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Combined Small Cell Carcinoma of the Lung: Is It a Single Entity?

Open ArchivePublished:October 31, 2017DOI:https://doi.org/10.1016/j.jtho.2017.10.010

      Abstract

      Background

      SCLC accounts for 15% and 20% of all lung cancers, with combined SCLC (CSCLC) comprising 2% to 5%. Little is known about the clinical characteristics and molecular changes associated with the various histologic components.

      Methods

      A total of 205 SCLC cases were resected between 2005 and 2015. Clinical and pathologic features were analyzed. All CSCLC cases were confirmed by histologic examination and immunohistochemistry. The individual components were microdissected using a novel automated dissection system, and DNA was extracted and subjected to targeted exome sequencing.

      Results

      A total of 10 cases of CSCLC were identified out of 170 cases with adequate histologic material; squamous cell carcinoma comprised the second component in half of these (n = 5). There were no significant differences between CSCLC and pure SCLC with respect to clinical features. The median follow-up time was 36 months. The median survival times of patients with pure SCLC and CSCLC were 58 months and 26 months, respectively (p = 0.030). The different components of three cases of CSCLC were deemed adequate for microdissection and sequencing. Approximately 75% of the identified somatic mutations were present in both components. There were also 15 gene mutations or six amplifications unique to only one of the components.

      Conclusions

      We identified no significant clinical or pathologic differences between pure SCLC and CSCLC; CSCLC was associated with decreased overall survival compared with pure SCLC. The histologic components of CSCLC had high genetic concordance but also showed divergent genotypes. These findings may suggest a common precursor with subsequent acquisition of oncogenic changes in CSCLC.

      Keywords

      Introduction

      Lung cancer is the leading cause of cancer death worldwide,
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      Cancer statistics in China, 2015.
      with SCLC accounting for approximately 15% to 20% of cases.
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      • Fennell D.A.
      • De Ruysscher D.K.
      Small-cell lung cancer.
      • Byers L.A.
      • Rudin C.M.
      Small cell lung cancer: where do we go from here?.
      Combined SCLC (CSCLC) is a rare subtype of SCLC, defined by the combination of SCLC and NSCLC components. The NSCLC component is typically composed of adenocarcinoma, squamous cell carcinoma, or large cell carcinoma, but it can also rarely involve sarcomatoid or giant cell carcinoma.
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      • Brambilla E.
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      Small cell lung carcinoma (SCLC): a clinicopathologic study of 100 cases with surgical specimens.
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      The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification.
      • Inamura K.
      Lung cancer: understanding its molecular pathology and the 2015 WHO classification.
      Furthermore, more than two components can also be observed.
      To date, the cell of origin of CSCLC remains unclear and the literature thus far has been based on limited but older techniques such as loss of heterozygosity (LOH), immunophenotype, or comparative genomic hybridization analysis.
      • Buys T.P.
      • Aviel-Ronen S.
      • Waddell T.K.
      • Lam W.L.
      • Tsao M.S.
      Defining genomic alteration boundaries for a combined small cell and non-small cell lung carcinoma.
      • Murase T.
      • Takino H.
      • Shimizu S.
      • et al.
      Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung.
      A few studies have concluded that the different components in CSCLC may have a common cellular origin, and that tumor stem cells undergo divergent differentiation during proliferation or, rather, one of the two components arises by random genetic mutations in the other component.
      • Murase T.
      • Takino H.
      • Shimizu S.
      • et al.
      Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung.
      • Fukui T.
      • Tsuta K.
      • Furuta K.
      • et al.
      Epidermal growth factor receptor mutation status and clinicopathological features of combined small cell carcinoma with adenocarcinoma of the lung.
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      A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer.
      Exposure to tyrosine kinase inhibitors (TKIs) against the EGFR may lead to the transformation from EGFR-mutant adenocarcinoma to SCLC in less than 10% of EGFR TKI–resistant cases, suggesting plasticity between the different histologic subtypes.
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      Small cell lung cancer transformation and T790M mutation: complimentary roles in acquired resistance to kinase inhibitors in lung cancer.
      An increase in CSCLC after neoadjuvant chemotherapy has been observed and is thought to be due to a positive selection of NSCLC by chemotherapy.
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      Histologic changes in small cell lung carcinoma after treatment.
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      A prospective randomized trial to determine the benefit of surgical resection of residual disease following response of small cell lung cancer to combination chemotherapy.
      Alternatively, CSCLC could represent the synchronous apposition of two malignant tumors (collision tumor).
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      Collision carcinoma at the esophagogastric junction: report of two cases.
      The prevalence of CSCLC is variable, being reported in 2% to 28% of all SCLC cases.
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      Combined small-cell and non-small-cell lung cancer.
      This variation may reflect ongoing changes in the classification of SCLC, as well as the nature of the specimen obtained (e.g., resection versus small biopsy versus fine-needle aspiration specimen). There are few studies assessing the clinical and prognostic features of CSCLC after surgery. Furthermore, few studies have addressed the molecular changes in the different histologic components. Here, we have investigated the clinicopathologic features of a large series of resected SCLCs and performed next-generation sequencing (NGS) on the different components in three CSCLC cases.

