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The Use of Immunohistochemistry Improves the Diagnosis of Small Cell Lung Cancer and Its Differential Diagnosis. An International Reproducibility Study in a Demanding Set of Cases

Open ArchivePublished:December 17, 2016DOI:https://doi.org/10.1016/j.jtho.2016.12.004

      Abstract

      Introduction

      The current WHO classification of lung cancer states that a diagnosis of SCLC can be reliably made on routine histological and cytological grounds but immunohistochemistry (IHC) may be required, particularly (1) in cases in which histologic features are equivocal and (2) in cases in which the pathologist wants to increase confidence in diagnosis. However, reproducibility studies based on hematoxylin and eosin–stained slides alone for SCLC versus large cell neuroendocrine carcinoma (LCNEC) have shown pairwise κ scores ranging from 0.35 to 0.81. This study examines whether judicious use of IHC improves diagnostic reproducibility for SCLC.

      Methods

      Nineteen lung pathologists studied interactive digital images of 79 tumors, predominantly neuroendocrine lung tumors. Images of resection and biopsy specimens were used to make diagnoses solely on the basis of morphologic features (level 1), morphologic features along with requested IHC staining results (level 2), and all available IHC staining results (level 3).

      Results

      For the 19 pathologists reading all 79 cases, the rate of agreement for level 1 was 64.7%, and it increased to 73.2% and 77.5% in levels 2 and 3, respectively. With IHC, κ scores for four tumor categories (SCLC, LCNEC, carcinoid tumors, and other) increased in resection samples from 0.43 to 0.60 and in biopsy specimens from 0.43 to 0.64.

      Conclusions

      Diagnosis using hematoxylin and eosin staining alone showeds moderate agreement among pathologists in tumors with neuroendocrine morphology, but agreement improved to good in most cases with the judicious use of IHC, especially in the diagnosis of SCLC. An approach for IHC in the differential diagnosis of SCLC is provided.

      Keywords

      Introduction

      The current WHO classification of lung cancer recognizes four major neuroendocrine (NE) tumors, namely, typical carcinoid tumor (TC), atypical carcinoid tumor (AC), SCLC, and large cell NE carcinoma (LCNEC).
      • Travis W.
      • Brambilla E.
      • Burke A.P.
      • Marx A.
      • Nicholson A.G.
      WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart.
      Although the diagnosis of SCLC has defined diagnostic criteria, sampling issues, fixation artifact, and the morphologic variability of pure SCLC tumor cells can all potentially make diagnosis of SCLC challenging, with a wide differential diagnosis that includes TC, AC,
      • Pelosi G.
      • Rodriguez J.
      • Viale G.
      • Rosai J.
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens.
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      • Teirstein A.S.
      • Nieburgs H.E.
      • Kleinerman J.
      Problems in the diagnosis of small cell carcinoma of the lungs by fiberoptic bronchoscopy.
      LCNEC,
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      • Hendriks L.E.
      • Buikhuisen W.A.
      • et al.
      Clinical features of large cell neuroendocrine carcinoma: a population-based overview.
      basaloid squamous cell carcinoma, small round cell sarcoma (e.g., primitive neuroectodermal tumor),
      • Marchevsky A.M.
      • Wick M.R.
      Diagnostic difficulties with the diagnosis of small cell carcinoma of the lung.
      metastatic breast carcinoma,
      • Marchevsky A.M.
      • Wick M.R.
      Diagnostic difficulties with the diagnosis of small cell carcinoma of the lung.
      and non-Hodgkin's lymphoma. This is emphasized by reproducibility studies showing pairwise κ scores ranging from 0.35 to 0.81 for LCNEC versus for SCLC,
      • Ha S.Y.
      • Han J.
      • Kim W.-S.
      • Suh B.S.
      • Roh M.S.
      Interobserver variability in diagnosing high-grade neuroendocrine carcinoma of the lung and comparing it with the morphometric analysis.
      • den Bakker M.A.
      • Willemsen S.
      • Grünberg K.
      • et al.
      Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability.
      • Travis W.D.
      • Gal A.A.
      • Colby T.V.
      • Klimstra D.S.
      • Falk R.
      • Koss M.N.
      Reproducibility of neuroendocrine lung tumor classification.
      although κ values are much higher for high-grade NE carcinoma versus for carcinoid tumors (0.8–1).
      • Travis W.D.
      • Gal A.A.
      • Colby T.V.
      • Klimstra D.S.
      • Falk R.
      • Koss M.N.
      Reproducibility of neuroendocrine lung tumor classification.
      Recent studies have demonstrated that immunohistochemistry (IHC) stains are useful in refining diagnoses of non–small cell carcinoma specimens to either adenocarcinoma or squamous cell carcinoma in the case of both cytologic and biopsy specimens and in many cases of large cell carcinoma when resected.
      • Kayser G.
      • Csanadi A.
      • Otto C.
      • et al.
      Simultaneous multi-antibody staining in non-small cell lung cancer strengthens diagnostic accuracy especially in small tissue samples.
      • Thunnissen E.
      • Noguchi M.
      • Aisner S.
      • et al.
      Reproducibility of histopathological diagnosis in poorly differentiated nsclc: an international multiobserver study.
      The aim of the current study was to test whether the use of IHC leads to greater diagnostic reproducibility when distinguishing SCLC from its differential diagnoses. To this end, interactive digital images of pulmonary NE tumors and other morphologic differential considerations, including IHC, were studied by a group of lung pathologists of the International Association for the Study of Lung Cancer.

