The bone are phenomenon during the treatment of bone metastases of lung adenocarcinoma

Background: The bone flare phenomenon, defined as an increase in bone lesion activity, is a kind of benign bone change in response to ongoing tumor treatment. The purpose of this study was to investigate the time, incidence, and clinical significance of bone flare in lung adenocarcinoma patients with bone metastases, as well as to observe the levels of serum alkaline phosphatase (AAAAAA) and serum calcium (CCCC2+) in patients with bone flare phenomenon. Methods: Fifty-seven patients with advanced lung adenocarcinoma who participated in the anti-tumor clinical trials from December 2017 to December 2019 in Linyi Cancer Hospital were included in the study. The CT (computed tomography, CT) images, serum AAAAAA and CCCC2+ from all patients were analyzed retrospectively. Results: Among a total of 57 patients, 28 were male, and 29 were female. The median age was 62 years (33-75 years), and 30 of them had bone metastases at baseline. Forty-six EGFR negative patients received platinum-based dual-drug chemotherapy or bevacizumab or combined with PD-1 antibody inhibitors, and 11 EGFR positive patients received targeted EGFR inhibitors. The bone flare was detected in 7 out of 30 patients with bone metastases at baseline (5 patients in the combined chemotherapy group and two patients in the targeted treatment group). The incidence of bone flare was 23.33% (7 / 30). The median time was 45 days after treatment (from 41 days to 120 days), and most of them occurred in the early stages of the treatment. Of the 30 patients with bone metastases at the beginning of treatment, twenty had AAAAAA and CCCC2+levels observed over five months, seven of them with bone flare, 13 with benefit (partial response,PR/stable disease,SD), and 5 with progression (progressive disease, PD). Analysis of AAAAAA from patients with different curative effects showed significant changes compared with the median percentage of baseline levels. The bone flare group and the beneficial group showed a decreasing trend. The bone flare group showed a more significant decreasing trend, AAAAAA levels in the progressive group increased continuously. The percentage change of serum CCCC2+during the treatment was not noticeable. Conclusion: The bone flare phenomenon was observed among patients with lung adenocarcinoma with bone metastasis. Most of them occur in the early stage of treatment. Combined with clinical evidence such as AAAAAA, physicians and radiologists should avoid a misinterpretation of bone flare as the progression of metastatic bone cancer and stopping the effective treatment too early.

Lung cancer is one of the most common malignant tumors in the world [1] . According to the histological classification, lung cancer was classified as small-cell carcinoma (SCLC, about 15% of lung cancers) and non-small-cell lung carcinoma (NSCLC, about 85% of lung cancers) [2] . Among them, lung adenocarcinoma was the most common subtype in NSCLC, accounting for about 40% of all lung cancers [3] , and the incidence is increasing [4] .
Some studies have shown that the incidence of bone metastasis in NSCLC patients is about 30-40% [5,6] , of whom 60% of the patients had bone metastasis at the time of diagnosis [7] , and most of them were osteolytic, and a few were osteogenic or mixed [8] .
With the development of cancer science, chemotherapy, targeted therapy, and immunotherapy have greatly improved the efficacy and survival time of advanced lung adenocarcinoma. However, in clinical practice, we have found that some patients in the early stage of treatment, primary lesions or other metastatic lesions all suggest a reduction or stabilization, but the density or number of metastatic bone lesions increased. Some patients have changed the treatment regimen according to their disease progression, but the lesions in other sites increased rapidly; some patients continued to be treated according to the original regimen to keep the tumor under control.
Upon inquiry, it was found that this treatment for Central Plain Foci or other metastatic foci was well controlled and resulted in a "progression of bone metastases," known as "bone flare" [9] . Bone flare is a manifestation of systemic therapy for malignant tumors, not a progression of metastatic bone tumors. As treatment continues, bone lesions remain stable or improve. Also, serum ALP and 2+ as markers of bone metabolism are closely related to bone metastasis of lung cancer [10,11] . Based on this, we retrospectively analyzed 57 patients with newly diagnosed advanced lung adenocarcinoma from 5 clinical trials. In the 7 cases, BMD (bone material density , BMD) or BMD number increased while other lesions were relieved or stable during anti-tumor therapy. We compared the changes in bone window by CT imaging, serum ALP and 2+ levels before and after treatment to observe the bone lesions in lung adenocarcinoma patients with bone metastasis during anti-tumor therapy, to distinguish the progression of bone metastasis or bone flare, and to look for the markers related to bone metabolism.

