicians blinded to other clinical and lifestyle information. In rare patients who died as a result of colon cancer not previously reported, we obtained medical records with permission from next of kin. More than 98% of deaths in the cohorts were identified by these methods.(31, 32)

Exposure assessment

Since 1986, leisure-time physical activity has been assessed every 2 years in both cohorts, as previously described and validated against subject diaries (33, 34). Subjects reported duration of participation (ranging from 0 to 11 or more hours per week) on walking (along with usual pace); jogging; running; bicycling; swimming laps; racket sports; other aerobic exercises; lower intensity exercise (yoga, toning, stretching); or other vigorous activities.

We have previously reported that women with colon cancer who were more physically active had a statistically significant improvement in colon cancer-specific mortality compared to those engaging in minimal leisure-time physical activity (15). In that analysis, as well as the present one, to avoid bias due to declining activity, physical activity was not updated (only a single post-diagnosis measurement was utilized).

Each activity on the questionnaire was assigned a metabolic equivalent task (MET) score (35). One MET is the energy expenditure for sitting quietly. MET scores are defined as the ratio of the metabolic rate associated with specific activities divided by the resting metabolic rate. The values from the individual activities were summed for a total MET-hours per week score. Based on prior studies of physical activity in colon cancer survivors, patients who engaged in at least 18 MET-hours per week had significantly improved colon cancer-specific mortality (14, 15). For primary analyses, we dichotomized physical activity to less than 18 MET-hours per week and greater than or equal to 18 MET-hours per week.

Since our prior studies have found an association between physical activity after diagnosis and survival (14, 15), the first physical activity assessment collected at least 1 year but no more than 4 years after diagnosis (median 17 months) was used to avoid assessment during the period of active treatment.

Covariates

Stage of disease, grade of tumor differentiation, year of diagnosis and location of tumor were extracted from the medical record. The time interval between cancer diagnosis and assessment of activity was also adjusted for in these analyses. Body mass index (BMI) was also obtained from the biennial questionnaire at the time of the respective physical activity assessment.

Immunohistochemistry for fatty acid synthase (FASN), p53, p21 and p27

Tissue microarrays (TMAs) were constructed and immunohistochemistry for FASN, p53, p21 and p27 was performed as previously described.(36-39) Appropriate positive and negative controls were included in each run for each marker’s immunohistochemistry. All immunohistochemically-stained slides were interpreted by a pathologist (S.O.) blinded from any other laboratory data.

FASN expression was categorized as negative (no or weak expression) or positive (strong expression). For p53, we visually estimated the fraction of tumor cells with strong and unequivocal nuclear staining, by examining at least two tissue cores in TMAs, or the whole tissue section in each case for which there was not enough tissue for TMAs or results were equivocal in TMAs. p53 positivity was defined as 50% or more of tumor cells with moderate or strong staining. For p21 immunohistochemistry, normal colonic mucosa or rare mesenchymal cells served as internal positive control. We visually estimated the fraction of tumor cells expressing p21, using the whole tissue section on a single slide for every case. p21 expression was interpreted as ‘loss’ in < 20% of cells were positive and ‘expressed’ if ≥ 20% of cells were positive. The extent of nuclear p27 expression was visually estimated using whole tissue sections, and interpreted as ‘loss’ (no staining, only weakly staining, or <20% of tumor cells positive for moderate/strong staining) or ‘expressed’ if moderate/strong positive in ≥ 20% of cells.

A random selection of 114-246 cases was re-examined for each marker by a second pathologist (p53 and FASN by K.N.; p21 and p27 by K.S.) unaware of other data, and concordance rates and κ coefficients between the two pathologists were as follows: 0.87 (κ=0.75; N=118) for p53; 0.93 (κ=0.57; N=246) for FASN; 0.83 (κ=0.62; N=179) for p21; and 0.94 (κ=0.60; N=114) for p27.

