Clinical features and KRAS mutation in colorectal cancer with bone metastasis

Bone metastasis is known as a poor prognostic factor in colorectal cancer (CRC), but its clinical manifestations and outcomes are uncertain. CRC with bone metastasis was searched from January 2006 to April 2016. Of 11,551 CRC patients, 321 (2.7%) patients had bone metastasis. Bone-only metastasis was found in only 8.7% of patients. Synchronous bone metastasis was present in 147 (45.8%) patients. In patients with metachronous bone metastasis, the median time from CRC diagnosis to bone metastasis (TTB) was 27.2 months. KRAS mutation status was a marginally significant factor affecting TTB (median TTB, KRAS wild-type or mutation: 29 or 25.8 months, respectively, P = 0.068). Skeletal-related events (SREs) were noted in 200 (62.3%) patients. Median overall survival (OS) from diagnosis of bone metastasis was 8.0 months. On multivariate analysis, multi-organ metastasis, peritoneal metastasis, neutrophil-to-lymphocyte ratio (NLR) ≥ 2.7, and alkaline phosphatase (ALP) ≥ 123 were independent factors for OS. Palliative chemotherapy prolonged survival in CRC patients with bone metastasis (HR 0.25, 95% CI 0.2–0.33). In conclusion, bone metastasis of CRC is rare, but it is related to SREs. Most patients have other organ metastasis and survival is 8.0 months. Attention should be paid to bone metastasis in CRC patients.


Prognostic model analysis.
We divided patients into two subgroups based on four independent prognostic factors for OS: 171 patients with 0 to 2 adverse prognostic factors (low-risk group) and 122 patients with three to four adverse prognostic factors (high-risk group). These risk groups were significantly associated with OS (median OS for low-and high-risk groups: 11.9 months and 4.1 months, respectively, P < 0.001) (Fig. 3A). Regardless of the risk group, palliative chemotherapy had a benefit for OS in patients with bone metastasis. Among 171 low-risk patients, 123 (71.9%) patients received palliative chemotherapy. As shown in Fig. 3B, patients treated with palliative chemotherapy had longer OS than patients who received the best supportive care, including radiotherapy (median OS: 16.7 months vs. 4.3 months respectively, P < 0.001). Among 122 highrisk patients, OS was also longer in the palliative chemotherapy group (median OS: 6.8 months vs. 2.3 months, P < 0.001) (Fig. 3C).

Discussion
Bone metastasis of CRC is rare, but it is related to SREs. Most patients have other organ metastasis and survival is 8.0 months in unselected patients. KRAS mutation affected different clinical manifestations in these patients. We showed that patients with KRAS mutation had a tendency to have shorter time to bone metastasis than patients with wild-type KRAS, but there were no significant survival differences between patients with KRAS wild-type and those with KRAS mutation. Several prognostic factors were noted in this study. Patients who received palliative chemotherapy showed prolonged survival compared to patients with supportive care, especially in patients with less adverse factors.
The observed frequency of bone metastasis in CRC was 1.2-12%, with a wide range 3,4 . Each study included CRC with bone metastasis in various clinical settings dealing with resected patients or metastatic patients. It might be related to the wide range of bone metastasis incidence in CRC patients. Recently, systemic chemotherapy was more effective and survival was prolonged. In addition, diagnostic modality such as positron emission tomography (PET) coupled with computed tomography (CT) scan (PET-CT) was developed and possibility of bone metastasis detection would be increased. Asymptomatic bone metastasis could be detected on routine follow-up. It might help prevent SREs, but it has a possibility of overtreatment, such as chemotherapy regimen change or radiotherapy. Therefore, the role of asymptomatic bone metastasis detection should be investigated in In clinical practice, at the time of bone metastasis, multi-organ metastases were frequently observed. Boneonly metastasis was found in only 8.7% of subjects, and 91.3% of patients had other organ metastasis in this study. Commonly observed multi-organ metastases might be related to the poor prognosis in bone metastasis 5 . KRAS mutation has been studied as a metastasis promoting factor in CRC. The results of our study also showed that patients with KRAS mutation had a higher frequency of both lung and liver metastases at the time of bone metastasis. In addition, patients with KRAS mutation more frequently had high ALP and high CEA levels. Regarding KRAS mutation type (codon 12 vs. codon 13), there were no significant clinical differences between these two groups, except the NLR level.
The median time to bone metastasis was 27.2 months in metachronous patients. Time to bone metastasis was shorter in patients with KRAS mutation compared to patients with wild type KRAS, although it was not statistically significant. It has been reported that KRAS mutation can lead to shorter time to metastasis in liver, lung, and bone 17 . Initial CRC stage was also a significant factor for time to bone metastasis. Based on our data, first surveillance for identification of bone metastasis might be considered within 2 years after primary CRC diagnosis and attention is needed, especially in patients with initial stage 4. Additional evaluation and close monitoring 2 years after CRC diagnosis should also be performed.
Risk factors for bone metastasis in CRC have been reported in many studies 8 . Primary tumor location, tumor stage and histologic type were revealed as significant risk factors for bone metastasis. Regarding tumor location,  Multiple bone metastases, primary site of colon/lung metastasis, and elevated CEA level have been identified as poor prognostic factors. In our study, four prognostic factors were revealed: multi-organ metastasis, peritoneal metastasis, high NLR (≥ 2.7), and high ALP (≥ 123 IU/L). Of these factors, whether a patient had bone-only metastasis was a prognostic factor with the highest hazard ratio for OS (HR 2.43; 95% CI 1.39-4.25). The median survival was 7.8 months for patients with other organ metastasis and 20.4 months for those with bone-only metastasis. We also made a prognosis scoring model using the four independent factors. Risk group was categorized as low-risk (score 0-2) and high-risk (score 3-4) groups. The median survival of the low-risk group was 11.9 months. It was almost 1.5-fold (4 months longer) than that of unselected CRC patients with bone metastasis.
Bone metastasis is an important factor for quality of life and survival. Proper treatment, including radiotherapy, surgery and medical therapy, improves bone metastasis symptoms, such as pain, and bisphosphonates could delay the development of SREs 8 . In addition, bone metastasis has been known to be a poor prognostic factor in metastatic CRC. In our study, palliative chemotherapy provided patients a chance to increase survival up to 16.7 months in the low-risk group. The high-risk group also had a survival gain from palliative chemotherapy, although the survival benefit was less than the low-risk group. We had to pay attention to the interpretation of this survival data analyzed by the presence of chemotherapy, because patients who did not receive chemotherapy might have poor performance status or comorbidities, which might be related to poor prognosis. Our results suggest the possibility of chemotherapy benefit in bone metastasis patients, and predictive markers for chemotherapy should be further studied. A prognosis scoring model might help predict the survival for CRC patients with bone metastasis and guide treatment decisions, including palliative chemotherapy, or the best supportive care according to their benefits.
This study had several limitations. First, it was a retrospective study. Thus, the performance status could not be identified. Second, this study did not evaluate the impact of bisphosphonates on SREs. Bisphosphonates are not reimbursed in Korea. They are only permitted in hypercalcemia secondary to CRC. However, this study also has its strengths. Large numbers of patients were enrolled, and KRAS mutation status and tumor sidedness were analyzed to identify their roles in bone metastasis of CRC. In conclusion, bone metastasis of CRC is rare, but it is related to SREs. Most patients have other organ metastasis and survival is 8.0 months in unselected patients. It should receive more attention from clinicians. Time to bone metastasis and prognosis were not different according to KRAS mutation status in our study. Surveillance for bone metastasis might be considered in the first 2 years after CRC diagnosis, and additional evaluation and close monitoring 2 years after the CRC diagnosis should be performed. According to our prognosis scoring model, a patient's prognosis could be predicted, and it may help to guide palliative chemotherapy in CRC with bone metastasis.

