Journal of Thoracic Oncology:
State of the Art: Concise Review
Ki-67 Antigen in Lung Neuroendocrine Tumors: Unraveling a Role in Clinical Practice
Pelosi, Giuseppe MD, MIAC*†; Rindi, Guido MD, PhD‡; Travis, William D. MD§; Papotti, Mauro MD‖
*Department of Pathology and Laboratory Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy; †Department of Biomedical and Clinical Sciences “Luigi Sacco,” Università degli Studi, Milan, Italy; ‡Division of Anatomic Pathology, Gemelli Hospital and Università Cattolica del Sacro Cuore, Rome, Italy; §Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York; and ‖Division of Anatomic Pathology, San Luigi Gonzaga Hospital and University of Turin, Orbassano, Italy.
Disclosure: The authors declare no conflict of interest.
Address for correspondence: Giuseppe Pelosi, MD, MIAC, Dipartimento di Patologia Diagnostica e Laboratorio, Fondazione IRCCS Istituto Nazionale dei Tumori e Università degli Studi, Via G. Venezian 1, I-20133 Milano, Italy. E-mail: email@example.com
Classification of lung neuroendocrine (NE) tumors is a step-wise process with four tumor categories being identified by morphology, namely typical carcinoid (TC), atypical carcinoid, large-cell NE carcinoma, and small-cell lung carcinoma (SCLC). Ki-67 antigen or protein (henceforth simply Ki-67) has been largely studied in these tumors, but the clinical implications are so far not clear. A well-defined role has regarded the diagnostic use in the separation of TC and AC from SCLC in nonsurgical specimens, with monoclonal antibody MIB-1 resulting in the most used reagent after antigen retrieval procedures. Uncertainties, however, have arisen in its assessment, usually expressed as Ki-67 labeling index, because of some variability in obtaining either value of the fraction. A diagnostic role is currently lacking, even though there are significant differences in most cases between TC and AC, less so between large-cell NE carcinoma and SCLC. In addition, the prognostic role of Ki-67 is debated, likely due to methodological and biological reasons. The last challenge would be to identify an effective lung-specific grading system based on Ki-67 labeling index. In this review article, five relevant issues to Ki-67 have been addressed by using a question-answer methodology, with relevant key points discussing major interpretation issues. The conclusion is that Ki-67 is a feasible and potentially meaningful marker in lung NE tumors, but more data are needed to determine its ideal function in this setting of tumors.
In the lung, neoplasms with neuroendocrine (NE) morphology and differentiation encompass four histologically defined variants, namely typical carcinoid (TC), atypical carcinoid, large-cell NE carcinoma (LCNEC), and small-cell carcinoma (SCLC).1,2 According to epidemiologic, genetic, and clinical data, pulmonary NE tumors may be assembled for prognosis and therapy purposes into a three-tier clinicopathological scheme, according to which TC are low-grade malignant tumors with long life expectation and usual surgical treatment; AC, intermediate-grade malignant tumors with more aggressive clinical course and multimodality therapy; and LCNEC and SCLC, high-grade malignant tumors with overlapping dismal prognosis and multimodality or exclusive medical treatment.1–8
Subtyping pulmonary NE tumors is a step-wise process in which the four histologic variants are primarily separated by the number of mitoses per 2 mm2 and the presence of necrosis. Immunohistochemistry (IHC) for NE markers along with NE morphology is required to separate LCNEC from conventional non–small-cell lung carcinoma (NSCLC), and the distinction from SCLC is primarily based on cytological characteristics, including cell size.1–3,9–11 NSCLC lacking NE morphology such as adenocarcinoma or squamous cell carcinoma but with NE differentiation by IHC or electron microscopy has not been shown consistently to have different prognosis or response to treatment, so this is not accepted as a distinct class of lung cancer.1,2
Ki-67 antigen, also known as simply Ki-67 or MKI67 antigen identified by monoclonal antibody Ki-67, is a 359-kD non-histone nuclear protein with short half-life, which is encoded by the 15 exon-spanning MKI67 gene mapping to chromosome 10q26.2. This protein plays an essential role in the control and timing of cell proliferation,12–17 which undergoes a complex mechanism of post-translational phosphorylation and dephosphorylation by cell cycle key regulators leading to its subcellular redistribution from the interior of the nucleus/nucleolus to the perichromosomal layer and heterochromatin during mitosis and meiosis and vice versa18–22 Functionally, Ki-67 expression is finely tuned by specific microRNAs23 and produced during the entire cell cycle with a maximum in the G2 and M phases.24,25 However, it may be found at sites linked to the ribosomal RNA transcription machinery with a tight chromatin-associated function in both interphase and mitotic cells, although this finding does not argue against its valuable function as proliferation marker.26,27 The name Ki-67 derives from the city of Kiel in Germany where the antibody was first raised and the number 67 from the clone position in the original 96-well plate generated immunizing mice with nuclei of the lymphoma cell line L428.28,29 As the original monoclonal antibody to Ki-67 worked on frozen or fresh material only, subsequent antibodies have been developed to react with Ki-67 in formalin-fixed and paraffin-embedded material in a huge variety of malignancies, among which endocrine tumors in different anatomical sites.30 These reagents included polyclonal Ki-6731 and monoclonal MIB-1-3-5,32–34 IND.64,35 JG-67-2a,34 Ki-S1,36 Ki-S3,37 Ki-S5,36,38 and Ki-S1139 antibodies, with most studies confirming the validity of results obtained with these reagents in the measurement of proliferative activity in routinely processed tissues and even cytological samples.40–43 In more recent years, clone Mib-1 has been emerging as the most reliable and consistent reagent to recognize Ki-67 in paraffin sections32,33 and its wide commercial availability allowed many investigative studies and meta-analyses to be performed on the clinical implications of Ki-67 to assess proliferative activity in different malignancies,44,45 including lung.46
In NE tumor pathology, Ki-67 was first clinically investigated as prognostic factor in the pancreas,47–49 then exported to many other types of intestinal NE tumors50,51 until it was incorporated into the grading system of digestive tract NE neoplasms in the 2010 World Health Organization (WHO) classification.52,53 However, Ki-67 has entered the clinical practice of other tumors, such as breast cancer, this outlining its role in the molecular classification54 and clinical management of these oncologic patients.55 In this evolving scenario of increasing clinical appraisal, it is not surprising that Ki-67 has been widely studied even in NE tumors of the lung.56–72 However, clarifying its limits and defining practical applications can help clinicians and pathologists to better understand the potential lesson of Ki-67 in the management of NE lung tumor patients.