      Methods

      Fourteen members of the International Association for the Study of Lung Cancer pathology committee and pathologists from this network involved in lung cancer diagnosis in North America, Asia, and Europe submitted 35 biopsy and 44 resection samples, for a total of 79 cases. The hematoxylin and eosin (HE)-stained glass slides of NE lung tumors and other differential considerations along with IHC performed during the work-up of the cases were previewed by two pathologists (G. P. and E. T.) to confirm diagnostic adequacy. Each case was scanned, and one digital image was uploaded (by N. K., Y. M., and M.N.) to a password-protected website in Tsukuba, Japan. IHC stains varied from case to case. In 77 of 79 cases, a Ki67 (MIB1) stain along with one or more NE markers (CD56 [n = 44], chromogranin A [n = 55], and synaptophysin [n = 70]) were available. Other stains such as cytokeratin (CK) (AE1/3, CK7, CK8/18, CK5/6 [n = 22 in total]), p40/63 (n = 26), thyroid transcription factor 1 (TTF1) (n = 44), CD45 (n = 3), CD99 (n = 1), and vimentin (n = 1) were occasionally submitted.
      The cases were read in two rounds, the first involving resection specimens (n = 44) and the second involving biopsy specimens (n = 35), by 25 pathologists during each period. Overall, 19 pathologists studied all 79 cases. The pathologists were not informed about the composition of the case mix and were not provided with any clinical data or a list of available IHC stains. Participants were required to render diagnoses of each case three separate times: on morphologic grounds alone (level 1); on the basis of morphologic features plus at least one IHC stain, of which the reviewing pathologist could request up to a maximum of five (level 2); and with all available IHC stains (level 3). At each level, the pathologist was permitted to list up to three diagnostic possibilities and state the likelihood for each, totaling 100% (see Table 1). IHC stain assessment was captured in the following categories: (1) negative, (2) focal positive, and (3) positive greater than 50%, except for MIB1 (a proliferation marker), in which case the choice was one of four categories (0% to 5%, 6% to 20%, 21% to 50%, and greater than 50%).
      Table 1Categories for Diagnosis
      DiagnosisPercentage (100%)
      The sum in each column should add up to 100. For one diagnosis the score equals 100. For two or three diagnoses, the preferred diagnosis should have the highest percentage and the remaining diagnoses should add up to 100%.
      Percentage (100%)
      The sum in each column should add up to 100. For one diagnosis the score equals 100. For two or three diagnoses, the preferred diagnosis should have the highest percentage and the remaining diagnoses should add up to 100%.
      1. SCLC30
      2. Combined SCLC
      This option was not available in levels 2 and 3 of the first round.
      0
       2.1. SCLC0
       2.2. Squamous cell carcinoma0
       2.3. LCNEC0
       2.4. Adenocarcinoma0
      3. LCNEC70
      4. Atypical carcinoid0
      5. Typical carcinoid0
      6. Poorly differentiated NSCLC0
      7. Small round cell sarcoma0
      8. Non-Hodgkin's lymphoma0
      9. Other0
      Note: The categories for diagnosis are shown, now filled in with 3’ and 70 for two options. The likely diagnosis should be scored in percentages adding up to 100%. Maximally, three diagnostic scores were allowed in each level. Equal percentages for the most likely diagnoses were not allowed.
      LCNEC, large cell neuroendocrine carcinoma.
      a The sum in each column should add up to 100. For one diagnosis the score equals 100. For two or three diagnoses, the preferred diagnosis should have the highest percentage and the remaining diagnoses should add up to 100%.
      b This option was not available in levels 2 and 3 of the first round.

      Statistics

      κ Scores were calculated for (1) all nine categories (see Table 1 for the categories), (2) combined categories, and (3) one diagnostic category versus all other categories. Only readers with complete readings were included for calculating κ scores (n = 19). A κ value less than zero indicated no agreement, whereas κ values from 0 to 0.20, from 0.21 to 0.40, from 0.41 to 0.60, from 0.61 to 0.80, and from 0.81 to 1 indicated poor, fair, moderate, good, and almost perfect agreement, respectively. A reader with mean κ value of 0.40 or less in the first-round analysis was defined as an outlier and excluded from the κ score calculations. One and two outliers, respectively, were excluded from κ score calculations for the first and second rounds.
      At each level, the agreement diagnosis of all pathologists for each case was defined by the mode of main diagnosis. For each case, the highest percentages (diagnostic scores) in levels 1 and 2 were compared with the Wilcoxon signed rank test for paired data, as well as in levels 2 and 3. For each case, the rate of agreement was defined as the highest number of pathologists with the same main diagnosis divided by the total number of pathologists. A “unanimous” result was arbitrarily defined when 13 of 19 pathologists had the same diagnostic score. The differential diagnoses per case and at each level were evaluated as well. We considered a differential diagnosis for a case to be relevant if at least five (of 19) pathologists assigned a positive score to an alternative diagnostic category. For evaluation of staining interpretation, the scores per case for all pathologists were examined. A p value less than 0.05 was considered significant.

      Results

      For the 19 pathologists reading all 79 cases, the rate of agreement for level 1 was 64.7%; it increased to 73.2% and 77.5% in levels 2 and 3, respectively. The mean κ score range of all readers in the first round (n = 25) was 0.42 to 0.59 (mean 0.51) and that of all readers in the second round (n = 26) was 0.53 to 0.65 (mean 0.56) (Table 2). The κ scores for the two rounds read separately by 25 pathologists were similar and are shown in Supplementary Tables 1 and 2. The κ scores in level 3 for the (combined) SCLC, LCNEC, and AC/TC groups were 0.60, 0.49, and 0.75, respectively.
      Table 2κ Scores Calculated for All Diagnoses
      κ AnalysisLevel 1 κ ScoreLevel 2 κ ScoreLevel 3 κ ScoreNo. Cases
      Level 1Level 2Level 3
      Diagnoses per diagnostic category (vs. remaining diagnosis)
       (Combined) SCLC
      In round 1 for levels 2 and 3, only the option (combined) SCLC was available; therefore, both categories were calculated together.
      0.550.580.60283433
       LCNEC0.340.450.49141011
       Atypical carcinoids0.180.270.30776
       Typical carcinoids0.530.670.74222222
       Carcinoids (typical and atypical)0.600.710.75292928
       Poorly differentiated NSCLC0.140.390.53422
       Small round cell sarcoma0.090.030.81001
       Non-Hodgkin's lymphoma0.420.550.96333
       Other0.100.090.05110
      For all diagnoses
       All 8 categories
      In round 1 for levels 2 and 3, only the option (combined) SCLC was available; therefore, both categories were calculated together.
      separately
      0.410.500.58
       4 categories (combined): SCLC, LCNEC, (a)typical carcinoid, others0.480.580.64
      Note: The κ scores were calculated for all diagnoses in 79 cases by 19 observers in eight categories, as well as per diagnostic category versus the other categories. The categories SCLC and combined SCLC are clustered into the category (combined) SCLC. In addition, separate κ values were calculated for combining the categories typical carcinoid and atypical carcinoid into (a)typical carcinoid. For the individual categories, the number of cases with an assigned diagnosis for that category is shown for the corresponding level.
      a In round 1 for levels 2 and 3, only the option (combined) SCLC was available; therefore, both categories were calculated together.

      Levels 2 and 3: IHC Stains

      The available IHC stains are shown in Table 3. The most frequently requested stains were the NE markers (synaptophysin [∼80%], chromogranin A [∼66%], CD56 [∼60%], TTF1 [∼53%], and MIB1 [Ki67] [∼75%]). CK and p63/40 were requested at lower rates (∼31%). The other staining options (CD45, CD3, CD20, CD99, and vimentin) were requested occasionally (<9%).
      Table 3Cases with Stain Available
      Cases with StainIHC Staining
      CD56 (n = 44)Chrom (n = 55)Synapt (n = 70)Ck (n = 22)MIB1 (n = 77)TTF1 (n = 44)
      1 category32 (73%)28 (51%)50 (71%)11 (50%)33 (43%)24 (55%)
      2 categories8 (18%)25 (45%)18 (26%)7 (32%)27 (35%)16 (36%)
      ≥3 categories4 (9%)2 (4%)2 (3%)4 (18%)17 (22%)4 (9%)
      Note: The number of cases in which this stain was available is shown for the major stains, as is the distribution of the immunohistochemistry scores in terms of absolute number and percentages of the individual stains. Note that the distribution in one category implies a unanimous score.
      IHC, immunohistochemistry; Chrom, chromogranin A; Synapt, synaptophysin; CK, cytokeratin; MIB1, Ki67; TTF1, thyroid transcription factor 1.
      To examine whether adding additional IHC stains improved the diagnostic process, the results of using additional stains in level 2 were compared with those of level 1 for all cases. A significant (p < 0.05) increase in the diagnostic score was reached in 55 of the 79 cases (70%). In level 3, when all available stains were provided, the percentage of the highest diagnostic score increased significantly in comparison with that in level 2 in eight of the 79 cases (10%), with two of the eight cases also showing a significant increase in level 2 compared with in level 1.
      To evaluate the confidence of the pathologists, only cases in which the main diagnosis remained the same throughout the two consecutive readings were examined. The use of additional IHC stains in level 2 compared with in level 1 significantly (p < 0.05) increased the percentage of the highest diagnostic score in 48 of the cases (61%). In level 3 compared with in level 2, the percentage of the highest diagnostic score significantly increased in five cases (6%).
      To examine the reproducibility of the interpretation of the IHC stains, the distribution of the IHC scores for the individual stains was established. The distribution of the major IHC scores over one, two, or more categories is shown in Table 3. In the case of the NE markers, staining for both CD56 and synaptophysin was frequently scored with complete agreement, but with chromogranin A, a dotlike staining pattern led to a greater variation in scoring. The distribution over three or more categories was highest (∼20%) for MIB1 and CK.