Pyrosequencing for K-ras and PIK3CA

Genomic DNA was extracted from dissected tumor tissue sections using QIAmp DNA Mini Kit (Qiagen, Valencia, CA) (40). Normal DNA was obtained from colonic tissue at resection margins. Whole genome amplification of genomic DNA was performed by polymerase chain reaction (PCR) using random 15-mer primers. Polymerase chain reaction and Pyrosequencing were performed as previously described (40, 41).

Statistical Analysis

Cox proportional hazards models were used to calculate hazard ratios of colon cancer-specific death from colon cancer, adjusted for other risk factors for cancer survival. Death from colon cancer was the primary endpoint and deaths from other causes were censored. Participants were followed from the date of return of post-diagnosis physical activity assessment to either death or June 2006, whichever came first. Modeling was performed by entering physical activity in the model and stratifying by the molecular marker and by entering the molecular marker and stratifying by physical activity. Tests of interactions between physical activity categories and molecular markers were assessed by entering in the model the cross product of the dichotomized physical activity variable and the dichotomized molecular marker. No formal adjustments for multiple hypothesis testing were done but considered when interpreting results. All analyses utilized SAS version 8.0 (SAS Institute Inc, Cary, NC).

RESULTS

Baseline characteristics

At the time of analyses of these 2 cohorts, 1024 subjects had available tumor blocks. Of those, 678 were colon cancers. Eight patients were excluded due to having another cancer diagnosis within 3 years of the colon cancer, 25 patients were excluded for not having a diagnosis of colon in the time frame of 1986-2006 (physical activity assessments began in 1986) and 81 patients were excluded for stage IV colon cancer (eligible sample size based on blocks was 564). Of those patients, 488 had a measurement of physical activity within 4 years of diagnosis (median time to assessment 17 months with 95% within 30 months after diagnosis) but 4 patients died within 6 months of the activity assessment and thus were excluded (consistent with our prior analyses). Thus, 484 colon cancer patients without evidence of metastatic disease at diagnosis were included in these analyses (Table 1). When considering all subjects in the cohort that meet the inclusion/exclusion criteria (colon cancer, not stage IV at diagnosis, time frame of study, having appropriate post diagnosis physical activity assessment), no significant changes were seen in baseline characteristics with and without blocks available for analysis (data not shown). Sixty-three percent (n = 307) reported physical activity levels less than 18 MET-hours per week while 37% (n = 177) engaged in 18 or greater MET-hours per week. The median age at diagnosis, median BMI, year of diagnosis distribution and median time from diagnosis to physical activity assessment were similar between the two exercise categories. Those engaging in less than 18 MET-hours per week were more likely to be female while those with at least 18 MET-hours per week of exercise were more likely to be male. Stage distribution was fairly similar though there was a higher percentage of stage I versus II patients in the less active cohort and higher percentage of stage II versus I patients in the more active cohort.

Table 1.

Baseline characteristics of patients with tumor samples included in this study by level of physical activity

Baseline characteristics	MET-hours / week < 18 N=307	MET-hours / week ≥ 18 N = 177
Median age at diagnosis (years)	68	67.5
Body mass index (kg/m2)	25.9	24.9
Gender (%)
Male	41	64
Female	59	36
Stage at diagnosis (%)
I	31	24
II	38	47
III	26	25
Unknown	5	4
Year of diagnosis (%)
Before 1990	13	9
1990-1995	31	32
After 1995	56	59
Median time from diagnosis to physical activity assessment (months)	16	18

Impact of physical activity on outcomes by molecular markers

We have previously reported that higher levels of physical activity after colon cancer diagnosis was associated with better colon cancer-specific and overall mortality (14, 15). We performed subgroup analyses by individual molecular markers comparing less than 18 MET-hours / week of exercise to at least 18 MET-hours / week on colon cancer-specific mortality (Table 2). A protective association for increased physical activity was detected regardless of FASN, K-ras, p53 or p21 status; no significant interactions were detected for these markers. In contrast, the effect of physical activity on patient outcome appeared to differ significantly according to p27 status (p for interaction = 0.03). For patients with loss of p27, regular physical activity conferred no benefit whereas among patients with tumoral expression of p27 intact, physical activity was associated with a significant reduction in colon cancer-specific mortality (HR 0.32 [95% CI 0.12 – 0.85]). The benefit associated with physical activity appeared to be absent among patients with PI3KCA mutations, although a test for statistical interaction was not significant.