      Materials and Methods

      This study included a total of 170 patients with resected SCLC at the Tianjin Medical University Cancer Institute and Hospital between 2005 and 2015 for which adequate material was available. Details of this series are described elsewhere (X. Zhao, Tianjin Medical University Cancer Institute and Hospital, personal/written communication, 2017). All specimens were reviewed and independently confirmed by two board-certified pathologists. Ten cases of CSCLC were identified, accounting for 5.9% of the SCLC in this series (Table 1). All cases were restaged according to the seventh edition of American Joint Committee on Cancer TNM staging system for lung cancer.
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      Clinical data on all patients were extracted from medical records and follow-up (as of March 2017) was obtained. Overall survival (OS) was defined as the interval between the date of the operation and the time of death or the date of the last follow-up.
      Table 1Clinicopathologic Characteristics of CSCLC (n = 10)
      CaseAgeSexSmoking indexLocationpStageSurgery TypeR ResectionDiameter, cmAdjuvant chemotherapy
      174M0RLIAWedgeR12Yes
      263M1600RLIIALobectomyR07Yes
      370F0RMIVLobectomyR06No
      459M1200LUIIIALobectomyR05Yes
      563M3200RLIALobectomyR02Yes
      660M800LUIALobectomyR02No
      762M800LUIIIAWedgeR12.2No
      858M1200LLIIIALobectomyR03Yes
      944M40RLIIIALobectomyR05Yes
      1049M400LUIIIBLobectomyR03.5No
      Note: Smoking index is cigarettes per day multiplied by years smoked.
      CSCLC, combined SCLC; M, male; F, female; LLL, left lower lobe; RLL, right lower lobe; RUL, right upper lobe; LUL, left upper lobe; RML, right middle lobe.

      Immunohistochemistry

      All samples were formalin-fixed and paraffin-embedded (FFPE); 4-μm tissue sections were stained with hematoxylin and eosin (HE) and analyzed using a select panel of immunohistochemical (IHC) markers. Pretreatment of the FFPE sections with heat-induced epitope retrieval was performed. Optimal results were obtained by pretreating tissues with heat-induced epitope retrieval using diluted Envision FLEX Target Retrieval Solution, HIGH pH (50×) (Agilent Technologies, Santa Clara, CA). Deparaffinization, rehydration, and epitope retrieval were performed in DAKO PT Link (PT100/PT101) (Agilent Technologies). The following parameters were used for PT Link: preheat temperature, 65°C; epitope retrieval temperature and time, 97°C for 20 minutes; and cool down to 65°C. Racks were placed in diluted Envision Flex Wash Buffer (20×) (Agilent Technologies) for 5 minutes.
      Slides were treated with Flex Peroxidase Blocking solution for 5 minutes and then with the first antibody with an incubation time of 20 minutes, Flex Mouse Linker for 15 minutes, Flex HRP for 20 minutes, Flex DAB with Substrate-Chromogen for 10 minutes, and Flex hematoxylin for 5 minutes. Wash buffer was applied before the addition of each reagent for 5 minutes to guarantee that no residual remains of any reagent were left on the slide.
      All the antibodies were run on the Dako/Agilent Autostainers Link 48, and the antigen retrieval for all the antibodies was performed on the Dako/Agilent PT Links.
      The following prediluted antibodies were used: CD56 (123C3), synaptophysin (DAK-SYNAP [Agilent]), thyroid transcription factor 1 (8G7G3/1), cytokeratin 7 (OV-TL12/30), Ki-67 (MIB-1), chromogranin A (1:50 dilution, Clone LK2H10, Cell Marque, Rocklin, CA), Napsin A (Pre-Diluted, Cell Marque), p63 (Pre-Diluted, Clone 4A4, Ventana Medical Systems, Tucson, AZ) and GNAS complex locus (GNAS) (1:200 dilution, Clone PA5-22261, Invitrogen, Carlsbad, CA) (see Supplementary Table 6 for details on the antibodies).