      Differential Diagnostic Considerations

      In level 1, without additional stains, many pathologists included the “final” diagnosis rendered in level 3 as a part of the possible diagnoses (the scored diagnostic categories). A clear exception was a case of sarcoma included in the biopsy specimens, in which the main diagnosis evolved after evaluation of the requested stains in level 2 or the provided stains in level 3, resulting in a high κ score (0.84).
      A unanimous result in level 3 was present in 55 of the 79 cases (63%), including 20 of the 55 cases diagnosed as SCLC (or combined SCLC [divided over four resection and 16 biopsy specimens]) (see Fig. 1), 10 diagnosed as LCNEC (seven resection samples [see Fig. 2] and three biopsy specimens), 17 diagnosed as TC (11 resection and six biopsy specimens), two diagnosed as AC (both resection specimens), two diagnosed as NSCLC (both biopsy specimens [see Fig. 3]), one diagnosed as sarcoma (a biopsy specimen), and three diagnosed as lymphoma (two resection specimens and one biopsy specimen).
      Figure thumbnail gr1
      Figure 1(A and B) Example of biopsy specimen with uniform diagnosis of SCLC in level 3 (hematoxylin and eosin stain; original magnification, ×10). (C) CD56 immunohistochemistry (IHC) staining with abundant membranous pattern (original magnification, ×40). (D) Chromogranin A IHC staining with dotlike cytoplasmic pattern (original magnification, ×40). (E) Synaptophysin IHC staining with diffuse cytoplasmic pattern (original magnification, ×40). (F) Strong MIB1 IHC staining in most tumor cell nuclei (original magnification, ×40).
      Figure thumbnail gr2
      Figure 2Example of resection specimen with high agreement regarding diagnosis of LCNEC (level 3: 16 of 19 diagnoses were LCNEC and three of 19 were SCLC). (A and B) Hematoxylin and eosin stain (original magnification, ×40). (C) Synaptophysin IHC stain (original magnification, ×40). (D) MIB1 IHC with nuclear staining in most of the neoplastic nuclei (original magnification, ×40).
      Figure thumbnail gr3
      Figure 3Example of biopsy specimen with uniform diagnosis of NSCLC in level 3. (A and B) Hematoxylin and eosin stain (original magnification, ×40). (C) CD56 IHC showing no staining (original magnification, ×40). (D) p40 IHC with abundant staining in most tumor cell nuclei (D, ×40).
      In the remaining 24 cases the differential diagnosis was as follows: between SCLC and carcinoid (TC and AC) in three cases (two resection specimens and one biopsy specimen [see Fig. 4]); between SCLC and LCNEC in seven cases (four resection specimens and three biopsy specimens); between TC and AC in eight cases (seven resection specimens and one biopsy specimen); between SCLC and NSCLC in two cases (one resection specimen and one biopsy specimen); between SCLC, LCNEC, and carcinoid (TC and AC) in one case (a resection specimen); and between LCNEC and AC in one case (a resection specimen). In two cases, not only was the unanimous threshold not reached, but none of the differential diagnostic categories was scored by at least five pathologists.
      Figure thumbnail gr4
      Figure 4Example of biopsy specimen with differential diagnostic scores (level 3: eight of 19 diagnoses were SCLC, eight of 19 were atypical carcinoid, and three of 19 were LCNEC). (A and B) Routine hematoxylin and eosin stain (original magnification, ×40). (C) CD56 IHC showing distinct membranous staining (original magnification, ×40). (D) Chromogranin A IHC with abundant cytoplasmic staining (original magnification, ×40). (E) Synaptophysin IHC with diffuse cytoplasmic staining (original magnification, ×40). (F) MIB1 IHC with nuclear staining in minority of tumor cell nuclei (original magnification, ×40). Note that low MIB1 favors atypical carcinoid.