MATERIALS AND METHODS
A general overview of articles thus far published on the issue of Ki-67 as an operational IHC marker of cell proliferation in lung NE tumors is shown in Table 1. The term “NE tumor” will be synonymously and interchangeably used with the more correct alternative “NE neoplasm” to encompass the whole spectrum of lung NE tumors.73 Only articles dealing with the 1999 or 2004 WHO classifications3,4 or equivalent systems58,70 have been considered because they are more homogeneous for the definition of NE tumor categories.1,2,4,74 A list of key questions was developed with regard to technical issues, diagnostic and prognostic implication, tumor grading, and relevance to therapy, and these formed the basis for the literature review. Our research was limited to the English literature available in PubMed by variably crossing different research terms, such as Ki-67 or Ki67 (either antigen or protein), MIB1, MIB-1, antibody, NE, tumor, neoplasm, pulmonary, lung, carcinoid, typical, atypical, LCNEC, SCLC, prognosis, survival, or therapy. As a whole, 2067 lung NE tumors were retrieved corresponding to 25 independent studies (Table 1). Our work did not intend to perform a quantitative meta-analysis but rather to provide a critical reappraisal of the literature addressing frequently asked questions on NE lung tumor pathology and Ki-67 in daily clinical practice. Accordingly, a question-answer methodology has been pursued in the article, with relevant key points summing up major interpretation issues at the end of each answer. In the literature, the term hot spot has been used to indicate tumor areas with the highest concentration of nuclear decoration for Ki-67, whereas the term cold spot has been exploited by some studies to indicate the opposite phenomenon of tumor areas showing the minimal concentration of Ki-67 immunoreactive tumor cells75 (Table 2).
Question 1. Are there relevant technical issues to Ki-67 IHC and evaluation of results?
Answer: Yes, there is no uniform methodology for Ki-67 IHC and evaluation of results, but most studies pinpointed monoclonal antibody MIB-1 on paraffin sections after antigen retrieval procedures and the assessment of a Ki-67 labeling index (LI) as the most widely agreed-upon methodologies, which have been optimized within each laboratory by longstanding experience on this marker.
Although there are no systematic investigations comparing different antibodies against Ki-67 in clinically worked up NE lung tumors, clone MIB-1 on paraffin sections has been used in all but three articles,57,68,76 with different antibody dilutions (ranging from 1:25 to 1:1800) and antigen retrieval procedures being adopted within each laboratory usually by utilizing heat-induced unmasking systems in saline buffer and/or following specific manufacturer’s instructions (Table 1). Although two articles used prediluted reagents,57,77 five others did not provide details on antibody dilutions being applied to58,59,61,62,68 and four lacked information about the antigen retrieval procedures in use.59,61,67,78 Quantification of Ki-67 expression has been accomplished on surgical resection specimens by manual counting in all but three studies, in which automated systems of assessment were exploited.58,68,79 A high overall agreement of manual Ki-67 LI evaluation and an automated evaluation method upon scanned slides have recently substantiated the value, reproducibility, and easiness of Ki-67 LI upon manual counting.79 Small biopsy and cytology samples were used in six57,64,66,76,80,81 and two studies,62,77 respectively, to witness the applicability of these materials to accomplish Ki-67 evaluation. Although two works have imaginatively expressed Ki-67 results as either cumulative score81 or staining index78 by including the intensity of immunoreactivity or the density of stained cells in tumor tissue areas, respectively, all the remaining studies used the percentage of nuclear-stained tumor cells to substantiate a Ki-67 LI.56–72,75–77,79,80,82 However, the way to select immunoreactive tumor cells differed somewhat among the diverse studies, with six of them even not providing useful contributory information for further evaluation.57,70,72,77,80,81 Briefly, Ki-67 LI was assessed in nine studies pinpointing hot spot63,64,68,69,71,75,78,79,82 or average labeling frequency fields60 after scanning the entire tumor area at low magnification, whereas the quantification of positive tumor cells per 1 mm2 76 or 2 mm2 58 or randomly selected areas61 was declared by others to better accomplish Ki-67 LI. In another study, both hot and cold tumor areas were screened, in the same tumor samples, at low magnification to count 1000 tumor nuclei and a separate evaluation was provided for comparison.75 Evaluation of stained tumor cells on whole tissue sections of biopsy samples has also been used to maximize information obtainable from small material and avoid selection biases.64 No particular