      Tumor Enrichment and DNA Extraction

      All CSCLC cases were carefully reviewed using HE staining and IHC markers. Only cases with a clear morphologic boundary between the SCLC and NSCLC components were selected for microdissection. CSCLC samples with highly mixed components that could not be confidently separated by the dissection method were not included.
      Tumor enrichment was achieved using a prototype novel software-guided dissection system from Roche Sequencing Solutions (Avenio Millisect System, Roche, Pleasanton, CA) that utilizes an automated platform to guide tissue dissection, and is capable of dissecting areas of interest as small as 200 μm in diameter. Serial 5-μm sections were cut from FFPE tissue blocks. Reference HE and IHC stains were used to assist with accurate demarcation of selected tumor regions to accurately separate and enrich for the different tumor components. The reference slides were converted to whole slide images using an Aperio Scanscope XT (Leica Biosystems Imaging, Buffalo Grove, IL) scanned at ×40 magnification. Areas of interest were digitally annotated using Imagescope (Leica Biosystems) to designate the dissection regions. The annotated reference images were imported to the Millisect instrument and overlayed with live images of unstained slides situated on an automated stage. Annotated tumor areas were then dissected using 200-μm, 400-μm, or 800-μm Millisect Milling tips directly into ATL (lysis) buffer (Qiagen, Valencia, CA), and DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Q). Proteinase K was added to the excised tissue lysis buffer mixture, and the samples were incubated at 56°C on a heater/shaker for 3 hours or until the tissue was completely digested. After incubation, the tubes were heated to 90°C and then brought back to room temperature. The samples were centrifuged to eliminate particulate undigested material, after which the supernatants were processed on a QIAcube robotic workstation (Qiagen) and the DNA was eluted in a volume of 40 μL. Optical density was measured using a NanoDrop microvolume spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), to determine DNA concentration and quality for subsequent analysis.

      NGS Library Construction and MiSeq Sequencing

      Before NGS library construction, DNA quality and quantity were analyzed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and the Quantifluor ONE dsDNA system (Promega, Madison,WI), respectively. Briefly, 200 to 300 ng of each DNA sample was sheared to a target size of 200 base pairs on the Covaris M220 focused ultrasonicator (Covaris, Woburn, MA). The sheared DNA fragments were repaired using the NEBNext End Repair Module (New England Biolabs, Ipswish, MA) and purified with Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN). Libraries were constructed by ligating Illumina adapters and unique indexes (Personal Genome Diagnostics, PGDx, Baltimore, MD) to the DNA fragments with the NEB Next Quick Ligation Module (New England Biolabs). Library amplification was performed by a 12-cycle polymerase chain reaction protocol with primers complimentary to the Illumina adapters. Each sample was hybridized to custom RNA baits from our PGDx custom gene panel (PGDx) for 24 hours and captured using streptavidin C1 beads (ThermoFisher Scientific). A final 16-cycle polymerase chain reaction amplification was performed to enrich for the indexed libraries. Quantity and quality of the libraries were assessed after each step by using either the DNA 1000 kit or the High Sensitivity DNA kit on the Bioanalyzer (Agilent Technologies). The indexed libraries were combined into a 4-nM equimolar pool for MiSeq (Illumina, San Diego, CA) sequencing preparation. The 4-nM library pool was diluted further to 8-pM with HT1 buffer (Illumina), and 5% of 12.5 nM PhiX v3 control (Illumina) was spiked into the diluted library pool. Six hundred microliters of the 8-pM pooled library with 5% PhiX control was loaded into the MiSeq Reagent Kit v2 (300 cycle) cartridge (Illumina). A 1 × 150 -base pair paired-end read was executed on the MiSeq sequencer (Illumina) according to the manufacturer’s user guide.