Subjects
The patients included in the study were treated at the Linyi Cancer Hospital, Linyi.

Imaging examination:
All CT scans were performed with an enhanced 64-slice spiral scanner to define the extent of bone lesions.Scanning parameters: Tube Voltage: 120 ; tube current:150 ; layer thickness: 5 ; layer distance:5 ; the contrast medium was iohexol; the dosage was from 1.5 to 2.0 / . The injection rate was from 2.5 to 3.5 /ℎ through the median elbow vein with a high-pressure syringe.
Follow-up was performed every six weeks, and enhanced CT was used to evaluate the therapeutic effect. The efficacy was assessed based on RECIST1.1 criteria.

Laboratory examination:
Serum ALP and Ca 2+ levels were measured at baseline and before treatment in the next cycle. Three milliliters of fasting venous blood was collected and detected by Beckman Coulter AU5800 automatic biochemical analyzer. The reference value of ALP was from 45 to 125 / ; the reference value of 2+ level ranged from 2.03 to 2.60 / .

Statistical analysis
All experiments are presented as the mean ±S. Student's t-test was used for statistical evaluations of two group comparisons. One-way analysis of variance (ANOVA) for more than two groups was performed for statistical analysis.

2.Results
Thirty patients of the 57 subjects had bone metastases at baseline, 7 had bone flare after treatment (5 cases in the combined group and 2 cases in the targeted group), the incidence rate was 23.33% (Table 1) Erlotinib [15] . Heng-sheng Chao et al. reported that bone flare occurred in 7 of 33 NSCLC patients with bone metastatic treated with gefitinib, with a median time of 34 days [9] . Yelena Krupitskaya, et al. also reported four patients with NSCLC who experienced a solitary deterioration of bone metastases during bevacizumab combined with standard chemotherapy. Subsequent scans confirmed that the deterioration was transient and consistent with flare reaction [16] . In summary, bone flare is not an adverse reaction, but a phenomenon of osteoblast activation during bone repair [17][18][19] .
It is characterized by rapid bone tissue repair around bone lesions and is an effective marker for systemic treatment of malignant tumors, which is not a tumor bone metastases progression [20] . Research data show that cancer cells migrate to the bone and release soluble mediators, activate osteoclasts and osteoblasts, and the cytokines released by osteoclasts further promote cancer cells to secrete osteolytic mediators, forming a vicious circle, and then there's osteolytic change [21] . Other studies have reported that many factors can promote the growth and differentiation of osteoblasts [16] .
We hypothesized that the bone flare phenomenon might be caused by the reduction of tumor load in bone metastases, the reduction of osteolytic mediators released by cancer cells, and the reduction of osteoclast activity. On the other hand, when anti-tumor therapy is effective, the changes of tumor-derived factors caused by changes in the tumor microenvironment can promote the proliferation and differentiation of osteoblasts and lead to increased uptake of bone imaging, thus leading to rapid bone repair and healing, and the bone flares formed. If the bone lesions continue to respond to treatment, the increase in uptake will eventually decrease [22] . Previous studies have found that bone flares typically occur in the early months of treatment [23] , usually from 2 weeks to 3 months after treatment, but rarely 6 months after treatment, and it was included as part of the MD Anderson Cancer CT Bone window examination can provide anatomical information, and can clearly show the shape, contour, density, and other abnormalities of bone lesions, high resolution, sensitivity can reach 77% [24] . As mentioned in the MDA standard, the definition of PR should pay attention to the phenomenon of osteoblast bone flare [25] , in the case of other lesions PR, osteosclerosis or osteolytic lesions during treatment of marginal sclerosis, it does not represent tumor progression but healing of bone lesions that were not evident prior to treatment [26] . The 7 patients with bone flare in our study were all patients with bone metastasis at baseline. After treatment, Osteogenic sclerosis reaction occurred in the osteolytic destruction lesions.