      Discussion

      This study demonstrates that in a selected set of difficult biopsy and resection specimens encompassing the differential diagnosis of SCLC, the reproducibility of histopathologic diagnosis markedly improves with the use of IHC.
      The κ scores for diagnoses based on HE staining only are comparable with previous data.
      • Ha S.Y.
      • Han J.
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      • Suh B.S.
      • Roh M.S.
      Interobserver variability in diagnosing high-grade neuroendocrine carcinoma of the lung and comparing it with the morphometric analysis.
      • den Bakker M.A.
      • Willemsen S.
      • Grünberg K.
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      Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability.
      The use of IHC led to an increase of approximately 0.2 in the κ score, which is impressive when the following factors are taken into account: (1) the lack of clinical information, (2) the 19 observers from various parts of the world, (3) the classification in up to nine categories, (4) the selected study set with great diagnostic difficulty, and (5) the use of digital images as opposed to glass slides. Therefore, this study provides a strong argument for use of IHC in the differential diagnosis of SCLC.
      The κ score on level 1 (HE staining only) reflects classification on the basis of a combination of architectural and cytologic features. The architectural features of NE morphology comprise rosettes, organoid nesting, formation of pseudorosettes, trabeculae, and palisading.
      • Travis W.
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      • Marx A.
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      WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart.
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      Neuroendocrine neoplasms of the lung: concepts and terminology.
      Cytologic criteria are used to split SCLC from LCNEC. However, several studies have shown morphologic overlap between these criteria, making this separation quite subjective and difficult to perform, especially in the case of small biopsy specimens with crush artifacts. Morphometric studies demonstrated considerable nuclear size overlap in high-grade pulmonary NE neoplasms
      • Ha S.Y.
      • Han J.
      • Kim W.-S.
      • Suh B.S.
      • Roh M.S.
      Interobserver variability in diagnosing high-grade neuroendocrine carcinoma of the lung and comparing it with the morphometric analysis.
      • Marchevsky A.M.
      • Gal A.A.
      • Shah S.
      • Koss M.N.
      Morphometry confirms the presence of considerable nuclear size overlap between “small cells” and “large cells” in high-grade pulmonary neuroendocrine neoplasms.
      and between SCLC and NSCLC.
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      Further evaluation of quantitative nuclear image features for classification of lung carcinomas.
      The rule that a tumor cell–to–lymphocyte size ratio greater than 3 helps to distinguish “large” from “small” neoplastic cells
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      holds true in only a minority of cases.
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      Morphometry confirms the presence of considerable nuclear size overlap between “small cells” and “large cells” in high-grade pulmonary neuroendocrine neoplasms.
      Moreover, an unsolved methodologic issue is that nuclear size in biopsy specimens of SCLC seems to be smaller than in resection specimens.
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      The effect of cell size on the pathologic diagnosis of small and large cell carcinomas of the lung.
      Overall, a small nuclear size may point to SCLC, but a larger nuclear size is not a reliable feature to distinguish SCLC from LCNEC and other diagnoses.
      Chromatin pattern analysis may aid in distinguishing SCLC from NSCLC.
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      Thus, the classic NE nuclear chromatin pattern of “salt and pepper” chromatin supports a diagnosis of SCLC or carcinoid rather than non–small cell carcinoma or LCNEC, but overlap remains. Some cases may have many nuclei with salt and pepper chromatin and a few nuclei with vesicular chromatin and prominent nucleoli or vice versa.
      The recently published WHO classification indicates on the one hand that the diagnosis of SCLC can be made reliably on the basis of routine histologic and cytologic preparations,
      • Travis W.
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      • Nicholson A.G.
      WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart.
      implying that the use of IHC stains may not be required. However, it further states that IHC may be needed in some cases for confirmation of the epithelial nature and NE phenotype of the tumor cells, especially in the case of crushed and small biopsy specimens. Furthermore, the proliferation activity of SCLC as assessed by Ki-67 antigen immunostaining can be performed to help avoid misdiagnosing carcinoid tumors in the presence of crush artifact.
      For pathologists, IHC may therefore be useful in the process of diagnosing SCLC (1) in cases in which histologic features are equivocal and IHC stains may help in the differential diagnosis and (2) in cases in which the pathologist favors a particular diagnosis but wants to increase his or her confidence in that diagnosis. For example, some observers feel uncomfortable in rendering a diagnosis of SCLC without confirmation that the lesion in question is, at minimum, a carcinoma.
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      In our study the significant increase in the percentage of the diagnostic score in 70% or more of the cases with use of IHC in the second level demonstrates a gain in diagnostic confidence by the pathologist, although it should be noted that the diagnostic difficulty of the specimens in the study set may indicate a level of bias when compared with standard cases.
      A strategy for IHC in the diagnosis of SCLC could therefore contain a two-tiered approach, as illustrated in Table 4.
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      • Papaxoinis G.
      • Mansoor W.
      Diagnostic utility of orthopedia homeobox (OTP) in pulmonary carcinoid tumors.
      • Swarts D.R.A.
      • Henfling M.E.R.
      • Van Neste L.
      • et al.
      CD44 and OTP are strong prognostic markers for pulmonary carcinoids.
      • Oliveira A.M.
      • Tazelaar H.D.
      • Myers J.L.
      • Erickson L.A.
      • Lloyd R V
      Thyroid transcription factor-1 distinguishes metastatic pulmonary from well-differentiated neuroendocrine tumors of other sites.
      • Epstein J.I.
      • Amin M.B.
      • Beltran H.
      • et al.
      Proposed morphologic classification of prostate cancer with neuroendocrine differentiation.
      • Saqi A.
      • Alexis D.
      • Remotti F.
      • Bhagat G.
      Usefulness of CDX2 and TTF-1 in differentiating gastrointestinal from pulmonary carcinoids.
      • Rekhtman J.
      • Pietanza M.
      • Hellmann M.
      • et al.
      Next-generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma–like and non–small cell carcinoma–like subsets.
      First, a pro-SCLC panel consisting of one or more of the following may be used: CD56, low-molecular-weight CK (e.g., CK 7, CK8, or CK18), TTF1, MIB1, and perhaps p16.
      Table 4IHC Stains in Differential Diagnosis of SCLC
      DifferentialIHCSCLCOther DiagnosisReferences
      Pro-SCLCCytokeratin (types 8,18,7)Dotlike cytoplasmic staining in SCLCDiffuse cytoplasmic staining does not exclude SCLC but is also found in LCNECs and other SqCCs, crushed carcinoids, metastatic carcinoma
      • Marchevsky A.M.
      • Wick M.R.
      Diagnostic difficulties with the diagnosis of small cell carcinoma of the lung.
      ,
      • Nagashio R.
      • Sato Y.
      • Matsumoto T.
      • et al.
      Significant high expression of cytokeratins 7, 8, 18, 19 in pulmonary large cell neuroendocrine carcinomas, compared to small cell lung carcinomas.
      ,
      • Sun L.
      • Sakurai S.
      • Sano T.
      • Hironaka M.
      • Kawashima O.
      • Nakajima T.
      High-grade neuroendocrine carcinoma of the lung: comparative clinicopathological study of large cell neuroendocrine carcinoma and small cell lung carcinoma.
      ,
      • Nitadori J.
      • Ishii G.
      • Tsuta K.
      • et al.
      Immunohistochemical differential diagnosis between large cell neuroendocrine carcinoma and small cell carcinoma by tissue microarray analysis with a large antibody panel.
      MIB1/Ki6750% of the nuclei are positive>50% also in LCNEC, e.g. metastases CRC; <25% in carcinoid
      • Pelosi G.
      • Rindi G.
      • Travis W.D.
      • Papotti M.
      Ki-67 antigen in lung neuroendocrine tumors. Unraveling a role in clinical practice.
      ,
      • Skov B.G.
      • 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.
      ,
      • Zheng G.
      • Ettinger D.S.
      • 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.
      ,
      • Pelosi G.
      • Fabbri A.
      • Cossa M.
      • Sonzogni A.
      • Valeri B.
      • Righi L.
      What clinicians are asking pathologists when dealing with lung neuroendocrine neoplasms?.
      TTF1Positive in 90% of SCLCs
      TFF1 may also be positive in thyroid cancer39 and SCLC of the bladder.23,40 Gastrointestinal neuroendocrine tumors do not stain with TTF1.39,41 Merkel cell tumor is TTF1 negative and CK7 negative but positive for CK20, typically with a perinuclear dotlike expression.24
      Lack of TTF1 may be present in LCNEC and in many carcinoids
      • Du E.
      TTF-1 expression is specific for lung primary in typical and atypical carcinoids: TTF-1-positive carcinoids are predominantly in peripheral location.
      ,
      • Carlson J.W.
      • Nucci M.R.
      • Brodsky J.
      • Crum C.P.
      • Hirsch M.S.
      Biomarker-assisted diagnosis of ovarian, cervical and pulmonary small cell carcinomas: the role of TTF-1, WT-1 and HPV analysis.
      ,
      • Sidiropoulos M.
      • Hanna W.
      • Raphael S.J.
      • Ghorab Z.
      Expression of TdT in Merkel cell carcinoma and small cell lung carcinoma.
      ,
      • Zhang H.
      • Liu J.
      • Cagle P.T.
      • Allen T.C.
      • Laga A.C.
      • Zander D.S.
      Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach.
      ,
      • Wu M.
      • Wang B.
      • Gil J.
      • et al.
      p63 and TTF-1 immunostaining. A useful marker panel for distinguishing small cell carcinoma of lung from poorly differentiated squamous cell carcinoma of lung.
      CD56Membranous abundant staining in 98%Lack of membranous staining fits with many other Dx (e.g LCNEC)
      • Zheng G.
      • Ettinger D.S.
      • 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.
      ,
      • Kontogianni K.
      • Nicholson A.G.
      • Butcher D.
      • Sheppard M.N.
      CD56: a useful tool for the diagnosis of small cell lung carcinomas on biopsies with extensive crush artefact.
      Chromogranin AFocal positivity in occasional tumor cell, sensitivity 46%–90%If strong diffuse staining, consider carcinoid; if focal or diffuse staining, few NSCLCs, nonepithelial tumors; negative staining fits with many other Dx
      • Zheng G.
      • Ettinger D.S.
      • 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.
      ,
      • Sidiropoulos M.
      • Hanna W.
      • Raphael S.J.
      • Ghorab Z.
      Expression of TdT in Merkel cell carcinoma and small cell lung carcinoma.
      ,
      • Kaemmerer D.
      • Specht E.
      • Sänger J.
      • et al.
      Somatostatin receptors in bronchopulmonary neuroendocrine neoplasms: new diagnostic, prognostic, and therapeutic markers.
      SynaptophysinFocal or diffuse staining, sensitivity 100%Focal or diffuse staining, few NSCLC, nonepithelial tumors; negative staining fits with many other Dx
      • Zheng G.
      • Ettinger D.S.
      • 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.
      ,
      • Sidiropoulos M.
      • Hanna W.
      • Raphael S.J.
      • Ghorab Z.
      Expression of TdT in Merkel cell carcinoma and small cell lung carcinoma.
      p16Nuclear staining 95%–100%Nuclear staining in LCNEC in 95%.

