Correlation between the Ki-67 proliferation index and response to radiation therapy in small cell lung cancer

The WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart (fourth edition; 2015) was the first classification system to adopt the Ki-67 proliferation index for the differentiation of neuroendocrine tumours. This classification indicates that, based on previous studies involving biopsy samples or surgical specimens [7, 8], the Ki-67 proliferation index in SCLC is typically >50%, ranging from 50% to 100%; in addition, cell proliferation is prominent in SCLC [6]. However, in the diagnosis of SCLC, the differentiation of this tumour from carcinoid tumours remains a challenge. Because the standard treatment for SCLC is chemoradiotherapy, large tumour samples are rarely obtained during surgery and the diagnosis is instead routinely made based on small tumour samples obtained using bronchoscopy or other procedures. As a result of limitations such as the presence of crush artefacts and poor tissue preservation, the use of cytoplasmic markers may lead to inaccurate diagnoses. The nuclear marker Ki-67, which is well preserved in samples with an extensive crush artefact, can effectively differentiate SCLC from carcinoid tumours [7, 8]. A further challenge associated with the diagnosis of lung tumours is the potential tumour heterogeneity observed between biopsy samples and surgical specimens. However, in NSCLC, high correlation has been reported between the expression of Ki-67 in biopsy samples and surgical specimens [9]. An additional limitation associated with the Ki-67 proliferation index is the lack of consistent counting methods, which has caused variations among pathologists in the index calculated [10]. Thus, to reduce these variations as much as possible in the present study, we used e-Count, a cell counting software program that counts cells with a nucleus that is positive for Ki-67 immunostaining on the basis of pixel colour intensity. Our study is the first to use e-Count (e-Path, Kanagawa, Japan). A recent study has reported the use of digital image analysis in Ki-67 immunostaining; in this study, digital image processing software was used to reduce variations in the Ki-67 proliferation index [11].

Regarding the association between the Ki-67 proliferation index and lung cancer, many studies on NSCLC (including meta-analyses) have indicated that Ki-67 expression is a poor prognostic factor for survival [4, 5]. In contrast, only two studies to date have evaluated the association between SCLC and the Ki-67 proliferation index, one of which reported that the outcome was poor in patients with biopsy samples yielding a Ki-67 proliferation index lower than the median [12]. Another study has reported that there was no association between survival and the Ki-67 proliferation index in SCLC [13]. Thus, the significance of the Ki-67 proliferation index in SCLC has been controversial. In NSCLC, the excision repair cross-complementation group 1 (ERCC1) protein is reported to be associated with resistance to platinum-based chemotherapy. In SCLC, however, ERCC1 was not related to survival or to chemoradiation therapy response, and no association was observed between Ki-67 and ERCC1 [13]. In vitro, SCLC cell lines are sensitive to radiation, and the dose-response curves for these cells lack a shoulder. Furthermore, relatively low radiation doses per fraction have been shown to be lethal to SCLC cell lines [14]. On the basis of these radiobiological features, it is known that twice-daily thoracic radiation therapy improves overall survival when this therapy is initiated with the first cycle of chemotherapy or less than 30 days after the start of the first cycle of chemotherapy [15, 16]. Moreover, according to the “law of Bergonié and Tribondeau” (1906), which established a link between cell proliferation and cellular radiosensitivity, cells that more frequently undergo cell division are more sensitive to radiation. Proliferating cells have been reported to be more sensitive to radiation than quiescent cells in vitro [17]. Ki-67 was originally obtained from monoclonal antibodies to the nuclear antigen in Hodgkin and Sternberg-Reed cells. The nuclear antigen detected by Ki-67 is expressed in almost all human cell lines, but is not expressed in normal human cells in the resting stage. Ki-67 thus recognizes a nuclear antigen associated with cell proliferation [1].

Thus, we expected that tumour cells would have a higher rate of proliferation, more frequently undergo cell division, and be more sensitive to radiation in patients with a higher Ki-67 proliferation index before treatment; when we compared the mean Ki-67 proliferation index of the two groups (≥79.77% vs. <79.77%), tumour responses in terms of the CR rate were greater in the group with a Ki-67 proliferation index equal to or higher than the mean, as we expected. Few previous reports have evaluated the association between the Ki-67 proliferation index and tumour responses to radiation therapy. A study on uterine cervical cancer demonstrated that patients with a higher Ki-67 proliferation index at the time of diagnosis showed a significantly better histological response to radiation therapy at a total dose of 30 Gy [18]. Another study on oral squamous cell carcinoma (OSCC) reported that the Ki-67 proliferation index at the time of diagnosis had no significant correlation with the response to radiation therapy; in contrast, the reduction in the growth fraction (decrease in proliferation index) after radiation therapy at a total dose of 10 Gy was significantly correlated with the CR rate [19]. A study of OSCC after curative resection and postoperative radiation therapy reported that low Ki-67 proliferation index tumours had a significantly shorter time to recurrence than high proliferation index tumours [20]. This study concluded that tumours with a high Ki-67 proliferation index might respond better to radiation therapy as a result of increased radiosensitivity. Two studies on rectal cancer have reported results that were contradictory: one showed that there was no correlation between the Ki-67 proliferation index and the rate of response to radiation therapy, while the other reported that there was a good correlation between high Ki-67 proliferation index and improved response to radiation therapy [21, 22]. Our study is the first to evaluate the correlation between the Ki-67 proliferation index and the rate of response to radiation therapy in SCLC. Our findings suggest that a higher Ki-67 proliferation index might represent a predictive factor for higher radiosensitivity. Although we also examined whether the Ki-67 proliferation index was a prognostic factor in SCLC, as observed for NSCLC in previous studies, no difference in the survival rate was observed between groups 1 and 2. This may have been attributable to the fact that all three patients who were unable to receive chemotherapy were included in group 2 and had a Ki-67 proliferation index that was lower than the mean; as mentioned, a higher Ki-67 proliferation index might be associated with a higher survival rate.

A challenge for future studies is the low number of tumour cells in each sample. In the present study, the mean number of tumour cells was 420, which is lower than the number required (≥500 cells) for the assessment of Ki-67 in breast cancer, for example, according to the Ki-67 proliferation index guidelines [23]. Given that large SCLC samples are rarely obtained from surgery, there is a need to develop a technique that consistently obtains an adequate number of tumour cells, even from small samples acquired using bronchoscopy or other procedures. Further studies among SCLC patients are needed to assess the significance of the Ki-67 proliferation index in the treatment of SCLC.

To the best of our knowledge, our study is the first to evaluate the correlation between Ki-67 proliferation index and SCLC tumour response to radiation therapy. Our findings suggest that a higher Ki-67 proliferation index might represent a predictive factor for increased tumour radiosensitivity.