Bone metabolic biomarkers can reflect the rate of bone resorption and the speed of bone formation during bone metastasis and indicate the degree of bone destruction and repair [27] . As a marker of bone metabolism closely related to bone metastasis of lung cancer, serum alkaline phosphatase and serum calcium ( 2+ ) are often used to assist the diagnosis and follow-up of bone metastasis. ALP is an enzyme expressed on the surface of osteoblasts, which can affect the activity of osteoblasts. In osteolytic bone metastasis, the activity of ALP increased after local bone formation reaction in order to compensate for the major osteoclast lesions, while in osteogenic bone metastasis, the activity of ALP increased after local stimulation by osteoblasts [29] .
Huang et al. (meta-analysis) found that ALP level was higher in patients with bone metastasis of lung cancer than in patients without bone metastasis, suggesting that serum ALP level is an indicator for bone metastasis in lung cancer [3] . Karhade Elevated serum 2+ levels has been found in a wide range of soluble osseous bone metastases. On one hand, tumor cells produce protein decomposing enzyme can cause bone matrix dissolution, destroy [21] ; on the other hand, the cancer bone metastases can produce a variety of promote the activity of bone absorption and bone calcium release factor [16] , eventually dissolved bone calcium release exceeds the ability of the kidney and intestine removing calcium, which may lead to increased calcium in the blood.
Bone damage caused by lung cancer metastasis is usually osteolytic, leading to an increase in serum 2+ when bone metastasis occurs [27] . However, in this study, during the whole treatment period, 25 patients with bone metastasis at baseline and complete serum 2+ data were not observed with significant changes in the percentage of 2+ compared with the median baseline in different curative effects, and the number of cases should be expanded for further observation.

Declarations:
Ethics approval and consent to participate: This study was approved by the Ethics Committee of the Linyi Cancer Hospital and all the patients in the study signed informed consent forms.

Consent for publication:
All authors agreed to publish the draft and transfer the copyright to the publisher.

Availability of data and material: Not applicable
Competing interests: The authors declare no competing financial interests.   (2) SD (2) PD (1) CR (1) EGFR-TK1 11 PR (4) 1 SD (5)     The changes of bone destruction of Thoracic Vertebra 5 in #1 patient with typical bone are after treatment. (A). No obvious bone abnormalities were found in the T5 vertebrae before treatment. (B). After 2-cycle treatment, the bone destruction of T5 vertebral body was found and bone metastasis was developped. (C). After 4-cycle treatment, bone destruction of the T5 vertebral body was similar to that of before treatment. (D). After 6-cycle treatment, bone destruction of the T5 vertebral body was similar to that of before treatment.  Changes of bone metastases lesions in #2 patient with typical bone are before and after the treatment. (A). At baseline PELVIC CT images showed bony destruction and soft tissue formation in the lumbar spine, which was dignosed bone metastases. (B). After 6 weeks of targeted therapy, PELVIC CT showed bone destruction and soft tissue formation in lumbar vertebrae, and bone metastasis, which was larger than before.(C). After 12-week targeted therapy, PELVIC CT showed bone destruction and soft tissue formation of lumbar vertebrae, which was larger than before. (D). After 18-week targeted therapy, PELVIC CT showed bone destruction and soft tissue formation of lumbar vertebrae, which was larger than before Serial changes in serum ALP changes before and after treatment in 7 patients with bone are The triangular symbols in this gure show the time and ALP levels of bone are found in 7 patients during treatment.