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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0b013e318227142d
Original Articles: Pathology

Subtyping of Non-small Cell Lung Carcinoma: A Comparison of Small Biopsy and Cytology Specimens

Sigel, Carlie S. MD*; Moreira, Andre L. MD, PhD*; Travis, William D. MD*; Zakowski, Maureen F. MD*; Thornton, Raymond H. MD†; Riely, Gregory J. MD, PhD‡; Rekhtman, Natasha MD, PhD*

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*Department of Pathology, †Interventional Radiology Service, Department of Radiology, and ‡Thoracic Oncology Service, Division of Solid Tumor, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York City, New York.

Disclosure: The authors declare no conflicts of interest.

Address for correspondence: Natasha Rekhtman, MD, PhD, Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. E-mail:

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Background: There is growing evidence that lung adenocarcinoma and squamous cell carcinoma (SQCC) have distinct oncogenic mutations and divergent therapeutic responses, which is driving the heightened emphasis on accurate pathologic subtyping of non-small cell lung carcinoma (NSCLC). The relative feasibility and accuracy of NSCLC subtyping by small biopsy versus cytology is not well established, particularly in current practice where immunohistochemistry (IHC) is becoming routinely used to aid in this distinction.

Methods: Concurrent biopsy and cytology specimens obtained during a single procedure and diagnosed as NSCLC during a 2-year period (n = 101) were reviewed. Concordance of diagnoses in the two methods was assessed. Accuracy was determined based on subsequent resection or autopsy diagnosis (n = 21) or IHC for thyroid transcription factor 1 and p63 on a subset of cases (n = 43).

Results: The distribution of definitive versus favored versus unclassified categories (reflecting the degree of diagnostic certainty) was similar for biopsy (71% versus 23% versus 6%, respectively) and cytology (69% versus 19% versus 12%, respectively), p = 0.29. When results from paired specimens were combined, the rate of definitive diagnoses by at least one method was increased to 84% and the unclassified rate was decreased to 4%. NSCLC subtype concordance between biopsy and cytology was 93%. Kappa coefficient (95% confidence interval) for agreement between methods was 0.88 (0.60–0.89) for adenocarcinoma and 0.76 (0.63–0.89) for SQCC. In pairs with discordant diagnoses (n = 7) the correct tumor type was identified with a similar frequency by biopsy (n = 4) and cytology (n = 3). Factors contributing to mistyping were poor differentiation, necrosis, low cellularity, and lack of supporting IHC. All concordant diagnoses for which verification was available (n = 57) were correct. IHC was used more frequently to subtype NSCLC in biopsy than cytology (32% versus 6%; p = 0.0001).

Conclusions: Small biopsy and cytology achieve comparable rates of definitive and accurate NSCLC subtyping, and the optimal results are attained when the two modalities are considered jointly. The lower requirement for IHC in cytology highlights the strength of cytomorphology in NSCLC subtyping. Whenever clinically feasible, obtaining parallel biopsy and cytology specimens is encouraged.

The practice of subtyping of non-small cell lung carcinoma (NSCLC) as adenocarcinoma (ADC) versus squamous cell carcinoma (SQCC) in pathologic specimens has undergone a major paradigm shift in recent years.1,2 Specifically, subtyping of NSCLC is no longer viewed as unimportant because of the discovery that several targetable molecular alterations, particularly EGFR mutations3,4 and EML4-ALK rearrangements,5 are largely restricted to ADC and because of a non-squamous indication for bevacizumab and pemetrexed.6,7 Other important molecular differences between ADC and SQCC are increasingly identified,8,9 suggesting that future targeted therapies will be increasingly “histology specific.” Selection of patients for appropriate molecular tests and histology-based therapies necessitates accurate pathologic distinction of ADC versus SQCC. As a result, a great emphasis is currently placed on accuracy of NSCLC subtyping in pathologic specimens.