      Lack of p16 positivity fits with many other Dx (e.g, NSCLC [also with NE staining])
      • Sun L.
      • Sakurai S.
      • Sano T.
      • Hironaka M.
      • Kawashima O.
      • Nakajima T.
      High-grade neuroendocrine carcinoma of the lung: comparative clinicopathological study of large cell neuroendocrine carcinoma and small cell lung carcinoma.
      ,
      • Carlson J.W.
      • Nucci M.R.
      • Brodsky J.
      • Crum C.P.
      • Hirsch M.S.
      Biomarker-assisted diagnosis of ovarian, cervical and pulmonary small cell carcinomas: the role of TTF-1, WT-1 and HPV analysis.
      ,
      • Zhang H.
      • Liu J.
      • Cagle P.T.
      • Allen T.C.
      • Laga A.C.
      • Zander D.S.
      Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach.
      ,
      • Hiroshima K.
      • Iyoda A.
      • Shida T.
      • et al.
      Distinction of pulmonary large cell neuroendocrine carcinoma from small cell lung carcinoma: a morphological, immunohistochemical, and molecular analysis.
      ,
      • Leversha M.A.
      • Fielding P.
      • Watson S.
      • Gosney J.R.
      • Field J.K.
      Expression of p53, pRB, and p16 in lung tumours: a validation study on tissue microarrays.
      ,
      • Yuan J.
      • Knorr J.
      • Altmannsberger M.
      • et al.
      Expression of p16 and lack of pRB in primary small cell lung cancer.
      ,
      • Peng W.-X.
      • Sano T.
      • Oyama T.
      • Kawashima O.
      • Nakajima T.
      Large cell neuroendocrine carcinoma of the lung: a comparison with large cell carcinoma with neuroendocrine morphology and small cell carcinoma.
      ,
      • Dosaka-Akita H.
      • Cagle P.T.
      • Hiroumi H.
      • et al.
      Differential retinoblastoma and p16(INK4A) protein expression in neuroendocrine tumors of the lung.
      pRbNegative in SCLCLCNEC positive or negative, AC positive
      • Leversha M.A.
      • Fielding P.
      • Watson S.
      • Gosney J.R.
      • Field J.K.
      Expression of p53, pRB, and p16 in lung tumours: a validation study on tissue microarrays.
      ,
      • Yuan J.
      • Knorr J.
      • Altmannsberger M.
      • et al.
      Expression of p16 and lack of pRB in primary small cell lung cancer.
      ,
      • Dosaka-Akita H.
      • Cagle P.T.
      • Hiroumi H.
      • et al.
      Differential retinoblastoma and p16(INK4A) protein expression in neuroendocrine tumors of the lung.
      Differential diagnostic stains depending of cytokeratin
      Negative cytokeratin stain result
      VimentinNegative in SCLCPositivity supports Dx other than carcinoma but also sarcomatoid carcinoma
      • Pelosi G.
      • Melotti F.
      • Cavazza A.
      • et al.
      A modified vimentin histological score helps recognize pulmonary sarcomatoid carcinoma in small biopsy samples.
      CD45Negative in SCLCPositivity supports non-Hodgkin's lymphoma
      Some plasmacytoid lymphomas may have dot-like cytokeratin positivity.
      CD99Negative in SCLCPositivity supports Ewing sarcoma
      S100Negative in SCLCPositivity supports melanoma
      Positive cytokeratin result
      p40Negative in SCLCPositivity supports
      Focal p40 positivity in a minority of tumor cells is reported on few SCLC-like LCNECs with concurrent KEAP1-NFE2L2, likely underling a putative squamous cell differentiation.42
      (basaloid) SqCC, metastases of SqCC
      ER, PRNegative in SCLCPositivity supports metastatic breast carcinoma
      PSANegative in SCLCPositivity supports metastatic prostate carcinoma
      p63Occasionally focally positive in SCLCFocal positivity may be present in LCNEC, carcinoid; positivity in >80% of nuclei favors SqCC.
      • Zhang H.
      • Liu J.
      • Cagle P.T.
      • Allen T.C.
      • Laga A.C.
      • Zander D.S.
      Distinction of pulmonary small cell carcinoma from poorly differentiated squamous cell carcinoma: an immunohistochemical approach.
      ,
      • Wu M.
      • Wang B.
      • Gil J.
      • et al.
      p63 and TTF-1 immunostaining. A useful marker panel for distinguishing small cell carcinoma of lung from poorly differentiated squamous cell carcinoma of lung.
      ,
      • Au N.H.
      • Gown A.M.
      • Cheang M.
      • et al.
      P63 expression in lung carcinoma: a tissue microarray study of 408 cases.
      ,
      • Thunnissen E.
      • Boers E.
      • Heideman D.A.
      • et al.
      Correlation of immunohistochemical staining p63 and TTF-1 with EGFR and K-ras mutational spectrum and diagnostic reproducibility in non small cell lung carcinoma.
      OTPNegative in SCLCPositive in typical carcinoid
      • Nonaka D.
      • Papaxoinis G.
      • Mansoor W.
      Diagnostic utility of orthopedia homeobox (OTP) in pulmonary carcinoid tumors.
      ,
      • Swarts D.R.A.
      • Henfling M.E.R.
      • Van Neste L.
      • et al.
      CD44 and OTP are strong prognostic markers for pulmonary carcinoids.
      Note: The various IHC stains in the differential diagnosis of SCLC are shown divided into pro-SCLC and other diagnosis. These stains can be used in equivocal cases or when the pathologist wants to gain more confidence for a diagnosis. If the pro-SCLC panel does not fit with SCLC, additional stains may be used depending on the outcome of cytokeratin stain. Clinical history may also be of value (e.g., in cases of other known primary tumor).
      IHC, immunohistochemistry; LCNEC, large cell neuroendocrine carcinoma; SqCC, squamous cell carcinoma; CRC, colorectal carcinoma; TTF1, thyroid transcription factor 1; Dx, diagnoses; NE, neuroendocrine; AC, atypical carcinoid; ER, estrogen receptor; PR, progesteron receptor; PSA, prostate-specific antigen; OTP, orthopedia homeobox.
      a TFF1 may also be positive in thyroid cancer
      • Oliveira A.M.
      • Tazelaar H.D.
      • Myers J.L.
      • Erickson L.A.
      • Lloyd R V
      Thyroid transcription factor-1 distinguishes metastatic pulmonary from well-differentiated neuroendocrine tumors of other sites.
      and SCLC of the bladder.
      • Carlson J.W.
      • Nucci M.R.
      • Brodsky J.
      • Crum C.P.
      • Hirsch M.S.
      Biomarker-assisted diagnosis of ovarian, cervical and pulmonary small cell carcinomas: the role of TTF-1, WT-1 and HPV analysis.
      • Epstein J.I.
      • Amin M.B.
      • Beltran H.
      • et al.
      Proposed morphologic classification of prostate cancer with neuroendocrine differentiation.
      Gastrointestinal neuroendocrine tumors do not stain with TTF1.
      • Oliveira A.M.
      • Tazelaar H.D.
      • Myers J.L.
      • Erickson L.A.
      • Lloyd R V
      Thyroid transcription factor-1 distinguishes metastatic pulmonary from well-differentiated neuroendocrine tumors of other sites.
      • Saqi A.
      • Alexis D.
      • Remotti F.
      • Bhagat G.
      Usefulness of CDX2 and TTF-1 in differentiating gastrointestinal from pulmonary carcinoids.
      Merkel cell tumor is TTF1 negative and CK7 negative but positive for CK20, typically with a perinuclear dotlike expression.
      • Sidiropoulos M.
      • Hanna W.
      • Raphael S.J.
      • Ghorab Z.
      Expression of TdT in Merkel cell carcinoma and small cell lung carcinoma.
      b Focal p40 positivity in a minority of tumor cells is reported on few SCLC-like LCNECs with concurrent KEAP1-NFE2L2, likely underling a putative squamous cell differentiation.
      • Rekhtman J.
      • Pietanza M.
      • Hellmann M.
      • et al.
      Next-generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma–like and non–small cell carcinoma–like subsets.
      c Some plasmacytoid lymphomas may have dot-like cytokeratin positivity.
      CK8, CK18, CK7, and CK19 may show dotlike staining, which is characteristic of SCLC and associated with a low score with H-score evaluation.
      • Marchevsky A.M.
      • Wick M.R.
      Diagnostic difficulties with the diagnosis of small cell carcinoma of the lung.
      • Nagashio R.
      • Sato Y.
      • Matsumoto T.
      • et al.
      Significant high expression of cytokeratins 7, 8, 18, 19 in pulmonary large cell neuroendocrine carcinomas, compared to small cell lung carcinomas.
      • Sun L.
      • Sakurai S.
      • Sano T.
      • Hironaka M.
      • Kawashima O.
      • Nakajima T.