Table 2.

Subgroup analyses by molecular markers for colon cancer-specific mortality comparing high to low levels of physical activity

	Events/ N	MET-hours / week ≥ 18 vs MET-hours / week < 18 Hazard ratio † (95% CI)	P interaction
All patients with tumor blocks *	50/484	0.64 (0.33-1.23)
FASN			0.77
Negative	41/390	0.61 (0.30-1.25)
Positive	4/66	0.95 (0.11-8.06)
K-ras			0.59
Wildtype	25/284	0.71 (0.28-1.82)
Mutation	22/169	0.42 (0.15-1.18)
p53			0.58
Negative	20/276	0.46 (0.16-1.35)
Positive	26/192	0.64 (0.26-1.59)
p21			0.19
Lost	37/360	0.87 (0.42-1.81)
Expressed	8/90	0.10 (0.01-0.98)
p27			0.03
Lost	17/195	1.40 (0.41-4.72)
Expressed	28/251	0.32 (0.12 – 0.85)
PI3KCA			0.96
Wildtype	33/340	0.59 (0.26-1.33)
Mutation	7/69	1.25 (0.25-6.40)

Abbreviations: MET = metabolic equivalent hours; N = total number of patients; CI = confidence interval

^*Block cohort represents subjects who had tumor blocks available, post diagnosis physical activity data and stages I-III at diagnosis. The number of patients varies for each marker since some samples were indeterminant for certain markers.

^†Adjusted for age, gender, stage, year of diagnosis, histology grade, body mass index, time of physical activity assessment

Increased levels of physical activity were associated with a statistically significant 40% improvement in overall mortality in this cohort of stage I — III colon cancer patients with tumor blocks and physical activity assessment at least 6 months post diagnosis (Table 3). These results were largely unchanged by status of FASN, K-ras, p53, p21, p27 and PIK3CA in the primary tumors.

Table 3.

Subgroup analyses by molecular markers for overall mortality comparing high to low levels of physical activity

	Events/ N	MET-hours / week ≥ 18 vs MET-hours / week < 18 Hazard ratio † (95% CI)	P interaction
All patients with tumor blocks *	152/473	0.60 (0.41-0.86)
FASN			0.62
Negative	128/390	0.65 (0.44-0.96)
Positive	18/66	0.28 (0.09-0.83)
K-ras			0.91
Wildtype	83/284	0.58 (0.34-0.98)
Mutation	62/169	0.56 (0.31-1.00)
p53			0.44
Negative	85/276	0.73 (0.45-1.19)
Positive	65/192	0.44 (0.24-0.80)
p21			0.76
Lost	112/360	0.65 (0.43-1.00)
Expressed	31/90	0.57 (0.24-1.36)
p27			0.37
Lost	57/195	0.67 (0.36-1.24)
Expressed	82/251	0.56 (0.34-0.93)
PI3KCA			0.71
Wildtype	106/340	0.55 (0.35-0.86)
Mutation	23/69	0.80 (0.31-2.03)

Abbreviations: MET = metabolic equivalent hours; N = total number of patients; CI = confidence interval

^*Block cohort represents subjects who had tumor blocks available, post diagnosis physical activity data

^†Adjusted for age, gender, stage, year of diagnosis, histology grade, body mass index, time of physical activity assessment

We tested whether any of the markers confounded the previously reported associations between physical activity and either colon cancer-specific mortality or overall mortality. Adding the status of these 6 markers, either individually or all in the same model, did not impact on the multivariate hazard models (data not shown).