      MiSeq Sequencing Data Analysis

      The PGDx custom gene panel (1.3–megabase pair region size) targets the exons of 206 cancer genes and interrogates microsatellite instability (MSI) and specific genes for copy number variation and translocations (Supplementary Table 1). Alignment to the human reference genome 19 (UCSC hg19), adapter trimming, and variant calling were automatically executed by the MiSeq Reporter software (version 2.6.2, Illumina). The MSI, copy number variation, and translocation analysis was completed by our PGDx custom analysis pipeline. The VariantStudio software (version 2.2.1, Illumina) was used to annotate and filter all the variants. The databases used to annotate the variants included SIFT, PolyPhen, Catalogue of Somatic Mutations in Cancer (COSMIC), dbSNP v137, and RefSeq. The variants were filtered on the basis of their passing score (not flagged with low genotyping, low quality, or strand bias), mapping quality score (Q score >30), read depth (>30), and consequence (only nonsynonymous mutations). The samples were analyzed by examining both the SCLC and NSCLC components together to report common mutations between both components in the same patient and by examining each component separately to reveal mutations unique to their SCLC or NSCLC component. All mutations were visually inspected in the Integrative Genomics Viewer (Broad Institute, Cambridge, MA). Variants could not be validated using Sanger sequencing because of limitations of tissue and lack of high quality DNA. However, there is high confidence in the variant calling from the quality scoring, coverage with a read depth greater than 30, and evidence of clean surrounding reads in Integrative Genomics Viewer.

      Statistical Analysis

      SPSS 19.0 software (IBM Corp., Armonk, NY) was adopted for statistical analysis of the data. OS was estimated by the Kaplan-Meier method and the log-rank test was used to compare survival curves. All tests were two sided, and p values less than 0.05 were considered statistically significant.

      Results

      Patient Demographics and Survival

      All patients with SCLC were followed up for 2 to 139 months, with a median follow-up period of 36 months. The median age was 61 years (range 25–78), with 133 male and 37 female patients. The distribution of clinicopathologic features was not statistically different between patients with pure SCLC and those with CSCLC (p > 0.05) (Table 2). The 1-, 3-, and 5-year survival rates of patients with pure SCLC and those with CSCLC were 85.6%, 59.2%, and 49.3% and 70%, 17.5%, and 17.5%, respectively. The OS of CSCLC was significantly shorter than that of pure SCLC: pure SCLC median OS was 58 months (95% confidence interval: 32.86–83.14), versus 26 months for CSCLC (95% confidence interval: 17.65–34.36) (log-rank χ2 = 4.729, p = 0.030) (Fig. 1).
      Table 2Distribution of Clinical and Pathologic Factors in Pure and Combined SCLC
      FactorPure SCLC (n = 160)Combined SCLC (n = 10)χ2p Value
      Age
       ≥608350.0001.000
       <60775
      Sex
       Male12490.2860.593
       Female361
      pStage
       I–II9732.7410.098
       III–IV607
      Smoking index
       ≥40010670.0001.000
       <400543
      Surgery type
       Lobectomy12980.0001.000
       Wedge resection312
      R resection
       R0 resection10680.0040.952
       R1 resection542
      Postoperative adjuvant chemotherapy
       Yes10960.0340.854
       No514
      NSE
       Normal7350.0001.000
       Abnormal705
      CEA
       Normal9770.0001.000
       Abnormal463
      NSE, neuron-specific enolase; CEA, carcinoembryonic antigen.
      Figure thumbnail gr1
      Figure 1Overall survival curves of pure versus combined SCLC (p = 0.030).