details on the selection criteria of tumor cells but only the global number of tumor cells being assessed were included in other investigations, which did not thus contribute to unveiling this issue.56,59,65–67 Interestingly, all studies dealing with LI determination were performed on biopsy or surgical specimens, except for two cytology investigations that expressed results either by quintiles62 or by Ki-67 LI.77 All tumor cells showing specific nuclear staining for Ki-67 were considered positive regardless of decoration patterns (diffuse, speckled, nucleolar, mitosis featuring), which are due to the differential expression of the protein during cell cycle progression.24,25 Expectedly, a significant correlation of Ki-67 LI with mitotic count59,65,69,75,76,79 or expression of cyclin B160 or histone H3 (a surrogate marker of mitoses)75 has been described in NE tumors of both the lung and the pancreas48 in virtue of the strong colinearity of the two indicators of cycling cells with variable correlation coefficients likely due to biological and technical reasons.75,79
More critical is the question of how Ki-67 LI should be calculated, because different methods have been provided to establish the optimal denominator, that is, the number of cells to be counted. Four to eight histological fields at ×2068 or ×4069,79 magnification, histological fields with average labeling incidence,60 2-mm2 tumor areas taken at ×25 magnification,58 1-mm2 tumor areas not otherwise specified,76 or 400 to 2000 tumor cells being consecutively counted56,59–61,63–67,71,75,78,82 have been used for assessing Ki-67 LI, which may account for some discrepant results and preclude a direct cross-study comparison.68
Another source of variability may derive from evaluating results of Ki-67 LI as mean56,58–61,63–71,75,77,81,82 or median57,64,79 thresholds, whereas other studies provided either poorly manageable categorical variables76 or no useful information.62,72,78,83 Reproducibility studies on Ki-67 LI evaluation by repeating the measurements in randomly selected carcinoid subsets68 or comparing manual mitotic count with Ki-67 LI by different pulmonary pathologists in the same tumor samples79 revealed encouraging results, with less than 1.5% of variability68 and an outperformance of Ki-67 LI over mitotic count with regard to interobserver agreement.79
Relevant key points: At variance with the gastroenteropancreatic system,84 there are no comparative studies evaluating different methods to perform and express Ki-67 results in lung NE tumors. However, most published investigations agreed on the opportunity of measuring Ki-67 LI in hot spot areas, taking into account all nuclear signals after visual scrutiny of the entire tumor. This would apply especially to TC or AC, whereas Ki-67 decoration is usually much more uniform in high-grade NE tumors. For practical purposes, Ki-67 LI should be calculated in surgical specimens by counting at least 2000 consecutive tumors cells in hot spot fields at ×40 magnification or 2 mm2 for consistency with the histological classification, possibly in the same tumor area as that used for assessing mitotic count. In biopsy or cytology samples, in which the number of tumor cells may be lower than 2000 and the 2-mm2 criterion unsuitable, it could be reasonable to calculate Ki-67 LI on all tumor cells. For experienced pathologists, manual counting of Ki-67 LI upon visual inspection or eyeball estimation differs little from more sophisticated, time-consuming, or cumbersome methods.84 Additional work and reproducibility studies are needed to address the optimal procedure for evaluating Ki-67 in lung NE tumors.
Question 2. Is there a diagnostic role for Ki-67 LI in lung NE tumors?
Answer: No, the classification on NE lung tumors is currently guided by morphology alone, but a practical utility for this marker has been emerging for separating TC/AC from high-grade NE tumors in limited diagnostic material.
As outlined in Table 2, the weighted average of Ki-67 LI values across different studies in which this evaluation could be done differed between TC and AC but not between LCNEC and SCLC when considering both surgical specimens only or biopsy/cytology and surgical specimens as a whole, with minor differences if hot spot values of Ki-67 LI were taken into account. The distribution of Ki-67 LI values across the different categories of lung NE tumors according to the type of specimens (only excised specimens or excised and biopsy samples) and the way to select tumor cells (declared hot spot areas versus not declared hot spot areas) is shown in Table 2. In three large studies8,64,85 accounting for 628 surgically excised NE tumors of the lung (one of which in abstract form only),85 the value of Ki-67 LI ranged from 2.3% to 4.15% in 211 TC, 9% to 17.8% in 131 AC, 47.5% to 70.0% in 153 LCNEC, and 64.5% to 77.5% in 133 SCLC, in substantial agreement with the expected proliferation rates of these tumors.