DISCUSSION

Prospective observational data suggest that physically active colon cancer survivors have lower rates of cancer recurrence and improved survival compared to inactive survivors (14, 15). However, as with any oncological intervention, it is likely that not all patients derive a benefit from exercise. We tested molecular pathways that have been associated with energy balance to determine if a population of colon cancer survivors particularly benefit from physical activity. Surveying a variety of molecular events, we found that the benefit associated with physical activity differed significantly according p27 expression. Patients with loss of p27 did not appear to benefit from physical activity but those with expression of p27 and were physically active (at least 18 MET-hours / week) had a 68% improvement in colon cancer-specific mortality compared to those with p27 expression but not physically active.

Personalized medicine is a growing goal in the treatment of cancer patients (42). It is clear that individual pharmacological interventions will not impact all patients with the same cancer type. As such, there is growing interest to find markers that better differentiate patients that are likely to benefit from a treatment from patients that have little to no chance of deriving benefit. Similarly, as evidence grows that non-drug therapies can influence patients with established cancer, there is a need to better delineate subpopulations of cancers that may or may not be more likely to be impacted by an intervention. Given the consistent evidence suggesting that physical activity reduces colon cancer recurrences in early stage patients (14, 15, 43), we hypothesized that certain characteristics of a patient’s tumor may interact with the biological effects of exercise. With the exception of p27, no such interaction was detected for colon cancer-specific or overall mortality. Further, while the p for interaction between p27 and physical activity was statistically significant for colon-cancer specific mortality (p = 0.03), there was no significant interaction for overall mortality (p = 0.37).

In preclinical models, higher levels of p27 expression were detected in chemically-induced malignancies in animals that were energy restricted compared to those not restricted (26-28). Such an effect will arrest cell cycle progression. Thus, the interaction detected in our data is consistent with a hypothesis that energy restriction by physical activity could influence p27 expressing tumors by cell cycle arrest inhibiting growth. Excess energy balance may have a much stronger impact on tumor behavior if tumor cells can upregulate p27 to arrest cell cycle, than if tumor cells have lost the ability to upregulate p27, possibly through the constitutive activation of the AKT1 pathway. However, it is not clear why no such benefit was detected for overall mortality. One explanation is that those with p27 expressing tumors clearly derive a benefit from exercise related to their colon cancer but that exercise still is beneficial to all patients irregardless of p27 status and equally protective for overall mortality related to non-cancer related causes (eg. cardiovascular disease). Another possibility is that the finding for colon cancer-specific mortality is by chance alone, a risk of multiple hypothesis testing.

The use of the Nurse’s Health Study and Health Professional Follow-up Study cohorts provides multiple advantages to study molecular-environment interactions. Diet and lifestyle are prospectively collected and entered into a database blind to a patient’s diagnosis. Data are updated every 2 years. Tumor block ascertainment has been fairly high (∼60 %). Subjects are treated at hospitals throughout the United States and represent diverse treatment approaches that could be considered generalizable on a population level. However, a limitation of this study is that cancer treatment data are not available for most patients in our cohorts. Nonetheless, it is unlikely that chemotherapy use differed according to molecular characteristics of the tumor beyond typical pathological features like stage of disease and grade of differentiation (which are adjusted for in multivariate models). In addition, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, given the median survival for metastatic colon cancer was approximately 10 to 12 months during much of the time period of this study (44), colon cancer-specific survival should be a reasonable surrogate for cancer-specific outcomes. Finally, these data are limited to patients that were alive to have their physical activity assessed after diagnosis (median 17 months). As such, conclusions are limited to that population.

In conclusion, this large prospective study of colon cancer patients confirms an association between physical activity and lower colon cancer-specific and overall mortality in colon cancer survivors. However, a molecular signature influencing this association was not clearly detected. While p27 status may be relevant, these findings require confirmation in independent populations of colon cancer patients.