      High-grade neuroendocrine carcinoma of the lung: comparative clinicopathological study of large cell neuroendocrine carcinoma and small cell lung carcinoma.
      • Nitadori J.
      • Ishii G.
      • Tsuta K.
      • et al.
      Immunohistochemical differential diagnosis between large cell neuroendocrine carcinoma and small cell carcinoma by tissue microarray analysis with a large antibody panel.
      CD56 is useful with crushed biopsy specimens in refining a differential diagnosis: in SCLC strong membranous positivity is almost always present.
      • Kontogianni K.
      • Nicholson A.G.
      • Butcher D.
      • Sheppard M.N.
      CD56: a useful tool for the diagnosis of small cell lung carcinomas on biopsies with extensive crush artefact.
      However, although CD56 is more sensitive in the context of SCLC than other NE markers, it is also positive in the case of many other tumor types and thus should not be viewed as specific.
      Estimating the mitotic index by simple examination of HE-stained sections microscopically is not always easy. Mitoses are very difficult (if not impossible) to count, especially in SCLC. Recent studies in lung cancer favor the use of MIB1/Ki67.
      • Rindi G.
      • Klersy C.
      • Inzani F.
      • et al.
      Grading the neuroendocrine tumors of the lung: an evidence-based proposal.
      In evaluating the proliferation rate with IHC, agreement within one laboratory seems feasible. However, agreement among different laboratories, at least in breast cancer, reveals insufficient analytical validity for application in daily practice.
      • Polley M.-Y.C.
      • Leung S.C.Y.
      • McShane L.M.
      • et al.
      An international Ki67 reproducibility study.
      • Polley M.-Y.C.
      • Leung S.C.Y.
      • Gao D.
      • et al.
      An international study to increase concordance in Ki67 scoring.
      In SCLC and LCNEC, the vast majority of tumor cells show high nuclear reactivity with MIB1/Ki67 in SCLC.
      • Rindi G.
      • Klersy C.
      • Inzani F.
      • et al.
      Grading the neuroendocrine tumors of the lung: an evidence-based proposal.
      In contrast, a low proliferation rate in this context points toward a carcinoid tumor.
      • Pelosi G.
      • Rodriguez J.
      • Viale G.
      • Rosai J.
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens.
      • Rindi G.
      • Klersy C.
      • Inzani F.
      • et al.
      Grading the neuroendocrine tumors of the lung: an evidence-based proposal.
      In our study it is likely that mitotic counting in the first round on the web system may have caused some variation in diagnosis, especially between TC and AC. Thus currently, Ki67 has great utility for discrimination of SCLC/LCNEC from carcinoids, but for discrimination between TCs and ACs more studies are needed on how to classify borderline cases
      • Pelosi G.
      • Rindi G.
      • Travis W.D.
      • Papotti M.
      Ki-67 antigen in lung neuroendocrine tumors. Unraveling a role in clinical practice.
      (i.e., those cases lacking conclusive mitotic indices) with intermediate proliferation indices (25%–50%) by MIB1/Ki67 staining (see Table 4 and Fig. 5).
      Figure thumbnail gr5
      Figure 5Diagnostic flow diagram integrating morphologic and immunohistochemical (IHC) stains for the diagnosis (Dx) of neuroendocrine (NE) lung cancer. Note that if the flow diagram had been available during this study, the case in might have been favored to be a carcinoid on the basis of limited proliferative activity. (1) Fields are characteristic of carcinoma and cytokeratin positive. In tumors with diffuse growth, in addition to undifferentiated carcinoma (dissociation) and sarcomatoid carcinoma, also consider sarcoma and lymphoma. (2) The number of mitoses in 2 mm2. (3) If using a biopsy specimen, think of crushed carcinoid, especially with small regular nuclei with unclear chromatin, and slightly too much cytoplasm for SCLC. In addition NE IHC may be ++. (4) Dense chromatin equates to salt and pepper chromatin. (5) NE architecture may be focally present in whole tumor but usually absent in small biopsy specimens (especially bronchoscopic biopsy specimens), which is the reason why the WHO states that a Dx of large cell neuroendocrine carcinoma (LCNEC) is to be made only on the basis of a resection specimen. If the biopsy specimen fits with a Dx of LCNEC, suggest LCNEC as a possible Dx, remarking that this is difficult to establish on the basis of very small biopsy specimens. (6) Chromogranin A/synaptophysin staining is diffusely positive in carcinoid tumors, but chromogranin A is usually present only focally in SCLC/LCNEC; strong membranous CD56 staining is present in SCLC. (7) In the WHO classification of lung cancer, it is stated more or less arbitrarily that an atypical carcinoid (Atyp C) morphologic pattern with more than 10 mitoses per 2 mm2 should be called LCNEC. (8) Necrosis in carcinoids is usually dotlike (punctate). More than dotlike necrosis is usually seen in SCLC, LCNEC, and NSCLC. (9) In Atyp C, there are many apoptotic figures in contrast to the low number of mitoses. (10) NE morphologic features include rosettes, organoid nesting, formation of pseudorosettes, trabeculae, and palisading. (11) In NSCLC further analysis (FA) of architecture/IHC (e.g., TTF1, mucin), if TTF1 and mucin staining result is negative, consider undifferentiated large cell (null phenotype). (12) In FA, think of lymphoma, sarcoma, melanoma, metastasis breast, and other. (13) If discernible, there are many mitoses; in any case, there are many apoptotic figures. (14) In the case of a resection specimen, nonkeratinizing squamous cell carcinoma (SqCC); in the case of biopsy specimens, NSCLC favor nonkeratinizing SqCC; and on the basis of “in-between” cytoplasm, think of basaloid carcinoma. (15) The mitoses threshold is 25 according to Pelosi et al.
      • Pelosi G.
      • Fabbri A.
      • Cossa M.
      • Sonzogni A.
      • Valeri B.
      • Righi L.
      What clinicians are asking pathologists when dealing with lung neuroendocrine neoplasms?.
      Ty C, typical carcinoma; NEC, neuroendocrine carcinoma. Diamond implies overlap in Dx.
      The biology and molecular biology of SCLC has recently been reviewed.
      • Bunn P.A.
      • Minna J.D.
      • Augustyn A.
      • Gazdar A.F.
      • et al.
      Small cell lung cancer: can recent advances in biology and molecular biology be translated into improved outcomes?.
      Reciprocal retinoblastoma suppressor protein inactivation and p16 expression/overexpression is present,
      • Hiroshima K.
      • Iyoda A.
      • Shida T.
      • et al.
      Distinction of pulmonary large cell neuroendocrine carcinoma from small cell lung carcinoma: a morphological, immunohistochemical, and molecular analysis.
      • Leversha M.A.
      • Fielding P.
      • Watson S.
      • Gosney J.R.
      • Field J.K.
      Expression of p53, pRB, and p16 in lung tumours: a validation study on tissue microarrays.
      • Yuan J.
      • Knorr J.
      • Altmannsberger M.
      • et al.
      Expression of p16 and lack of pRB in primary small cell lung cancer.
      • Shapiro G.
      • Edwards C.
      • Kobzik L.
      Reciprocal Rb inactivation and p16 INK4 expression in primary lung cancers and cell lines.
      probably because of a negative feedback loop thought to be futile in SCLC.
      • Shapiro G.
      • Edwards C.
      • Kobzik L.
      Reciprocal Rb inactivation and p16 INK4 expression in primary lung cancers and cell lines.
      • Serrano M.
      • Hannon G.J.
      • Beach D.
      A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4.
      In contrast, in NSCLC, retinoblastoma suppressor protein is frequently expressed and p16 deregulated, leading to loss of p16 protein expression.
      • Shapiro G.
      • Edwards C.
      • Kobzik L.
      Reciprocal Rb inactivation and p16 INK4 expression in primary lung cancers and cell lines.
      • Sakaguchi M.
      • Fujii Y.
      • Hirabayashi H.
      • et al.
      Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: an immunohistochemical study.
      • Geradts J.
      • Fong K.M.
      • Zimmerman P.V.
      • Maynard R.
      • Minna J.D.