      IHC Characteristics of Different Components of CSCLC

      Among the 10 CSCLC cases, squamous cell carcinoma was the most common NSCLC component (five of 10 [50%]), followed by large cell neuroendocrine carcinoma (LCNEC) (three of 10 [30%]), and adenocarcinoma (two of 10 [20%]). The SCLC and NSCLC components were clearly demarcated in six cases (five SCCs and one adenocarcinoma), whereas in the remaining four cases the two different components were highly mixed, without obvious tumor boundaries (Fig. 2 and Supplementary Fig. 1). CD56 staining was positive in all SCLC components and was negative in all NSCLC components except LCNEC. The SCC component was consistently positive for p63. Thyroid transcription factor 1 staining was positive in 30% of the SCLC component and almost completely negative in the NSCLC component, except for one LCNEC that was positive. The proliferation index (Ki-67) was higher in the SCLC component, with an average of 80% compared with 50% in the NSCLC component. LCNEC as the NSCLC component had a proliferation index similar to that of the SCLC component (Table 3).
      Figure thumbnail gr2
      Figure 2Immunohistochemical characteristics and boundary areas of different components in four selected cases of combined SCLC. Syn, synaptophysin; TTF-1, thyroid transcription factor 1.
      Table 3Immunohistochemistry for Different Histologic Components in Combined SCLC (n = 10)
      CaseComponentCD56ChrASynTTF-1CK7Ki67p63Napsin A
      1SCLC+focal/weak+strong+++∼100np
      GUH54Squamous80+np
      2SCLC+Patchy++∼100np
      GUH81Squamous80+np
      3SCLC+Focal+++Patchy+npn/a
      GUH95Poorly differentiated adenocarcinoma+npn/aFocal+
      4SCLC+npPatchy+80np
      GUH98Squamousnp+50–60+np
      5SCLCPatchy++npnpnp
      GUH90Squamousnpnpnp
      6SCLC++70–80np
      GUH93Squamous40–50+np
      7SCLC+++++n/a
      GUH68Adenocarcinoma30n/a+
      8SCLC+++++80n/anp
      GUH45Poorly differentiated LCNEC+Patchy++20n/anp
      9SCLC++++50n/anp
      GUH164LCNEC++++Dot-like+80n/anp
      10SCLC with clear cell change+Focal+80n/anp
      GUH167LCNEC+Focal+100n/anp
      ChrA, chromogranin A; Syn, synaptophysin; TTF-1, thyroid transcription factor 1; CK7, cytokeratin 7; GUH, Georgetown University Hospital; n/a, not applicable; np, not present; LCNEC, large cell neuroendocrine carcinoma.

      MiSeq Sequencing Mutations

      A targeted exome sequencing approach with a custom 206–cancer gene panel on the Illumina MiSeq platform was used to investigate the mutation profiles of CSCLC tumors. The MiSeq sequencing run yielded 5.17 gigabase pairs, with 95.4% greater than Q30 (5.0 gigabase pairs), and it produced 33,872,774 passing filter reads, with a mean total coverage and distinct coverage of 206 and 77, respectively. Six samples were microdissected and DNA successfully extracted (Fig. 2 and Supplementary Fig. 1); however, only three cases passed quality control and were sequenced successfully. The NSCLC component of these three cases was squamous carcinoma in each of them. A total of 81 mutations were found in all samples: most (77% [n = 62]) were missense mutations, whereas the remaining mutations (23% [n = 19]), were frameshift indels, stop gain, or splicing mutations. Moreover, of the 81 mutations, 23% (n = 19) were flagged as deleterious or damaging and 14% (n =11) were found in the COSMIC database. Of those mutations found in COSMIC, 64% (n = 7) were predicted as pathogenic according to the FATHMM predictor.
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      Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden Markov models.
      Subsequently, all missense mutations were retrieved in he Exome Aggregation Consortium database; 22 mutations and 9 mutations were found as common SNPs and rare SNPs, respectively. Excluding the common SNPs, common gene mutations found in both the SCLC and NSCLC components, represented approximately 75% of all mutations and were found in all samples: case 1 (n = 24), case 2 (n = 12), and case 3 (n = 8). Unique mutations found only in one component were identified in five out of the six samples: case 1-SCLC (n = 6), case 1-NSCLC (n = 2), case 2-SCLC (n = 2), case 2-NSCLC (n = 4), case 3-SCLC (n = 1) (Supplementary Tables 2 and 3 and Fig. 3A). Different tumor protein p53 gene (TP53) mutations were present in all three cases, but both histologic components shared the same mutation. Amplifications of GATA binding protein 2 gene (GATA2) and ret proto-oncogene gene (RET) were identified in both components in sample case 3. Amplifications of five genes (fibroblast growth factor receptor 3 gene [FGFR3], GNAS complex locus gene [GNAS], NK2 homeobox 1 gene [NKX2-1], H3 family member histone 3A gene [H3F3A], and nuclear receptor coactivator 3 gene [NCOA3]) were found in only one component of the tumor in samples case 3-SCLC (n = 2), case 1-NSCLC (n = 1), case 2-NSCLC (n = 1), and case 2-SCLC (n = 2) (Supplementary Tables 4 and 5 and Fig. 3B). There were 2 cases (cases 2 and 3) showing a unique GNAS amplification in only the SCLC components. We used IHC to verify the expression of GNAS in these two cases. Higher expression of GNAS with a diffuse pattern of distribution could be seen in the SCLC components than in the NSCLC components (Supplementary Fig. 2). Furthermore, the SCLC component of sample case 3 was flagged as MSI-positive with the microsatellite marker MONO27 with 30% of the reads being shorter than normal, indicating damage to the mismatch repair pathway. It should be noted that the components in case 3 had a lower number of mutations because of the low quality of input DNA and the low-quality sequencing reads and coverage. Case 3 showed a lower unique mutation percentage (19% [one of nine]) compared with case 1 (25% [eight of 32]) and case 2 (33% [six of 18]).
      Figure thumbnail gr3
      Figure 3(A) The number of mutations in common, unique mutations in the SCLC component and unique mutations in the NSCLC component in three cases of combined SCLC. (B) The number of gene amplifications in common, SCLC component unique amplifications and unique amplifications in the NSCLC component in three cases of combined SCLC.

