Although platinum-based chemotherapy has been recognized as the standard treatment for advanced NSCLC, the PFS only reaches 4–6 months. Antiangiogenic agents are believed to delay the recovery of residual tumor from chemotherapeutic impairment by suppressing blood supply to the tumor. Endostatin was capable of inhibiting endothelial cell proliferation and migration leading to inhibition of tumor growth and metastasis. Rh-endostatin, a novel recombinant humanized endostatin, improved stability of the protein. In clinical trials in China, the combination of rh-endostatin and chemotherapy was shown to have significantly improved OS and PFS of advanced NSCLC.[8, 9] However, doubts remained in those trials due to few therapeutic cycles (less than three cycles, without maintenance). Therefore, we designed a trial of longer therapy in order to confirm or modify the previous conclusion. Despite the unexpected end of the trial, in the present study the obtained CBR in the single chemotherapy arm and combined arms achieved 58.3% and 73.7% with PFS of 136 and 196 days, respectively, with insignificant differences (P = 0.220 and 0.059, respectively). The results other than that from a phase III trial of NP with or without rh-endostatin (PFS was 6.3 and 3.6 months in NP plus rh-endostatin and single NP, respectively, with P-value 0.00) might be ascribed to the limited number of subjects or more therapeutic cycles in our trial. However, the PFS for cases with a clinical benefit achieved 239.57 days (8 months) and 165.20 days (5.5 months) in the combined arm and single NP arm (P = 0.034), respectively, whereas the PFS for cases with PD was 52.00 and 48.00 days in the combined arm and single NP arm, respectively. This implies that only some “optimal individuals” could benefit from continuous antiangiogenesis when induced treatment was effective. It is necessary to distinguish the optimal group from the total patient population by way of predicting their response to therapy as early as possible to avoid insufficient or excessive antiangiogenic therapy. Because antiangiogenic drugs mainly suppress growth of tumor blood vessels rather than tumor tissue per se, tumor volume shrinkage always occurs after repression of tumor blood supply. Since both WHO and the RECIST criteria estimate efficacy for solid tumors based on volume change that is usual later events during treatment, much earlier predicting approaches are urgently needed to provide a more timely assessment of the treatment efficacy.
In the present study, we investigated early predictors of response and PFS, consideration of age, sex, histology, stage, prior therapy, ECOG, number of therapeutic cycles, aCEC and BV were incorporated into the multivariate Cox's proportional hazards model. However, significance was only found on the latter three ones. Therefore, they were reincorporated into the multivariate Cox's proportional hazards models and significance was only found on aCEC and BV. In addition, although it was not included in model 1 (P = 0.07), the number of therapeutic cycles is of significant importance and further study is needed to determine whether an increased number of therapeutic cycles can prolong PFS.
aCEC for predicting the efficacy of antiangiogenic therapy
Although antiangiogenic efficacy was proven in the E4599 trials, the endostatin and the thalidomide trial, it remains to be determined whether efficacy can be assessed based on changes of TAF, such as serum VEGF and bFGF levels.[25, 27, 29] This might be due to the unstable balance resulting from confrontation of the TAF and antiangiogenic factors, suggesting more downstream markers other than TAF should be searched.