Because 70% of NSCLC are unresectable at presentation,10 treatment is often based on diagnoses from small biopsy or cytology specimens. Until recently, NSCLC subtyping was based entirely on morphology with elective use of mucin stains. However, the classic morphologic criteria for differentiating ADC versus SQCC (glandular architecture and intracellular mucin versus keratinization and intercellular bridges, respectively) may be focal or subtle in small specimens, particularly in poorly differentiated tumors. This explains the historically high rate of unclassified NSCLC designated as NSCLC-not otherwise specified (NOS) in both small biopsy and cytology.11,12

An important recent development in NSCLC subtyping is mounting evidence that immunohistochemistry (IHC) is a powerful ancillary tool for the distinction of ADC and SQCC, which substantially lowers the rate of unclassified and misclassified NSCLC and decreases the interobserver variability in small specimens.13–21 In particular, several recent studies have shown that ADC and SQCC have virtually nonoverlapping coexpression profiles of thyroid transcription factor 1 (TTF-1) and p63—the master transcriptional regulators in glandular and squamous cell lineages, respectively.13,17,20–22 These robust markers are now increasingly incorporated into routine clinical practice to aid in the diagnosis of morphologically unclassifiable NSCLC (NSCLC-NOS). Recently, our group performed a large cytology/resection correlation study that showed that in a practice with routine utilization of IHC, the rate of unclassified NSCLC is low (3%), and accuracy of ADC versus SQCC subtyping is high—97% overall and 100% for IHC-aided diagnoses,23 suggesting that cytologic diagnoses in current practice are suitable for guiding the new NSCLC subtype-based therapeutic decisions. However, a direct comparison between the performance of cytology versus small biopsy for NSCLC subtyping in the current practice has not been performed.

In this study, we directly compared the rate with which specific subtyping is achieved, the concordance and accuracy of NSCLC diagnoses in cytology versus small biopsy specimens obtained during a single procedure in the context of our routine practice. As a means of verification of diagnostic accuracy, we used not only resection/autopsy (as has been done traditionally) but also IHC, based on the recent evidence that IHC profiles are an extremely accurate means of determining tumor cell lineage in NSCLC. In addition, we compared the frequency with which IHC is used to aid the diagnosis in biopsy versus cytology in our routine practice.

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A review of the Memorial Sloan-Kettering Cancer Center Department of Pathology database was conducted to identify all concurrent small biopsy and cytology specimens with a diagnosis of NSCLC during a 2-year period (January 1, 2006–January 1, 2008). The decision to obtain parallel specimens (as opposed to a sole biopsy or cytology) was made at the discretion of the individual interventional radiologist or bronchoscopist. Selection was limited to cases in which both biopsy and cytology were either diagnostic or suspicious for malignancy, and NSCLC was reported by at least one of the methods. Because the goal of the study was to analyze NSCLC subtyping in actual clinical practice, we recorded original diagnoses rendered during the initial case review without modification. For determination of accuracy, the diagnoses rendered by biopsy/cytology were compared with subsequent resection (n = 20) or autopsy (n = 1) when available. In addition, IHC for TTF-1 (SPT24, NovoCastra, 1:50 dilution) and p63 (4A4, Dako, 1:700 dilution) was used as another means of diagnosis verification. IHC was performed either as part of initial work-up (n = 38) or as part of this study to resolve discordant diagnoses (n = 5). Interpretation of TTF-1/p63 immunoprofiles was based on the algorithm established in recent studies.13,17,20,22 The morphologic features for all discordant and unclassified cases were reviewed by two pathologists with subspecialty expertise in cytopathology (N.R. and C.S.S.) and thoracic pathology (N.R.).