      Correlation of abnormal RB, p16 ink4a, and p53 expression with 3p loss of heterozygosity, other genetic abnormalities, and clinical features in 103 primary non-small cell lung cancers correlation of abnormal RB, p16 ink4a, and p53 expression with 3p.
      This suggests that in classic SCLC p16 will be strongly positive and possibly not in the LCNEC NSCLC–related and NSCLC cases with frequent loss of p16 expression. However, as for this and several IHC markers that were not included in this project (such as BAI3, CDX2, and VIL1
      • Bari M.F.
      • Brown H.
      • Nicholson A.G.
      • et al.
      BAI3, CDX2 and VIL1: a panel of three antibodies to distinguish small cell from large cell neuroendocrine lung carcinomas.
      ; STK11
      • Rekhtman J.
      • Pietanza M.
      • Hellmann M.
      • et al.
      Next-generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma–like and non–small cell carcinoma–like subsets.
      ; and other NE markers
      • Kaemmerer D.
      • Specht E.
      • Sänger J.
      • et al.
      Somatostatin receptors in bronchopulmonary neuroendocrine neoplasms: new diagnostic, prognostic, and therapeutic markers.
      • Specht E.
      • Kaemmerer D.
      • Sänger J.
      • Wirtz R.M.
      • Schulz S.
      • Lupp A.
      Comparison of immunoreactive score, HER2/neu score and H score for the immunohistochemical evaluation of somatostatin receptors in bronchopulmonary neuroendocrine neoplasms.
      ), additional studies are warranted before they become established in clinical practice.
      If a pathologist considers the use of IHC for the diagnosis of SCLC, the questions of how many and which IHC markers to use are open for debate. For example, in a case favored to be SCLC on the basis of HE staining in which an initial IHC staining pattern emerges that is inconsistent with SCLC (particularly negative staining for CK), several alternative diagnoses may be considered and assessed by using a wider panel that includes vimentin, CD45, CD138, for lymphoma, CD99 for sarcoma, and S100 and Melan A for melanoma, respectively. If CK positivity is more than dotlike, testing with the following markers can be performed: (1) p40/p63 for (basaloid) squamous cell carcinoma (where p40 is more specific), (2) estrogen and progesterone receptor for metastases of (for example) the breast, (3) prostate-specific antigen for metastases of the prostate, and (4) orthopedia homeobox for TCs.
      In relation to concerns regarding the frequency of SCLC being negative for all three commonly used NE markers, a survey of authors revealed that this was a rare finding, even in a referral setting, for 22 of 24 respondents.
      A diagnostic flow diagram integrating morphology and IHC stains for the diagnosis of NE lung tumors is shown in Figure 5. This flow diagram shows three possible morphologic ways to reach the diagnosis of LCNEC: (1) differentiating from SCLC, (2) differentiating from ACs with fewer than 10 mitosis per 2 mm2, and (3) differentiating from NSCLC (in particular adenocarcinoma). Indeed, in this study, several cases with panel overlap diagnoses comprised the first two scenarios. Of note, the flowchart is supported by molecular data in which a cohort of LCNECs consisted of three categories: a large group biologically similar to SCLC and two smaller groups fitting more toward NSCLC and carcinoids.
      • Rekhtman J.
      • Pietanza M.
      • Hellmann M.
      • et al.
      Next-generation sequencing of pulmonary large cell neuroendocrine carcinoma reveals small cell carcinoma–like and non–small cell carcinoma–like subsets.
      • Fernandez-Cuesta L.
      • Heifer M.
      • George G.
      • et al.
      Genomic characterization of large-cell neuroendocrine lung tumors.
      In the process of diagnosis of NE lung carcinomas, application of uniform nomenclature among all physicians is advocated, as otherwise, differences in interpretation between clinicians and pathologists are likely to occur.
      • Derks J.L.
      • Jan van Suylen R.
      • Thunnissen E.
      • et al.
      A population-based analysis of application of WHO nomenclature in pathology reports of pulmonary neuroendocrine tumors.
      SCLC is generally treated with a combination of platinum and etoposide.
      • Bunn P.A.
      • Minna J.D.
      • Augustyn A.
      • Gazdar A.F.
      • et al.
      Small cell lung cancer: can recent advances in biology and molecular biology be translated into improved outcomes?.
      For LCNEC, prospective treatment data are lacking and the amount of retrospective data is currently too small for a confident recommendation.
      • Naidoo J.
      • Santos-Zabala M.L.
      • Iyriboz T.
      • et al.
      Large cell neuroendocrine carcinoma of the lung: clinico-pathologic features, treatment, and outcomes.
      Of note, the U.S. Food and Drug Administration recently approved everolimus for treating inoperable, locally advanced, or metastatic, well-differentiated NE tumors of the lung (TCs and ACs), so such tissue diagnoses are clinically important.
      • Yao J.C.
      • Fazio N.
      • Singh S.
      • et al.
      Everolimus for the treatment of advanced, non-functional neuroendocrine tumours of the lung or gastrointestinal tract (RADIANT-4): a randomised, placebo-controlled, phase 3 study.
      Our study has several limitations. First, the lack of clinical information (i.e,. age, sex, previous history, and radiographic appearances) would likely affect the judgment of the clinical pathologists, as it has been shown that the most accurate diagnosis is obtained when clinical context is available.
      • Thunnissen F.B.
      • Peterse J.L.
      • van Pel R.
      • et al.
      Reliability of fine needle aspiration cytology for distinguishing between carcinoma, lymphoma and sarcoma; the influence of clinical information.
      Second, the IHC procedure was not standardized, nor was a list of available IHC stains provided. In particular, at level 2, because no more than five stains were allowed to be requested, a study participant could have requested a particular NE stain that was not available but did not request a NE stain that was available. Third, in daily practice, in cases with both cytologic and histologic samples, the diagnosis of SCLC may be equivocal on the basis of the biopsy specimen whereas the cytologic specimen may well provide sufficient confidence for a reliable diagnosis of SCLC. Fourth, the process of sampling (i.e., by biopsy and/or cytologic examination) inherently interferes with the reliability of the diagnosis compared with when resection specimens are used. For example, in a small sample there may be a lack of NE morphologic features or only one of the phenotypes in a combined carcinoma may be sampled, which would lead to underdiagnosis. Fifth, this study set had a high selection bias toward difficult cases in the differential diagnosis of SCLC. Therefore, the κ scores are likely to be higher in an unselected series and thus provide a strong argument for the use of IHC in the differential diagnosis of SCLC. Sixth, mitotic counting using the digital slides in round 1 was not performed in a standardized way.
      Our study does not deal with nonepithelial neoplastic lesions with NE differentiation (intrapulmonary paraganglioma, primitive neuroectodermal tumor, and neuroblastoma) nor with NSCLC with occult NE differentiation (with NE features shown only by IHC or ultrastructural studies).
      • Wick M.R.
      • Marchevsky A.M.
      Neuroendocrine neoplasms of the lung: concepts and terminology.
      The latter category is currently not recommended for use in the 2015 WHO classification.
      • Travis W.
      • Brambilla E.
      • Nicholson A.
      Testing for neuroendocrine immunohistochemical markers should not be performed in poorly differentiated NSCCs in the absence of neuroendocrine morphologic features according to the 2015 WHO classification.
      In conclusion, the HE staining–based diagnosis of SCLC or other pulmonary NE tumor is relatively straightforward, but IHC improves diagnostic reproducibility. IHC can aid the pathologist in cases in which histologic features are considered equivocal or in cases in which the pathologist is looking for additional support. An approach for IHC in the differential diagnosis of SCLC is provided and needs further confirmation, preferably linked to treatment outcome.