Significant differences in the Ki-67 LI distribution have been described in several studies between TC and AC,58–60,64–66,68,69,71,72,79 between LCNEC and SCLC,64 or across the entire spectrum of lung NE tumors,81 whereas other authors did not support this correlation at all56,70,75 or limited the failure to poorly differentiated NE tumors only.60 Proposed cutoff thresholds of Ki-67 LI ranged from 2.5 to approximately 30% for carcinoids,58,59,62,64–66,68,69,75,76,79,82 with two studies detecting 50% or more in few AC,68,70 whereas the separation of SCLC and LCNEC, if any,64 is of more limited clinical impact.1,86 In another study on 190 lung NE tumors published in an abstract form only, although there were differences in Ki-67 LI among diverse tumor categories, the incorporation of this marker as a primary criterion in the classification scheme of lung NE tumors was not further supported.85 A possible explanation why Ki-67 LI could not effectively split biologically adjacent tumor variants could be the imperfect correlation with mitotic count. This causes the frequency distributions of Ki-67 LI to consistently overlap between these adjacent tumor variants,68,69,75 also taking into account the high interobserver variability existing, for example, in high-grade NE tumor subclassification.87
One of the most agreed-upon uses of Ki-67 LI with important clinical implications deals with the distinction of low to intermediate grade from poorly differentiated NE tumors (especially SCLC) in small biopsy or cytology samples,57,62,64,71,77 especially in the presence of crush artifacts or poor tissue preservation,55,60,62 in which nuclear markers are more suitable for the diagnostic interpretation than cytoplasmic markers. In fact, nuclear markers are easier to scrutiny because there is no passive diffusion of cytoplasmic proteins into adjacent cells, but chromatin-related molecules, such as Ki-67, are likely to remain tightly associated to nuclear remnants even when filamentous changes occur due to tumor cell fragmentation. Thresholds up to 25% to 30% of Ki-67 LI have been quoted as a useful diagnostic adjunct to exclude poorly differentiated NE tumors, which are associated with an exceedingly high proliferation index,57,62,64,71 whereas thresholds of less than 3% would support a diagnosis of low-grade NE tumor and thresholds between 3% and 30% would indicate indeterminate tumors that most often consisted of AC with very few poorly differentiated tumors.75 A comparative assessment of Ki-67 LI in biopsy64 or cytology samples77 and paired surgical specimens was available from two studies, with similar but not perfectly overlapping results in the setting of low to intermediate malignant lung NE tumors in keeping with those obtained in pancreatic NE tumors,88 likely owing to either sampling or methodological issues.64,77 Although it is mandatory to avoid major pitfall in the management of lung cancer patients and Ki-67 LI assessment is effective to assist this task,64 worth noting, however, is that Ki-67 LI on small biopsy sample may represent the only available data of cell proliferation for clinical decisions in inoperable patients.89
Relevant key points: Ki-67 LI is not part of the current WHO diagnostic criteria for classifying lung NE tumors and should not be used to differentiate TC and AC owing to considerable overlapping in the distribution of Ki-67 indices between biologically adjacent NE tumor categories. The assessment of Ki-67 LI, however, is useful as a diagnostic adjunct in small biopsy or cytology specimens with poor preservation or crush artifact, to avoid misdiagnosing low- to intermediate-grade NE tumors as poorly differentiated NE carcinoma.57,62,64,71,77 Ki-67 LI does not serve to make specific diagnoses of lung NE subtypes, rather it very sensitively parallels the inherent proliferative properties of the tumors under evaluation.
Question 3: Is there a prognostic role for Ki-67 LI?
Answer: Possibly, Ki-67 LI has been emerging as a promising prognostic factor in excised specimens especially of low- to intermediate-grade lung NE tumors, although more data are needed to establish its ideal role.
As indicated in Table 1, at least 12 articles have investigated the prognostic inference of Ki-67 LI in diverse categories of lung NE tumors, especially TC and AC,58–60,63,65,66,68,69,76,78,82,83 but results are sometimes conflicting and not conclusive yet to authorize a well-recognized role as a prognostic factor for Ki-67 LI in lung NE tumors. Some authors denied any relevance for this marker to pinpoint differences in patients’ life expectancy inside individual tumors categories,60,66,76 whereas others indicated a worse prognosis in TC80 or purported a role as metastasis predictor alone78 or upon fascin overexpression (a protein involved in cell migration).82 Only six studies have indeed supported a prognostic role of Ki-67 LI in surgically excised TC and/or AC,58,59,65,68,69,80 but results are far from being conclusive. In fact, Ki-67 LI seemed to accurately segregate TC and AC into two distinct prognostic categories by cutoff values between 2.5% and 5.8%,58,59,65,68,69 which turned out independent of morphology in three studies totaling 220 carcinoids.58,59,65 In another study dealing with 43 TC, patients with increased Ki-67 expression had significantly shorter survival time.80 A note of caution, however, has been advanced on the limited role of Ki-67 LI in predicting survival of low to intermediate malignant lung NE tumors when lumping TC and AC, inasmuch as a threshold of 5% did not offer substantial better survival information over morphology, also within individual tumor categories.68 Similar conclusions on the lack of an independent prognostic efficacy of Ki-67 LI in lung NE tumors have recently been published in an abstract form only.85 Interestingly, however, several studies have revealed that a Ki-67 LI cutoff of 4% to 5% could differentiate between lower and higher malignant NE tumors in the setting of TC and AC,58,59,65,68,69,82 similar to what already demonstrated in analogous NE tumors of the pancreas.48,49 Although conceptually reasonable, no studies have so far addressed a role of Ki-67 LI in the prognostic stratification of poorly differentiated NE tumors, at variance with what has been proposed in other endocrine organs, such as the pancreas.90
Relevant key points: Ki-67 LI has been proposed as a prognostic factor in excised specimens of TC and AC, with cutoff values ranging from 2.5% to 5.8%, sometimes but not always independent of morphology. The existence of conflicting results and the lack of widely agreed-upon cutoff thresholds to stratify these tumors preclude making a recommendation at this time. Additional information is needed to establish the ideal role of Ki-67 LI in the prognostic assessment of lung NE tumors, ideally helping to predict prognosis within individual tumor categories. Because there is not much variability in survival for TC, LCNEC, and SCLC, the tumor category where there would be the greatest potential to predict prognosis is within AC. When lumping TC and AC together, it is not surprising that Ki-67 may help with prognosis, but this is not really adding anything to existing diagnostic capabilities.
Question 4: Is there an established role for Ki-67 LI in tumor grading?
Answer: No, in lung NE tumors, the concept of tumor grading as a biological continuum paralleling increasing malignancy is tautologically included into the current WHO classification, according to which TC are considered low malignant, AC intermediate malignant, and LCNEC and SCLC high malignant tumors.