The types of cytologic preparations included the following (according to standard specimen processing protocol in our cytology laboratory): (1) air-dried smear stained with Diff-Quik (used for immediate adequacy assessment), (2) smear fixed in 95% ethanol and stained with hematoxylin and eosin (H&E) or Papanicolaou stain, (3) ThinPrep monolayer prepared from the needle rinse in CytoLyt fixative, and (4) H&E-stained section from a paraffin-embedded cell block prepared by centrifugation of CytoLyt fluid. No cell blocks were available for cases with a very scant needle rinse, which yielded no visible cell pellet after centrifugation. All fine-needle aspirates (FNAs) were obtained with on-site adequacy assessment by a cytotechnologist or pathologist. As part of routine clinical practice during the study period, biopsy and cytology specimens were reviewed independently by different pathologists. Biopsy specimens were reviewed by pathologists with subspecialty expertise in thoracic pathology, and the cytology specimens were reviewed by experienced staff cytopathologists. A standard practice at our institution is for cytology specimens to be reviewed and reports finalized a day earlier than biopsy specimens due to a shorter processing time of cytologic specimens, and therefore, in most cases, cytologic diagnoses were rendered without the knowledge of biopsy diagnosis. However, a correlative review may have been performed at the discretion of individual pathologists, and therefore, we cannot exclude that for a subset of cases, diagnoses were rendered with the knowledge of the diagnosis in the other specimen.

During the study period, the use of the terms “definitive” versus “favored” versus “unclassified” reflected the degree of diagnostic certainty as judged on case-by-case basis by the individual pathologists. In general, definitive diagnoses (ADC or SQCC) were rendered when morphologic features were diagnostic of a tumor type (such as obvious glandular features or cytoplasmic mucin for ADC versus keratinization for SQCC) or when IHC was interpreted as diagnostic of a tumor type. The “favored” category was used when morphology and IHC were interpreted as suggestive of a tumor type but qualitatively or quantitatively insufficient for definitive diagnosis. An alternative use of the “favored” category has been recently proposed,24 but this study reflects the use of this category in clinical practice during the study period. The unclassified (NSCLC-NOS) category was used for samples where tumor type could not be determined by morphology, but cellularity was insufficient for IHC, or IHC results were judged as equivocal. In addition, in some underclassified cytology samples, IHC was intentionally withheld because of the deferral of IHC to the concurrent biopsy to avoid duplication of the workup. Because bronchoscopic specimens usually include several cytologic specimens (bronchial brush, wash, lavage, and transbronchial FNA), the most specific diagnosis reached by at least one of the methods was recorded for the study purposes. For example, if a bronchial brush was diagnosed as “ADC” and bronchial wash as “NSCLC-NOS,” the overall diagnosis for cytology was recorded as “ADC.”

The significance of associations was assessed by two-tailed Fisher's exact test, with a p value of ≤0.05 considered statistically significant. Agreement was analyzed by calculation of Cohen's kappa coefficient with the strength of association interpreted according to the following guidelines: very good (0.81–1.00), good (0.61–0.80), moderate (0.40–0.60), fair (0.20–0.40), and poor (<0.20).25

This study was performed with the approval of the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (New York, NY).

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Specimen Characteristics

One hundred one patients had a concurrent cytology and small biopsy specimens from the same diagnostic procedures with diagnosis of NSCLC during the study period. Specimens included concurrent transthoracic specimens—transthoracic core biopsies paired with transthoracic FNAs (n = 60), and concurrent bronchoscopic specimens—transbronchial biopsies paired with transbronchial FNAs (n = 4) and/or bronchial wash/brush/lavage (n = 37). Seven transthoracic core biopsies were paired with core touch preparations (core imprints); for the purposes of this study they were analyzed as a single group with transthoracic FNAs. Average follow-up was 14.6 months (range: 3 days–41 months). The patient characteristics were male:female ratio, 1:1.9; average age, 68 years; and age range, 37–91 years.

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Comparison of Degree of Diagnostic Certainty in Biopsy versus Cytology

The level of diagnostic certainty in NSCLC subtyping consisted of three categories: definitive, favored, and unspecified (NSCLC-NOS). As presented in Table 1, the distribution of definitive versus favored versus unclassified categories was similar for biopsy (71% versus 23% versus 6%, respectively) and cytology (69% versus 19% versus 12%, respectively), p = 0.29. This distribution was similar for ADC and SQCC in both methods. As presented in Table 2, a more definitive diagnosis was made by biopsy in 17% of cases and cytology in 13% of cases. By combining the results of the two methods, the number of patients with unclassified diagnoses was reduced to 4%, and the rate of definitive diagnosis by at least one modality was increased to 84%. We did not find a difference in the level of diagnostic certainty between various cytology methods (transthoracic FNA versus transbronchial FNA versus bronchial brush/wash/lavage) and between biopsy methods (transthoracic biopsy versus transbronchial biopsy) in this study.