      Acknowledgments

      Part of this study was funded by the International Association for the Study of Lung Cancer. The support for website application and statistical analysis is greatly appreciated.

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      Linked Article

      • Immunohistochemistry Improves the Diagnosis of Small Cell Lung Cancer; Statistical Issue on Reproducibility Analysis
        Journal of Thoracic OncologyVol. 12Issue 6
        • Preview
          I was interested to read the article by Thunnissen et al. published in the February 2017 issue of the Journal of Thoracic Oncology.1 The purpose of the authors was to examine whether judicious use of immunohistochemistry (IHC) improves diagnostic reproducibility for SCLC.1 Nineteen lung pathologists studied interactive digital images of 79 tumors. Images of resection and biopsy specimens were used to make diagnoses solely on the basis of morphologic features (level 1), on the basis of morphologic features along with requested IHC staining results (level 2), and on the basis of all available IHC staining results (level 3).
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      • Reply to Letter “The Use of Immunohistochemistry Improves the Diagnosis of Small Cell Lung Cancer and Its Differential Diagnosis. An International Reproducibility Study in a Demanding Set of Cases.”
        Journal of Thoracic OncologyVol. 12Issue 6
        • Preview
          The use of (weighted) κ is currently the standard method of reporting, and as such there should not be any controversy.1 The weighted κ is preferred when the data are ordinally categorized, which is not the case in our study, allowing use of the Fleiss κ, which suitable for our data. The limitations of κ statistics in relation to prevalence and number of categories are well known.2 However, we take issue with his statement “They concluded that the diagnosis using hematoxylin and eosin staining alone showed moderate agreement among pathologists in tumors with neuroendocrine morphologic features, but agreement improved to good in most cases with the judicious use of [immunohistochemistry], especially in the diagnosis of SCLC.
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