In this setting, tumor grade of lung NE tumors refers to the degree of biologic aggressiveness and is related to, but different from, differentiation that is in turn defined by morphology.73 The main reason why Ki-67 LI assessment cannot currently claim any primacy in the grading system of lung NE tumors over morphology to realize the clinical three-tier spectrum regards its suboptimal correlation with histological features used for classification, especially mitotic count75,79 and necrosis, which causes adjacent categories of traditionally assessed tumors to partially imbricate with each other. This also reflects the fact that morphology itself is insufficient in separating borderline/overlapping lesions for either low to intermediate malignant or high malignant tumors.3,7,91 To try to overcome the drawback of the largely expected close but not perfect colinearity of Ki-67 and mitotic count, the grading system devised for digestive NE tumors (G1: <2 mitoses per 2 mm2 and/or Ki-67 LI ≤2%; G2: 2–20 mitoses per 2 mm2 and/or Ki-67 LI >2% but ≤20%; G3: >20 mitoses per 2 mm2 and Ki-67 LI >20%)52,92 has been tested on 111 TC and 83 AC to identify tumor subpopulations from lower to higher aggressive biological behavior.69 All TC corresponded to G1 tumors using the mean (1.8%) but not hot spot (2.5%) values of Ki-67 LI, whereas all AC resulted in G2 tumors by either threshold (3.7% and 5.8%, respectively). Distribution of tumors across this scheme indicated that 72.3% more patients with G2 tumors (no tumor fit with G3 criteria) developed lymph node metastases, 18.2% more distant metastases, and 20% more died, when compared with traditionally assessed AC, thereby concluding that Ki-67 LI in addition to mitotic count could improve the prediction of clinical behavior of lung carcinoids.69 This work, however, was not supported by multivariate analysis to validate superiority of this grading system in lung NE tumors in comparison with the traditional criteria adopted in WHO 2004 classification, and in this data set, the method used to apply the 2004 WHO criteria resulted in an extraordinary finding of significantly greater lymph node metastases in TC (25.2%) compared with AC (13.4%).1 Other works have correlated the distribution of Ki-67 LI with tumor grade according to the usual diagnostic categories by light microscope,75,77 but they reflected variations in tumor cell differentiation along the clinicopathologic spectrum of lung NE tumors rather than introducing a grading system based on Ki-67 LI.
Relevant key points: Establishing a lung-specific grading system based on a widely agreed-upon marker, such as Ki-67 LI, alone or better in combination with other morphologic parameters, in analogy with other NE tumors elsewhere in the body, is a desirable and clinically warranted goal, also because this marker is familiar to most oncologists and pathologists. However, to date, the existing data do not support a recommendation to apply to the lung the grading systems devised for NE tumors in other anatomical sites, particularly the gastrointestinal tract. Nonetheless, the behavioral heterogeneities within individual lung NE tumor subtypes (especially AC and LCNEC) may be opportunities in future research to develop a specifically devised grading procedure for lung NE tumors where Ki-67 LI as defined by widely agreed-upon criteria according to a grading system could play a role even within individual tumor categories.
Question 5: Is there a predictive role for Ki-67 LI in therapeutic decisions?
Answer: No, the therapy of NE lung tumors is basically guided by morphology and tumor staging by tumor, node, metastasis (TNM) system, with TC being usually treated by surgery, AC, and LCNEC by multimodality approach especially in advanced stage and SCLC by almost exclusive chemoradiotherapy.5
The role of Ki-67 LI may be directed to improve diagnosis for better chance of cure, especially in challenging cases of small biopsy or cytology specimens,64 but its direct implications in establishing the type, timing, and results of therapy have not been evaluated by randomized trials, but at the moment, a role as dynamic biomarker of treatment efficacy has not been provided. A correlation between excision repair cross-complementation 1 expression, a resistance factor against platinum-based chemotherapy in lung cancer, and Ki-67 LI has been described in diverse lung NE tumors considered as a whole, although the correlation was weak and the significance disappeared within different tumor types.66
Another study investigated the relationship between Ki-67 LI and thymidylate synthase expression, an enzyme involved in DNA synthesis whose expression acts as a resistance factor to fluoropyrimidine therapy, but results showed that these two markers were independently regulated.93 Likewise, the expression of mammalian target of rapamycin signaling activation pathways, an attractive target for mammalian target of rapamycin inhibitors such as everolimus in NE tumors, did not correlate with Ki-67 LI.8 In conventional NSCLC, the predictive impact of Ki-67 to treatment has remained unclear.94
Relevant key points: There are no randomized trials documenting that establishing Ki-67 LI in lung NE tumors may guide therapy, beyond refining better diagnosis in difficult cases, usually with small crushed biopsy specimens. Future work will determine the role of evaluating Ki-67 in lung NE tumors other than SCLC (most often are AC and LCNEC, the less familiar categories of these tumors), for deciding chemotherapy intervention especially in symptomatic patients with clinically aggressive tumors.
CONCLUSIONS AND PERSPECTIVES
Three decades after its introduction in the medicine practice and 20 years after its proven prognostic relevance for pancreatic and digestive NE tumors, Ki-67 continues to be a protagonist marker also in lung NE tumors. Conflicting results may be stemming from several factors, including selection of patients, number and type of tumors under evaluation, histological criteria used for classification, variability in the choice of antibodies and immunostaining protocols, Ki-67 staining cutoff thresholds, assessment criteria (automated analysis, manual counting, eyeball estimation, field and cell selection, number of analyzed cells), length and accuracy of follow-up, and/or clinical parameters under evaluation, which may have prevented direct cross-study comparisons. Establishing a lung-specific and clinically meaningful grading system based on Ki-67 LI, alone or in combination with other parameters, with defined cutoff thresholds and uniform procedures for assessing Ki-67 LI is clinically warranted.
The most agreed-upon procedure to express Ki-67 is to calculate the percentage of stained tumor cells on at least 2000 cells in hot spot areas (Ki-67 labeling index).
Lung NE tumors are classified by morphology, and Ki-67 does not provide relevant information because of overlapping values in biologically adjacent tumor categories.