Table 1
Table 1
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Table 2
Table 2
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Unclassified biopsy (n = 6) and cytology (n = 12) specimens were distributed as follows: unclassified biopsy/classified cytology (n = 2), unclassified cytology/classified biopsy (n = 8), and both methods unclassified (n = 4) (Table 1). In particular, review of unclassified biopsy/classified cytology showed that in one instance, a tumor in the biopsy sample was highly necrotic, whereas the cells were better preserved in cytology, and in the second instance, a tumor had poorly differentiated histology and IHC was noncontributory, whereas morphologic features in concurrent cytology were diagnostic of a tumor type. In the unclassified cytology/classified biopsy group, the majority (7/8) of biopsy diagnoses were aided by IHC. Four cases unclassified by both methods were uniformly high-grade, poorly differentiated, necrotic tumors. In two cases, the possibility of neuroendocrine carcinoma was considered, one case had basaloid morphology and one case had pleomorphic/sarcomatoid morphologic feature. In all cases, IHC results were equivocal. Resection follow-up for these cases was not available.

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Concordance and Accuracy of NSCLC Subtyping in Biopsy versus Cytology

The concordance in the NSCLC subtypes between biopsy and cytology was 93% (Table 3). Kappa coefficients with 95% confidence intervals for the agreement of biopsy and cytology diagnoses were 0.88 (0.60–0.89) for ADC, 0.76 (0.63–0.89) for SQCC, and 0.40 (0.10–0.69) for NSCLC-NOS. NSCLC subtype was discordant in seven cases (7%). For discordant diagnoses, subsequent resection (n = 1), autopsy (n = 1), or additional IHC (n = 5) revealed that the correct diagnosis was rendered with a similar frequency by biopsy (n = 4) and cytology (n = 3) (Table 4). Misclassified cytologic specimens included an even number of SQCC (n = 2) and ADC (n = 2), whereas misclassified biopsies were all ADC morphologically mimicking SQCC due to solid growth pattern and abundant pink cytoplasm (n = 3). The morphologic pitfalls are illustrated in Figure 1. Overall, all misclassified case were scant specimens of poorly differentiated and/or necrotic tumors classified in the absence of IHC. All biopsy and cytology specimens with concordant diagnoses for which resection or IHC verification was available (n = 53) were correct.

Table 3
Table 3
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Table 4
Table 4
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Figure 1
Figure 1
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Utilization of IHC in Routine Practice

As summarized in Table 5, utilization of IHC to aid in the diagnosis of ADC and SQCC was significantly higher in biopsy than cytology (32% versus 6%, p = 0.0001). Diagnosis of ADC was more commonly aided by IHC in biopsy than cytology (21% versus 6%, p = 0.003), as was the diagnosis of SQCC (11% versus 0, p = 0.0015). The majority of IHC-aided diagnoses were classified as “definitive” in both biopsy (66%) and cytology (83%), and the rest were classified as “favored.”

Table 5
Table 5
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For paired specimens where the diagnosis of SQCC required IHC in biopsy but not cytology, definite evidence of keratinization was evident in Papanicolaou-stained cytological preparations but was inapparent on H&E-stained biopsies (Figures 2A-C). Similarly, clear evidence of glandular differentiation (polarized cells with a flat luminal edge and/or acinar structures) was readily identifiable in cytology specimens in which concurrent biopsy was indefinite for the tumor type and required IHC for diagnosis (Figures 2D-F).