The CEC, as discovered by Bouvier in the 1970s, are circulating endothelial cells that directly reflect injury to the vascular endothelium in vivo through its alteration in the blood levels. The CEC include mainly EPC that originate from the bone marrow, mature endothelial cells shed from blood vessel walls and cancer-derived cells with endothelial function. These cells need to be activated by TAF so that they can home to tumor sites, adhere and form new tumoral vasculature. Many markers have been used to identify CEC because of the diversity of their origin and complexity of their differentiation. The currently recognized markers are CD45−CD146+Flk1+.[33, 34] In addition, Beaudry introduced CD117 to identify EPC and Mancuso[19, 21, 23, 34] chose CD105 to distinguish activated functional CEC (aCEC), which are considered more relevant to angiogenesis than the total CEC. A previous study showed that EPC level is usually undetectable because of its scarcity in the circulation, but the correlation was observed between changes in CD105+ aCEC and efficacy. Given that activated mature endothelial cells (CD45−CD146+CD105+) are viable and continue to exhibit proliferative capacity despite their terminal differentiation, CD45−CD146+CD105+ was used to identify aCEC in the present study in accordance with the literature.[19-21]
Even though both CEC and EPC provide the potentiality to reveal the angiogenetic status and predict antiangiogenic therapeutic efficacy, controversy remains due to the variability of CEC and EPC levels in different studies. The aCEC decrease after a complete response to chemotherapy or mastectomy was proven by Mancuso.[16, 21] However, elevation of both CEC and EPC several days after antiangiogenic therapy and/or chemotherapy was reported, especially after taxane drug treatment in some studies. This is because of mobilization of EPC from bone marrow by platelet-released stromal cell-derived factor induced by taxane. Nevertheless, a decrease in both CEC and EPC was reported with longer PFS or OS in the majority of reports after effective antiangiogenic therapy and/or chemotherapy, such as bevacizumab with non-taxane and metronomic chemotherapy.[38, 39] Furthermore, after taxane treatment, a higher elevation in apoptotic/dead CEC compared with EPC was reported. These results imply a complex equilibrium between mobilization of EPC and apoptosis of CEC induced by different regimens and drugs.
Another probable explanation for a short increase in CEC after treatment might be “normalization” of tumoral vasculature. Normalization of tumor vasculature is a common effect of antiangiogenic agents, including a series of antiangiogenic events in which suppression of MMP by antiangiogenic agents induces decreased degradation and low permeability of the micrangium basal membrane, reduction of fluid leakage and interstitial fluid pressure. Thus, the dilated and tortuous vessels in the tumor are constricted and stretched, allowing chemotherapeutic drugs in the blood to enter tumors more easily. Because endothelial cells in tumoral vasculature are more vulnerable to damage by antiangiogenic agents and are easy to shed, both endothelial retraction/apoptosis and tumor vasculature shrinkage might simultaneously induce endothelial cells to shed from the blood vessel walls, allowing for easier penetration by chemotherapeutic agents.
In the present study, to eliminate possible disturbance from transient elevation of aCECs caused by chemotherapeutic or antiangiogenic drugs,[38, 42] the blood samples were obtained after completion of the previous therapeutic cycle and prior to the beginning of next therapeutic cycle until completion of therapy (other than three blood samples drawn in another trial) in order to obtain the ‘dynamic tendency’ and confirmed terminal data of aCECs during therapy. The results showed aCEC increased on tumor progression, especially in the single NP arm, but did not increase or slightly increased in SD and PR, which showed a tumor response. Apart from a possible deviation caused by the limited number of cases, this is probably due to the fact that effective antiangiogenic therapy also increases aCEC through “vasculature normalization” separately from decreasing aCEC by suppression of EPC mobilization and induction of aCEC apoptosis, which renders aCEC to be kept stable or slightly increased in the SD and PR patients. However, the “window” of vasculature normalization is only 1–2 weeks after antiangiogenic therapy, after which it turns into vascular insufficiency induced by suppression of tumor angiogenesis. These two effects of antiangiogenesis could alternate during therapy leading to CEC fluctuation and a gradual decline along with tumor shrinkage and decreasing circulating TAF after effective prolonged therapy, resulting in the inverse correlation between ∆aCEC and either PFS or BV in the present study, which also suggests a need for multiple inspections of CEC during therapy. These results are consistent with a previous report by Fürstenberger et al. showing that effective treatment induced robust apoptosis of CEC and that chemotherapy based on platinum and non-taxane drugs did not mobilize EPC from bone marrow.[14, 35] Furthermore, ∆aCEC was confirmed as a significant indicator of PFS in the combined antiangiogenesis regimen using Cox regression in model 1.
Value of CT perfusion imaging in predicting efficacy
Bellomi et al. proposed the concept of CT perfusion imaging whereby rapid dynamic scans are conducted at a single location after a bolus of contrast medium injection. The imaging data are calculated using an enhanced level of each pixel in each layer and are displayed in grayscale to form the quantitative or semi-quantitative perfusion images of the organ.
Computed tomography perfusion imaging has been used for early diagnosis of brain infarction and cardiovascular and renal diseases, using BF, BV, MTT and PS as the main indexes.[19, 44] The perfusion indexes have been proven to be higher in cancerous lung tissues than normal peripheral tissue and increasing BV indicates angiogenesis. Better efficacy has been reported in cases with higher baseline BV. Changes in the perfusion indexes, such as BV, indicate changes in tumor vasculature, suggesting that they could be used to monitor antiangiogenic therapy.