Ki-67 LI is useful to avoid misdiagnosing TC and AC as SCLC.
Avoid untested and untrusting grading systems devised for NE tumors of other anatomical sites because of the expected different biology.
Ki-67 LI correlates closely but not perfectly with mitotic count introducing more ample information on NE tumor cell population: a more sensitive grading system should hopefully include both parameters.
This work is dedicated to the memory of Carlotta, an extraordinarily lively girl who untimely died of cancer in the prime of life.
After this paper was accepted for publication, an innovative evidence-based proposal of a three-tier, morphology-independent grading system of NE lung tumors was reported on, which combined managerially Ki-67 LI, mitotic counting and necrosis assessment in a large series of surgically excised tumors.95 In particular, lung-specific cut-off thresholds were generated for these tumors, which provided an effective tool for accurately predicting prognosis and biological aggressiveness.
1. Travis W, Brambilla E, Muller-Hermelink H, Harris C Tumours of the Lung, Pleura, Thymus and Heart. 2004 Lyon IARC Press
2. Travis W, Colby T, Corrin B, Shimosato Y, Brambilla E Histological Typing of Lung and Pleural Tumours. 1999 Berlin, Heidelberg, New York Springer Verlag
3. Travis WD, Gal AA, Colby TV, et al. Reproducibility of neuroendocrine lung tumor classification. Hum Pathol. 1998;29:272–279
4. Travis WD, Rush W, Flieder DB, et al. Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid. Am J Surg Pathol. 1998;22:934–944
5. Gridelli C, Rossi A, Airoma G, et al. Treatment of pulmonary neuroendocrine tumours: state of the art and future developments. Cancer Treat Rev. 2013;39:466–472
6. McMullan DM, Wood DE. Pulmonary carcinoid tumors. Semin Thorac Cardiovasc Surg. 2003;15:289–300
7. Asamura H, Kameya T, Matsuno Y, et al. Neuroendocrine neoplasms of the lung: a prognostic spectrum. J Clin Oncol. 2006;24:70–76
8. Righi L, Volante M, Rapa I, et al. Mammalian target of rapamycin signaling activation patterns in neuroendocrine tumors of the lung. Endocr Relat Cancer. 2010;17:977–987
9. Franks TJ, Galvin JR. Lung tumors with neuroendocrine morphology: essential radiologic and pathologic features. Arch Pathol Lab Med. 2008;132:1055–1061
10. Rekhtman N. Neuroendocrine tumors of the lung: an update. Arch Pathol Lab Med. 2010;134:1628–1638
11. Litzky L. Pulmonary neuroendocrine tumors. Surg Pathol Clin. 2010;3:27–59
12. Duchrow M, Schlüter C, Key G, et al. Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins. Arch Immunol Ther Exp (Warsz). 1995;43:117–121
13. Duchrow M, Schlüter C, Wohlenberg C, Flad HD, Gerdes J. Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67. Cell Prolif. 1996;29:1–12
14. Fonatsch C, Duchrow M, Rieder H, Schlüter C, Gerdes J. Assignment of the human Ki-67 gene (MK167) to 10q25-qter. Genomics. 1991;11:476–477
15. Schlüter C, Duchrow M, Wohlenberg C, et al. The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J Cell Biol. 1993;123:513–522
16. Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells. Cell Prolif. 1992;25:31–40
17. Schonk DM, Kuijpers HJ, van Drunen E, et al. Assignment of the gene(s) involved in the expression of the proliferation-related Ki-67 antigen to human chromosome 10. Hum Genet. 1989;83:297–299
18. Endl E, Gerdes J. Posttranslational modifications of the KI-67 protein coincide with two major checkpoints during mitosis. J Cell Physiol. 2000;182:371–380
19. Kreitz S, Fackelmayer FO, Gerdes J, Knippers R. The proliferation-specific human Ki-67 protein is a constituent of compact chromatin. Exp Cell Res. 2000;261:284–292
20. Scholzen T, Endl E, Wohlenberg C, et al. The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure. J Pathol. 2002;196:135–144
21. Traut W, Endl E, Scholzen T, Gerdes J, Winking H. The temporal and spatial distribution of the proliferation associated Ki-67 protein during female and male meiosis. Chromosoma. 2002;111:156–164
22. Saiwaki T, Kotera I, Sasaki M, Takagi M, Yoneda Y. In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase. Exp Cell Res. 2005;308:123–134
23. Hou YY, Cao WW, Li L, et al. MicroRNA-519d targets MKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703. Cancer Lett. 2011;307:182–190
24. Baisch H, Gerdes J. Identification of proliferating cells by Ki-67 antibody. Methods Cell Biol. 1990;33:217–226
25. Gerdes J, Lemke H, Baisch H, et al. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984;133:1710–1715
26. Bullwinkel J, Baron-Luhr B, Ludemann A, et al. Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells. J Cell Physiol. 2006;206:624–635
27. Starborg M, Gell K, Brundell E, Höög C. The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression. J Cell Sci. 1996;109 (Pt 1):143–153
28. Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983;31:13–20
29. Gerdes J, Li L, Schlueter C, et al. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991;138:867–873
30. Pelosi G, Zamboni G. Proliferation markers and their uses in the study of endocrine tumors. Endocr Pathol. 1996;7:103–119
31. Key G, Petersen JL, Becker MH, et al. New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections. J Clin Pathol. 1993;46:1080–1084
32. Cattoretti G, Becker MH, Key G, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol. 1992;168:357–363
33. Key G, Becker MH, Baron B, et al. New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope. Lab Invest. 1993;68:629–636