      Discussion

      As an entity, SCLC has undergone significant changes in classification over the past 40 years. SCLC was initially divided into the four types of lymphocyte-like, polygonal, fusiform, and others. Later, the classification of SCLC was revised as oat cell, intermediate, and combined types by the WHO.
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      Following recommendations of the International Association for the Study of Lung Cancer in 1998, the oat cell type was changed to pure SCLC, CSCLC was retained, the intermediate type was removed, and mixed with large cell components changed to mixed type. Last revised in 1999, SCLC was divided into two types, pure SCLC and CSCLC, which could be a mixed tumor with any NSCLC components.
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      The definition of CSCLC has not changed even in the latest SCLC classification of 2015.
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      On the basis of morphologic features, spindle cell carcinoma, which is initially regarded as an NSCLC component, was reclassified to a subtype of SCLC. The change in CSCLC definitions is a potential reason for the variability in the proportion of CSCLC reported in older series. Furthermore, studies have shown that the diagnostic rate for CSCLC reflects the size of the biopsy specimen, the presence of crush artifacts, and the low frequency of cases. The material commonly used for diagnosis of SCLC is limited (fine-needle aspiration or bronchoscopic biopsy), and previous studies showed that CSLC accounts for approximately 14.3% in postmortem material, 8.6% in cytologic or biopsy specimens, and as high as 12% to 28% in surgical resection specimens.
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      To our knowledge, our series is the largest series of resected SCLC from a single institution. In the current study, the incidence of CSCLC was low at around 6%.
      To our knowledge, this is the first study utilizing NGS to investigate potential the various components of CSCLC. As previously mentioned, prior studies have utilized less sensitive and/or specific methods (e.g., IHC, LOH, comparative genomic hybridization).
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      A high frequency of LOH was shown to be unique to SCLC for alterations in 4p, 4q, 10q, 13q, 16q, and 17p.
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      Additionally, the SCLC component was characterized by an extremely high frequency of gains and losses.
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      A more recent study used NGS on 110 samples of fresh-frozen SCLC.
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      The most frequently identified mutation was of TP53 (92%), followed by mutation of the retinoblastoma 1 gene (RB1) (76%), and inactivating mutations in NOTCH family genes (25%); common genomic rearrangements of tumor protein p73 gene (TP73), SRY-box 2 gene (SOX2) (27%) and MYC family (16%) amplification were also documented. Like SCLC, squamous cell carcinoma is a smoking-related disease and shows a high frequency of TP53 mutations (91%),
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      as well as amplification of SOX-2 on chromosome 3q.
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      TP53 is also commonly mutated in adenocarcinoma (46%), followed by KRAS (33%).
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      Our sequencing results indicate that TP53 was the only gene that was mutated in all components of the three CSCLCs that were subjected to microdissection and sequencing. Nearly 75% of all mutations identified were present in both components of the CSCLC samples analyzed. In our study we found mutations in cyclin-dependent kinase inhibitor 2 gene (CDKN2A) and F-box and WD repeat domain containing 7 gene (FBXW7), aberrations that are more commonly reported in SCC.
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      Cancer Genomic Atlas Research Network
      Comprehensive genomic characterization of squamous cell lung cancers.
      We also identified mutations in insulin-like growth factor 2 receptor gene (IGF2R) in both components; these mutations have been more frequently reported in SCLC than in SCC.
      • George J.
      • Lim J.S.
      • Jang S.J.
      • et al.
      Comprehensive genomic profiles of small cell lung cancer.
      We confirmed by IHC analysis that GNAS, a G protein, has copy number gain in the SCLC component, which has not been reported before.
      • George J.
      • Lim J.S.
      • Jang S.J.
      • et al.
      Comprehensive genomic profiles of small cell lung cancer.