In the results of present study, decreased BV could indicate clinical benefits, particularly in the combined arm. Chemotherapy mainly causes shrinkage of tumor in addition to vasculature impairment. In contrast, effective antiangiogenic therapy leads to vasculature normalization by constricting dilated and tortuous tumor vessels to reduce the vasculature area. Considering the decreased blood volume from vascular normalization and the subsequent inadequacy resulting in vascular bed reduction, a significant decrease in BV was observed only in the combined arm but not in the single NP arm. In contrast, poor antiangiogenic efficacy could at least partially be ascribed to enhanced tumor angiogenesis induced by TAF and migration of tumor cells along the blood vessels (perivascular invasion) into normal tissues to evade antiangiogenesis. Clearly, predicting tumor invasion and progression based only on BV change in the regions of interest inside tumors is difficult in the above case. This might explain the non-significant increase in BV during tumor progression. Unlike previous reports, no significant connection between changes in other perfusion indexes, such as BF, MMT and PS, was found in the current study. This might be due to a limited sample pool or the fact that BV might be a more optimal indicator of antiangiogenic efficacy than the other parameters, which might warrant further investigation.
In the long-term observation, an inverse correlation was observed between ∆BV and PFS (r = −0.461, P = 0.005), suggesting that a continuous decrease of BV might indicate long-term efficacy, as does aCEC. The results show that ∆BV is a significant indicator of PFS in the combined therapy group (P = 0.019). In addition, no significant difference was found between PFS in cases of SD and PR, as defined by the RECIST criteria, implying that predicting antiangiogenic efficacy using ∆BV is more reliable than tumor volume and a longer stable disease duration could be achieved in some no-shrink tumors with significantly decreased BV.
Although some reports showed better responses to chemotherapy could be observed in cases with higher baseline levels of CEC and BV, no significant difference in baseline CEC and BV was found between cases of PD and non-PD in the present study.[46, 47] Further investigation is needed to clarify whether baseline CEC and BV can serve as ideal predictors.
Correlation between BV and aCEC
The correlation between ∆aCEC and ∆BV was only found in the combined arm (r = 0.430, P = 0.022); the positive but not very close correlation implies that their similar trend could reflect the progression and regression of tumoral angiogenesis and provide a way for accurate prediction of antiangiogenic efficacy through combined detection. It suggested that blood volume in tumor is “determined” by the growth of micrangium formed by aCEC. Considering that rh-endostatin could suppress many targets in endothelial cells compared with bevacizumab, which only inhibits VEGF, changes in the VEGF level alone might be insufficient to reflect rh-endostatin's efficacy, as demonstrated in a previous study, therefore multiple markers should be detected. Besides, suppression of micrangium around the tumor could have different effects on neoplastic cells, including depression or enhancement of growth. Therefore, not only baseline but dynamic detection of markers must be done during therapy and follow up. Nevertheless, further prospective study with a larger patient pool is needed for validation.
To eliminate disturbance from confounding factors due to insufficient subjects in the study and to determine real influential factors for PFS, we made a multivariate Cox's proportional hazards regression analysis to disclose all possible factors by inputting variables such as age, sex, history, TNM stage, prior therapy and ECOG performance into a model besides CEC and perfusion indexes, and only CEC and BV were defined as significant indicators of PFS in the combined therapy group.
Limits of present research
Some reports proposed that the exceptionally high CEC counts detected with the commonly used flow cytometry test (FCT)-based assay are attributed to the fact that the majority of cells designated as CEC are actually large platelets. To avoid such artifact, combined detection through both magnetic isolation and FCT assays has been initiated in our center to determine total CEC, aCEC and apoptotic CEC.
In conclusion, both aCEC and BV can predict antiangiogenic efficacy and their combined detection is helpful in predicting antiangiogenic efficacy more sensitively and reliably than plain or enhanced CT scans alone. Optimization of the detection method to elucidate changes in the subgroups of CEC during antiangiogenic therapy, as well as the connection between CEC and efficacy in further prospective trials, are necessary.