34. Kubbutat MH, Key G, Duchrow M, et al. Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein). J Clin Pathol. 1994;47:524–528
35. Key G, Meggetto F, Becker MH, et al. Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64. Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67. Virchows Arch B Cell Pathol Incl Mol Pathol. 1992;62:259–262
36. Rudolph P, Lappe T, Schubert C, et al. Diagnostic assessment of two novel proliferation-specific antigens in benign and malignant melanocytic lesions. Am J Pathol. 1995;147:1615–1625
37. Heidebrecht HJ, Buck F, Haas K, Wacker HH, Parwaresch R. Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the ‘Ki-67’ proteins. Cell Prolif. 1996;29:413–425
38. Kreipe H, Wacker HH, Heidebrecht HJ, et al. Determination of the growth fraction in non-Hodgkin’s lymphomas by monoclonal antibody Ki-S5 directed against a formalin-resistant epitope of the Ki-67 antigen. Am J Pathol. 1993;142:1689–1694
39. Rudolph P, Kellner U, Chassevent A, et al. Prognostic relevance of a novel proliferation marker, Ki-S11, for soft-tissue sarcoma. A multivariate study. Am J Pathol. 1997;150:1997–2007
40. Schwarting R, Gerdes J, Niehus J, Jaeschke L, Stein H. Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67. J Immunol Methods. 1986;90:65–70
41. Scott RJ, Hall PA, Haldane JS, et al. A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction. J Pathol. 1991;165:173–178
42. Sasaki K, Matsumura K, Tsuji T, Shinozaki F, Takahashi M. Relationship between labeling indices of Ki-67 and BrdUrd in human malignant tumors. Cancer. 1988;62:989–993
43. Pelosi G, Bresaola E, Manfrin E, et al. Immunocytochemical detection of cell proliferation-related antigens in cytologic smears of human malignant neoplasms using PC10, reactive with proliferating cell nuclear antigen, and Ki-67. A comparative study. Arch Pathol Lab Med. 1994;118:510–516
44. Chalkidou A, Landau DB, Odell EW, et al. Correlation between Ki-67 immunohistochemistry and 18F-fluorothymidine uptake in patients with cancer: A systematic review and meta-analysis. Eur J Cancer. 2012;48:3499–3513
45. de Azambuja E, Cardoso F, de Castro G Jr, et al. Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients. Br J Cancer. 2007;96:1504–1513
46. Martin B, Paesmans M, Mascaux C, et al. Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis. Br J Cancer. 2004;91:2018–2025
47. Pelosi G, Zamboni G, Doglioni C, et al. Immunodetection of proliferating cell nuclear antigen assesses the growth fraction and predicts malignancy in endocrine tumors of the pancreas. Am J Surg Pathol. 1992;16:1215–1225
48. Pelosi G, Bresaola E, Bogina G, et al. Endocrine tumors of the pancreas: Ki-67 immunoreactivity on paraffin sections is an independent predictor for malignancy: a comparative study with proliferating-cell nuclear antigen and progesterone receptor protein immunostaining, mitotic index, and other clinicopathologic variables. Hum Pathol. 1996;27:1124–1134
49. Rindi G, Falconi M, Klersy C, et al. TNM staging of neoplasms of the endocrine pancreas: results from a large international cohort study. J Natl Cancer Inst. 2012;104:764–777
50. Chaudhry A, Oberg K, Wilander E. A study of biological behavior based on the expression of a proliferating antigen in neuroendocrine tumors of the digestive system. Tumour Biol. 1992;13:27–35
51. Rindi G, Luinetti O, Cornaggia M, Capella C, Solcia E. Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: a clinicopathologic study. Gastroenterology. 1993;104:994–1006
52. Rindi G, Klöppel G, Alhman H, et al.All Other Frascati Consensus Conference Participants; European Neuroendocrine Tumor Society (ENETS). TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 2006;449:395–401
53. Bosman F, Carneiro F, Hruban R, Theise N WHO Classification of Tumours of the Digestive System. 2010 Lyon IARC Press
54. Guiu S, Michiels S, André F, et al. Molecular subclasses of breast cancer: how do we define them? The IMPAKT 2012 Working Group Statement. Ann Oncol. 2012;23:2997–3006
55. Dowsett M, Nielsen TO, A’Hern R, et al.International Ki-67 in Breast Cancer Working Group. Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. J Natl Cancer Inst. 2011;103:1656–1664
56. Arbiser ZK, Arbiser JL, Cohen C, Gal AA. Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis. Mod Pathol. 2001;14:1195–1199
57. Aslan DL, Gulbahce HE, Pambuccian SE, Manivel JC, Jessurun J. Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact. Am J Clin Pathol. 2005;123:874–878
58. Costes V, Marty-Ané C, Picot MC, et al. Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study. Hum Pathol. 1995;26:740–745
59. Grimaldi F, Muser D, Beltrami CA, et al. Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score. Front Endocrinol (Lausanne). 2011;2:20
60. Igarashi T, Jiang SX, Kameya T, et al. Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors. Mod Pathol. 2004;17:1259–1267
61. Iyoda A, Hiroshima K, Moriya Y, et al. Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity. Ann Thorac Surg. 2004;77:1891–1895
62. Lin O, Olgac S, Green I, Zakowski MF, Klimstra DS. Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms. Am J Clin Pathol. 2003;120:209–216
63. Pelosi G, Pasini F, Sonzogni A, et al. Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma. Cancer. 2003;97:2487–2497
64. Pelosi G, Rodriguez J, Viale G, Rosai J. Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients. Am J Surg Pathol. 2005;29:179–187