      In one case we also detected high microsatellite instability in the SCLC component only. This may have therapeutic implications.
      • Le D.T.
      • Durham J.N.
      Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.
      Losses of TP53 and RB1 in neuroendocrine cells of the airways are sufficient molecular events to generate SCLC in mouse models.
      • Sutherland K.D.
      • Proost N.
      • Brouns I.
      • Adriaensen D.
      • Song J.Y.
      • Berns A.
      Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung.
      In contrast, type II alveolar cells and club cells of the bronchoalveolar duct, and basal cells in the proximal airway are reported cells of origin for lung adenocarcinoma and squamous carcinoma, respectively.
      • Swanton C.
      • Govindan R.
      Clinical implications of genomic discoveries in lung cancer.
      • Desai T.J.
      • Brownfield D.G.
      • Krasnow M.A.
      Alveolar progenitor and stem cells in lung development, renewal and cancer.
      Murase et al. suggested that SCLC and SCC components of CSCLC are more closely related than adenocarcinoma, which is more likely to be derived from a different clone or from the same stem cell at a much earlier stage before occurrence of mutations in p53.
      • Murase T.
      • Takino H.
      • Shimizu S.
      • et al.
      Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung.
      It has also been suggested that SCLC is derived from neuroendocrine cells (Kulchitsky cells) but less frequently may also derive from type II alveolar epithelial cells.
      • Zhang H.
      • Liu J.
      • Cagle P.T.
      • Allen T.C.
      • Laga A.C.
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      Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach.
      Wagner et al. showed that the two different components of CSCLC had similar immunophenotype and gene phenotype.
      • Wagner P.L.
      • Kitabayashi N.
      • Chen Y.T.
      • Saqi A.
      Combined small cell lung carcinomas: genotypic and immunophenotypic analysis of the separate morphologic components.
      It has also been proposed that CSCLC is more closely related to pure SCLC than to NSCLC. Other investigators have suggested that the SCLC component originates through chromosomal changes in squamous cell carcinoma in early stages of development.
      • Murase T.
      • Takino H.
      • Shimizu S.
      • et al.
      Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung.
      In one study, one of six cases of CSCLC had an EGFR mutation (exon 21 L858R) in both components (adenocarcinoma and SCLC),
      • Fukui T.
      • Tsuta K.
      • Furuta K.
      • et al.
      Epidermal growth factor receptor mutation status and clinicopathological features of combined small cell carcinoma with adenocarcinoma of the lung.
      suggesting that both histologic forms may coexist, independent of transformation to SCLC observed as a mechanism of resistance to EGFR TKIs. Studies about the complexities of the SCLC metastases in mice have indicated that multiple metastases with different genomic profiles are driven by intratumoral heterogeneity.
      • McFadden D.G.
      • Papagiannakopoulos T.
      • Taylor-Weiner A.
      • et al.
      Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing.
      In spite of the small number of CSCLC cases analyzed, our results may indicate that CSCLC components are derived from common precursors. The different components shared nearly 50% of target gene mutations and amplifications. We hypothesize that poly-subclones of tumor stem cells may undergo different genetic mutations or amplifications under the influence of the tumor microenvironment, influencing the differentiation of tumor cells. Swanton et al. have pioneered the field of cancer heterogeneity and demonstrated the evolutionary process of polyclonal seeding of metastases from a primary tumor in adenocarcinoma of the lung.
      • Swanton C.
      • Govindan R.
      Clinical implications of genomic discoveries in lung cancer.
      They suggest that the early clonal mutational events, such as TP53 mutations and EGFR-activating mutations, that drive tumorigenesis occur at the very early time points, early in the trunk of the evolutionary tree. Subclonal driver events such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PI3KCA) mutations may occur later, in the branches of the evolutionary tree of the tumor. In our study, the different components of CSCLC shared nearly 75% common mutations, and we hypothesize that the different components have the same stem cell of origin because they had a similar genetic background, such as TP53 mutation. One component of CSCLC arises from the other component at a relatively late time point in the presence of a different microenvironment.