Figure 2
Figure 2
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The main finding of this study is that in our institutional experience paired small biopsy and cytology specimens achieve a comparable certainty and accuracy in NSCLC subtyping, and optimal results are obtained when the findings from both methods are combined. In particular, results from combined methods maximize the number of definitive diagnoses by at least one modality and decrease the rate of unclassified diagnoses (NSCLC-NOS).

The number of prior studies which have directly compared the efficacy of NSCLC subtyping in paired small biopsy versus cytology specimens is limited, and none have been performed in a practice where IHC is routinely used to subtype morphologically challenging cases. Similar to the findings of this study, prior studies found that the feasibility and accuracy of subtyping of malignant tumors overall was comparable in paired biopsy and cytology specimens.26–28 Of note, in prior studies, the ability to determine specific diagnoses for benign tumors and mass-forming lesions (such as organizing pneumonia) was superior for biopsy,26,27 which remains the main advantage of biopsy over cytology. The reported rate of unclassified specimens and accuracy of NSCLC subtyping in unpaired small specimens varies significantly among prior studies,11,12,29,30 with unclassified NSCLC rate of more than 30% reported in some studies.30 This is in stark contrast with the low rate of unclassified NSCLC by combined (4%) and individual methods in this series and recent cytology23 and small biopsy31 studies from our institution. The low unclassified rate (and high concordance and accuracy) of biopsy and cytology in this study is attributable at least in part to routine utilization of IHC for subtyping of difficult cases. This approach is becoming incorporated in routine practice in recent years and is recommended in the recent IASLC/ATS/ERS ADC classification proposal.24 In addition to the role of IHC, the high rates of NSCLC subtyping in this series may be partially reflective of practice in a subspecialized referral cancer center.

A key observation in this study is that morphologic evidence of differentiation as ADC or SQCC is frequently more apparent in cytologic specimens than in small biopsies. This is demonstrated by the significantly lower need for IHC to identify a tumor type in cytology compared with biopsy (6% versus 31%, respectively) despite a similar degree of certainty and accuracy of NSCLC subtyping by these methods. The main advantage of cytologic preparations in NSCLC subtyping is that the Papanicolaou stain (a routine stain in cytology) specifically highlights keratinizing cells, whereas H&E (a routine stain in histology) does not. This accounts for greater ability of routine cytology to identify squamous differentiation without the aid of IHC. Another advantage of cytology is that the three-dimensional cell arrangements of ADC may be better visualized in cytology than histology. Finally, cytology lacks formalin-fixation artifact and has less crush artifact compared with small biopsy, which allows for greater resolution of nuclear and cytoplasmic detail. This latter factor accounts for a well-known advantage of cytology over small biopsy in the diagnosis of small cell lung carcinoma.32,33 Our data support the conclusion that cytology offers a similar advantage in the subtyping of NSCLC.

A novel approach used in this study was to use IHC (in addition to final diagnosis in subsequent resection or autopsy) as a means of verifying the accuracy of the small specimen diagnoses. Several recent studies have shown that ADC and SQCC have distinct profiles of TTF-1 and p63 expression,13,17,20–22 and this can be used to aid in the diagnosis of morphologically challenging cases. The recently developed IHC algorithm is becoming increasingly incorporated into clinical practice (as discussed earlier) and provides an alternative gold standard for measuring the accuracy of small specimens. The validity of this approach is supported by recent studies showing that tumor type identified by IHC in preoperative specimens predicts with excellent accuracy the resection diagnosis of ADC versus SQCC.13,20,23

The study design, which included specimens obtained during a single procedure, was chosen to allow the most direct comparison between cytology and biopsy specimens; however, this approach has several limitations. The main disadvantage of this approach is that paired specimens may not be entirely representative of the individual, isolated performance of either method. For example, the total number of FNA passes performed by an interventional radiologist may be reduced if a core biopsy is obtained concurrently, resulting in a less cellular cytology specimen. Conversely, FNA may be more likely to be obtained in cases where a core biopsy is judged to be suboptimal, particularly if friable or minute. The second limitation is that for some paired specimens pathologists may elect to render a more generic diagnosis (such as NSCLC-NOS) in cytology, and defer the IHC work-up and further classification to a biopsy. Therefore, the relative rate of IHC utilization and the rate of unclassified specimens in this study may not reflect a full potential of cytology for NSCLC subtyping, which is more accurately reflected in a study where cytology is the only diagnostic modality.23 The third limitation of the study is that verification of diagnosis (in the form of resection, autopsy or IHC) was not available for nearly half of the patients. As a result, the primary conclusion from this study is the high level of concordance in the diagnoses, whereas the conclusion for comparable accuracy is based on only a subset of cases.