65. Rugge M, Fassan M, Clemente R, et al. Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients. Clin Cancer Res. 2008;14:149–154
66. Skov BG, Holm B, Erreboe A, Skov T, Mellemgaard A. ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival. J Thorac Oncol. 2010;5:453–459
67. Tsuta K, Kalhor N, Raso MG, Wistuba II, Moran CA. Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases. Hum Pathol. 2011;42:578–585
68. Walts AE, Ines D, Marchevsky AM. Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors. Mod Pathol. 2012;25:1258–1264
69. Zahel T, Krysa S, Herpel E, et al. Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach. Virchows Arch. 2012;460:299–308
70. Al-Khafaji B, Noffsinger AE, Miller MA, et al. Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors. Hum Pathol. 1998;29:992–999
71. Helpap B, Köllermann J. Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence? Virchows Arch. 2001;438:86–91
72. Laitinen KL, Soini Y, Mattila J, Pääkkö P. Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity. Cancer. 2000;88:1590–1598
73. Klimstra DS, Modlin IR, Coppola D, Lloyd RV, Suster S. The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems. Pancreas. 2010;39:707–712
74. Beasley MB, Thunnissen FB, Brambilla E, et al. Pulmonary atypical carcinoid: predictors of survival in 106 cases. Hum Pathol. 2000;31:1255–1265
75. Tsuta K, Liu DC, Kalhor N, Wistuba II, Moran CA. Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas. Am J Clin Pathol. 2011;136:252–259
76. Van E, eden S, Quaedvlieg PF, Taal BG, et al. Classification of low-grade neuroendocrine tumors of midgut and unknown origin. Hum Pathol. 2002;33:1126–1132
77. Zheng G, Ettinger DS, Maleki Z. Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors. Acta Cytol. 2013;57:281–290
78. Das-Neves-Pereira JC, Bagan P, Milanez-de-Campos JR, et al. Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors. Eur J Cardiothorac Surg. 2008;34:473–477
79. Warth A, Fink L, Fisseler-Eckhoff A, et al.Pulmonary Pathology Working Group of the German Society of Pathology. Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids. Virchows Arch. 2013;462:507–513
80. Granberg D, Wilander E, Oberg K, Skogseid B. Prognostic markers in patients with typical bronchial carcinoid tumors. J Clin Endocrinol Metab. 2000;85:3425–3430
81. Li F, Ye B, Hong L, Xu H, Fishbein MC. Epigenetic modifications of histone h4 in lung neuroendocrine tumors. Appl Immunohistochem Mol Morphol. 2011;19:389–394
82. Pelosi G, Pasini F, Fraggetta F, et al. Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids. Lung Cancer. 2003;42:203–213
83. Greenberg RS, Baumgarten DA, Clark WS, Isacson P, McKeen K. Prognostic factors for gastrointestinal and bronchopulmonary carcinoid tumors. Cancer. 1987;60:2476–2483
84. Tang LH, Gonen M, Hedvat C, Modlin IM, Klimstra DS. Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods. Am J Surg Pathol. 2012;36:1761–1770
85. Wang H, Iyoda A, Roh MS, et al. WHO histologic classification is an independent predictor of prognosis in lung neuroendocrine (NE) tumors but Ki-67 proliferation rate is not (abstract #1952). Mod Pathol. 2013;26:469A
86. Righi L, Volante M, Tavaglione V, et al. Somatostatin receptor tissue distribution in lung neuroendocrine tumours: a clinicopathologic and immunohistochemical study of 218 ‘clinically aggressive’ cases. Ann Oncol. 2010;21:548–555
87. den Bakker MA, Willemsen S, Grünberg K, et al. Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability. Histopathology. 2010;56:356–363
88. Larghi A, Capurso G, Carnuccio A, et al. Ki-67 grading of nonfunctioning pancreatic neuroendocrine tumors on histologic samples obtained by EUS-guided fine-needle tissue acquisition: a prospective study. Gastrointest Endosc. 2012;76:570–577
89. Volante M, Righi L, Berruti A, Rindi G, Papotti M. The pathological diagnosis of neuroendocrine tumors: common questions and tentative answers. Virchows Arch. 2011;458:393–402
90. Bastrurk O, Yang Z, Tang L, et al. Increased (>20%) Ki-67 proliferation index in morphologically well differentiated pancreatic neuroendocrine tumors (PanNETs) correlates with decreased overall survival (abstract #1761). Mod Pathol. 2013;26:423A
91. Marchevsky AM, Gal AA, Shah S, Koss MN. Morphometry confirms the presence of considerable nuclear size overlap between “small cells” and “large cells” in high-grade pulmonary neuroendocrine neoplasms. Am J Clin Pathol. 2001;116:466–472
92. Sobin L, Gospodarowicz M, Wittekind C TNM Classification of Malignant Tumours. 2010 New York Wiley-Blackwell
93. Ceppi P, Volante M, Ferrero A, et al. Thymidylate synthase expression in gastroenteropancreatic and pulmonary neuroendocrine tumors. Clin Cancer Res. 2008;14:1059–1064
94. Jakobsen JN, Sørensen JB. Clinical impact of ki-67 labeling index in non-small cell lung cancer. Lung Cancer. 2013;79:1–7
95. Rindi G, Klersy C, Inzani F, et al. Grading the neuroendocrine tumors of the lung: an evidence-based proposal. Endocr Relat Cancer. 2014;21:1–16
Ki-67; Antigen; MIB-1; Labeling index; Lung; Neuroendocrine; Carcinoid; Large-cell neuroendocrine carcinoma; Small-cell lung carcinoma; Diagnosis; Immunohistochemistry; Prognosis; Therapy
Copyright © 2014 by the European Lung Cancer Conference and the International Association for the Study of Lung Cancer.
Highlight selected keywords in the article text.