Methods
Patient selection. From January 2006 to April 2016, a total of 11,551 patients were diagnosed with CRC at Severance Hospital, Seoul, Korea. Diagnosis was made from surgical excision or tissue biopsy. Inclusion criteria were as follows: (1) age > 18 years, (2) histologically confirmed diagnosis of CRC, (3) radiologically or pathologically confirmed bone metastasis, and (4) available electronic medical records (including treatment information). Patients with other histology types, such as neuroendocrine tumors, small cell carcinoma, melanoma, or lymphoma, were excluded. A total of 321 patients fulfilled the inclusion and exclusion criteria. The Severance Hospital Institutional Review Board (IRB) approved this retrospective study, and the requirement to obtain

Data collection.
To diagnose bone metastasis, plain radiography, technetium-99m methylene diphosphonate (Tc-99m MDP) whole body bone scintigraphy, CT scan, PET-CT and magnetic resonance imaging (MRI) were performed. All available correlative radiographic studies were reviewed. The following baseline data were recorded at the time of bone metastasis: age, sex, tumor location, histologic type, initial stage, metastatic organ, and KRAS mutation status. For tumor sidedness determination, the splenic flexure was used for differentiation of the left side of the colon and right side of the colon. KRAS mutation status was identified by Sanger sequencing, the peptide nucleic acid (PNA)-mediated PCR clamping method 19 , or pyrosequencing. Sanger sequencing and the PNA-mediated PCR clamping method were used to detect mutations in codons 12 and 13. Pyrosequencing was performed to detect mutations at codons 12, 13, and 61. Metachronous bone metastasis was defined as the time to bone metastasis being greater than 3 months from initial CRC diagnosis.
Hematological and blood chemistry values included NLR, platelet count, ALP, and CEA. The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. Parameters related to bone metastasis included time to bone metastasis, site of bone metastasis, and SREs defined by pathologic fracture, spinal cord compression, surgery to bone, radiotherapy to bone, and hypercalcemia. After the diagnosis of bone metastasis, whether patients received palliative chemotherapy was also reviewed.
Statistical analysis. The demographic and clinical characteristics were compared using the Chi square or Fisher exact test. The Mann-Whitney U test was used for comparisons of continuous variables. In patients with metachronous bone metastasis, freedom from bone metastasis since CRC diagnosis was estimated using the Kaplan-Meier method. Overall survival (OS) was defined as the time from the date of bone metastasis diagnosis to death from any cause. Hematological and blood chemistry values were initially recorded as continuous variables and later transformed into categorical variables according to lower or upper normal values (platelet count, ALP level, and CEA level) or the best cut-off point (NLR) determined by the Contal and O'Quigley method, which calculates the maximization of the hazard ratio based on log rank statistics and estimates the best cut-off value 20 . Survival curves were generated using the Kaplan-Meier method and compared using the log-rank test.
Univariate analysis was performed to determine associations of OS with the following prognostic factors: age; sex; tumor location and sidedness; timing of bone metastasis (synchronous or metachronous); histologic type; bone metastasis pattern (bone-only or not); liver, lung, peritoneal, and/or brain metastasis; NLR; ALP level; CEA level; KRAS mutation status; and palliative chemotherapy treatment. A stepwise multivariate analysis with Cox proportional hazard model was performed using significant factors from the univariate analysis. Hazard ratio (HR), 95% CI, and χ 2 scores were obtained for all regressions. Statistical analyses were performed using PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA), SAS version 9.2 (SAS institute, Cary, NC, USA), and R version 3.1.3 (Institute for Statistics and Mathematics, Vienna, Austria www.R-proje ct.org).