In addition to tumor subtyping, another important consideration in the method comparison is diagnostic yield (i.e., the rate of successful sampling of a mass lesion). Because in this study only cases that were diagnostic by both modalities were selected, the comparative diagnostic yield of biopsy versus cytology cannot be addressed. However, prior studies show that diagnostic yield is comparable for transthoracic biopsy and FNA,26,27,34–36 whereas results vary for different bronchoscopic methods.28,36 Importantly, several studies show significant improvement in the diagnostic yield by combining biopsy and cytology specimens in a single bronchoscopic28,37–39 or transthoracic26,27,40 procedure. It is fairly standard in bronchoscopic procedures to obtain cytological preparations concurrently with a biopsy, whereas for transthoracic approaches, the material obtained is more variable.41 When a concurrent FNA is obtained with a transthoracic core biopsy, it is a common view that the utility of cytology is limited to on-site adequacy assessment to confirm the successful targeting of a lesion, but that cytology does not have an important role in reaching the final diagnosis. In contrast, we show a distinct value added by cytologic specimens, in that in a subset of cases a more specific and accurate diagnosis is achieved in cytology. Together with prior studies showing the advantage of paired specimens for maximizing the overall diagnostic yield, an attempt should be made to obtain paired specimens whenever clinically possible.

Another essential consideration for small specimens in the age of targeted therapies is suitability for molecular testing. Although this question was not addressed in the current study, several prior studies have shown that cytology specimens, particularly FNAs are highly suitable for EGFR (and KRAS) mutation analysis.16,23,42–45 Recent studies from our institution suggest that overall small biopsies and FNAs have a comparable rate of yielding sufficient material for molecular testing,23,46,47 although further studies are needed to directly compare specific specimen types. In particular, when deciding on a method of tissue sampling, an important consideration is that standard molecular studies and IHC generally require cellular paraffin-embedded material (formalin-fixed paraffin-embedded tissue for biopsy and cell block for cytology). Although FNAs typically yield enough material for a cell block, other cytologic specimens (sputum, bronchial brush/wash/lavage) are usually insufficient for a cell block and cannot be used for routine IHC or molecular studies. In particular, core touch preparations (core imprints),48 which are currently used at some institutions, including ours, for immediate on-site assessment of adequacy of a core biopsy, should not be regarded as an FNA-equivalent as they do not yield a cell block and cannot be used for routine ancillary studies. For both biopsies and FNAs, obtaining tissue in excess of what traditionally has been sufficient for a minimal morphologic diagnosis of non-small cell carcinoma versus small cell carcinoma (which may require only a few tumor cells) is now necessary to ensure that cellularity is sufficient for potential IHC or molecular studies. This can be achieved by performing additional FNA passes and maximizing the amount of material in the needle-rinse fluid, which is used as a source of a cell block, or by obtaining an extra biopsy, if clinically feasible. Close interdisciplinary communication is encouraged to ensure that the specimens are providing sufficient material for the optimal diagnosis and predictive marker testing.

In summary, in our routine practice, small biopsy and cytology specimens achieve comparable specificity and accuracy of NSCLC subtyping, and optimal results are obtained when the findings from both modalities are combined. We therefore recommend obtaining concurrent biopsy and cytology samples, whenever clinically feasible, and correlating the results from both methods to ensure the greatest diagnostic accuracy.

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Back to Top | Article Outline

Adenocarcinoma; Squamous cell carcinoma; Cytology; Biopsy

© 2011International Association for the Study of Lung Cancer


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