      The National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (National Comprehensive Cancer Network guidelines) do not make a distinction in the treatment recommendations for CSCLC and SCLC.
      • Johnson B.E.
      • Crawford J.
      • Downey R.J.
      • et al.
      Small cell lung cancer clinical practice guidelines in oncology.
      However, the NSCLC component of CSCLC shows decreased responsiveness to chemotherapy, which is a commonly used modality for pure SCLC (etoposide and cisplatin [EP]).
      • Wang X.
      • Jiang R.
      • Li K.
      Prognostic significance of pretreatment laboratory parameters in combined small-cell lung cancer.
      Attempts to supplement standard EP regimens by adding agents that are more active in NSCLC (such as paclitaxel or vinorelbine) to them have not been successful, with overall response rate, progression-free survival, and OS not significantly improved and side effects greater than those of the EP regimen.
      • Luo J.
      • Wu F.Y.
      • Li A.W.
      • Zheng D.
      • Liu J.M.
      Comparison of vinorelbine, ifosfamide and cisplatin (NIP) and etoposide and cisplatin (EP) for treatment of advanced combined small cell lung cancer (cSCLC) patients: a retrospective study.
      • Li Y.Y.
      • Zhou C.
      • Yang D.X.
      • et al.
      Paclitaxel-etoposide-carboplatin/cisplatin versus etoposide-carboplatin/cisplatin as first-line treatment for combined small-cell lung cancer: a retrospective analysis of 62 cases.
      • Zhang J.
      • Qi H.W.
      • Zheng H.
      • et al.
      Etoposide-cisplatin alternating with vinorelbine-cisplatin versus etoposide-cisplatin alone in patients with extensive disease combined with small cell lung cancer.
      In a report of 10 patients with CSCLC who underwent surgical treatment, OS was found to be longer in the patients with CSCLC than in those with pure SCLC.
      • Babakoohi S.
      • Fu P.
      • Yang M.
      • Linden P.A.
      • Dowlati A.
      Combined SCLC clinical and pathologic characteristics.
      Furthermore, the postoperative 5-year survival rate was 100% for patients with stage I and II disease. Whether survival of CSCLC is any different from survival of SCLC remains controversial. Evidence, including that presented here, indicates that CSCLC has a shorter OS and is less responsive to chemotherapy than pure SCLC.
      • Mangum M.D.
      • Greco F.A.
      • Hainsworth J.D.
      • Hande K.R.
      • Johnson D.H.
      Combined small-cell and non-small-cell lung cancer.
      • Hirsch F.R.
      • Matthews M.J.
      • Aisner S.
      • et al.
      Histopathologic classification of small cell lung cancer. Changing concepts and terminology.
      • Radice P.A.
      • Matthews M.J.
      • Ihde D.C.
      • et al.
      The clinical behavior of “mixed” small cell/large cell bronchogenic carcinoma compared to “pure” small cell subtypes.
      In conclusion, the results presented here indicate that CSCLC has a poorer OS compared with pure SCLC. There were no significant differences in clinicopathologic features between pure SCLC and CSCLC. The different histologic components of CSCLC had a similar mutational profile but also demonstrated divergent genotypes. The different components of CSCLC may share a similar cellular precursor. Further research involving larger numbers of dissected cases will be needed to confirm our findings.

      Acknowledgments

      This study was supported by the National Key R&D Program of China (grant) 2016YFC0905501 and National Institutes of Health grant P30 CA51008.

      Supplementary Data

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      Linked Article

      • Morphologic and Other Forms of Heterogeneity in Small Cell Lung Cancer: What Can We Learn from Them?
        Journal of Thoracic OncologyVol. 13Issue 2
        • Preview
          SCLC is a deadly, recalcitrant form of lung cancer that is strongly associated with tobacco exposure. Inactivation of the tumor protein p53 gene (TP53) and retinoblastoma 1 gene (RB1) genes in SCLC is almost universal and is believed to be the initiating molecular event.1,2 The WHO classification of lung cancers recognizes only one major morphologic form of SCLC, although elements of NSCLC cancers may be present (combined SCLC [CSCLC]).3 Historically, SCLC has been regarded as a “homogenous” disease (with little documented intertumor or intratumor heterogeneity with respect to histologic characteristics or molecular biology).
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