Originally published as JCO Early Release 10.1200/JCO.2009.24.0903 on August 31 2009

This Article Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Google Scholar Articles by Mansukhani, M. M. Articles by Rosenfeld, N. PubMed PubMed Citation Articles by Mansukhani, M. M. Articles by Rosenfeld, N. Related Articles Related Article Social Bookmarking                            What's this?

Reply to G. Rossi et al

Molecular Pathology Laboratory, Columbia University Medical Center, New York, NY; and Rosetta Genomics Ltd, Rehovot, Israel

We read with interest the response of Rossi et al^1a to our recent article on the use of microRNA expression for subtyping of non–small-cell lung cancer (NSCLC).¹ Without questioning any of our conclusions, they nevertheless state that morphological evaluation and a limited immunohistochemical (IHC) panel can reproduce the results obtained by microRNA profiling, with the advantage of being cheaper, faster, and requiring fewer tumor cells. They also fault us for failing to cite recent reports that, they claim, demonstrate the value of newer IHC markers.

The comments by Rossi et al^1a are well placed, and we do not discount the value of morphology or IHC; in fact, we used morphological examination as the gold standard against which we compared microRNA profiling. Our article did not directly compare the performance of microRNA profiling with that of IHC, but we need to take issue with a number of points made by Rossi et al. They provide three rules for using p63 and thyroid transcription factor-1 to distinguish adenocarcinoma (AC) from squamous cell carcinoma (SCC). However, Ring et al² found that this panel failed to classify 22% of cases in a 551-tumor tissue microarray, and a five-antibody panel failed to classify 11% of tumors. Desmocollin, mentioned by Rossi et al, appears to be a promising marker of squamous differentiation, but the article they refer to focuses on its use in large cell carcinomas.³ Napsin A (another marker mentioned by Rossi et al), although a sensitive marker of lung AC, appears to have been evaluated only for the differentiation of primary lung AC from metastases.⁴ Its value in distinguishing AC from SCC has not been evaluated.

IHC relies, to a large extent (also according to Rossi et al^1a), on the knowledge and experience of the specific pathologist. IHC is affected by preanalytic variables, differences in primary antibodies used to detect individual antigens, and variability in detection methodology, as well as lab-to-lab and even pathologist-to-pathologist variability in interpretive criteria. This lack of standardization can significantly reduce the accuracy of IHC, as underscored by a recent expert panel that reviewed human epidermal growth factor receptor 2 testing in breast cancers, which estimated that 20% of nonstandardized IHC testing may be inaccurate.⁵ The nearly 40% reclassification rate of estrogen and progesterone receptor status of 1,000 retested cases from Newfoundland may be an extreme example, but it serves to highlight these issues.⁶ For NSCLC the situation is more complicated, because the accuracy levels of the different stains themselves are not well defined; different studies report widely varying sensitivity and specificity values for each of the stains.^7–11 It is not clear whether this stems from variation in protocols or skills, biologic and/or epidemiologic differences, or the generally small number of samples in each study.

Classification of NSCLC using IHC stains may be significantly superior to classification using morphology alone, and accumulated practical experience may point toward relative advantages of the use of IHC, yet rigorous studies of the diagnostic reproducibility and accuracy of NSCLC subtyping using IHC are still lacking. Studies of interobserver variation for NSCLC classification based on morphology found rates of concordance of 70% and even lower.^12–17 In a recent large study of diagnostic reproducibility using morphology,¹⁸ only moderate rates of concordance were observed overall. On average, agreement was lower than thresholds generally acceptable for clinical diagnostic tests. The confidence in diagnosis was moderate or high for > 90% of slides, and requests for special stains were infrequent (10%; D. Hayes, personal communication, June 2009). Although this study may not entirely reflect clinical practice, it suggests that many pathologists feel comfortable rendering diagnoses without special stains. A clear use of special stains is not part of the WHO consensus criteria for diagnosis of tumors of the lung,¹⁹ and the extent of use of special stains in clinical practice is unknown. It would be important to find how IHC would fare in similarly rigorous tests of concordance and accuracy. In the absence of studies that assess the performance of IHC in a blinded fashion and, possibly, compare it head-to-head with microRNA expression analysis in a systematic manner, to state that one is equivalent (or superior) to the other is to confuse opinions with conclusions.

In summary, the reliability and accuracy of IHC as a method for subclassification of NSCLC, as well as the dependence on various preanalytic and analytic factors that vary from laboratory to laboratory, have yet to be assessed. Furthermore, the effective precision of this method may depend on the skill and experience of the practicing pathologist. In contrast, a standardized protocol to measure expression level of hsa-miR-205, performed at a qualified laboratory, demonstrated an accuracy of > 90% in subtyping NSCLC, when compared to the consensus diagnosis of two or three expert pathologists,¹ with a high rate of interlaboratory reproducibility. Therefore, although we expect IHC to be a valuable adjunct in the subclassification of NSCLC, the evidence available to date, and the arguments presented by Rossi et al,^1a do not support a conclusion of equivalence (or nonequivalence) of IHC to microRNA profiling. Ultimately, these approaches may become complementary, as they each can serve to improve diagnostic accuracy.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a"U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: Nitzan Rosenfeld, Rosetta Genomics Ltd (C) Consultant or Advisory Role: Mahesh M. Mansukhani, Rosetta Genomics Ltd (C) Stock Ownership: Nitzan Rosenfeld, Rosetta Genomics Ltd Honoraria: Mahesh M. Mansukhani, Rosetta Genomics Ltd Research Funding: Mahesh M. Mansukhani, Rosetta Genomics Ltd Expert Testimony: None Other Remuneration: None

REFERENCES

1a. Rossi G, Papotti M, Barbareschi M, et al: Morphology and a limited number of immunohistochemical markers may efficiently subtype non–small-cell lung cancer. J Clin Oncol 27:e141–e142, 2009.[Free Full Text]

1. Lebanony D, Benjamin H, Gilad S, et al: Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol 27:2030–2037, 2009.[Abstract/Free Full Text]

2. Ring BZ, Seitz RS, Beck RA, et al: A novel five-antibody immunohistochemical test for subclassification of lung carcinoma. Mod Pathol 22:1032–1043, 2009.[CrossRef][Medline]

3. Monica V, Ceppi P, Righi L, et al: Desmocollin-3: A new marker of squamous differentiation in undifferentiated large-cell carcinoma of the lung. Mod Pathol 22:709–717, 2009.[CrossRef][Medline]

4. Suzuki A, Shijubo N, Yamada G, et al: Napsin A is useful to distinguish primary lung adenocarcinoma from adenocarcinomas of other organs. Pathol Res Pract 201:579–586, 2005.[CrossRef][Medline]

5. Wolff AC, Hammond ME, Schwartz JN, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25:118–145, 2007.[Abstract/Free Full Text]

6. Hede K: Breast cancer testing scandal shines spotlight on black box of clinical laboratory testing. J Natl Cancer Inst 100:836–844, 2008.[Free Full Text]

7. Downey P, Cummins R, Moran M, et al: If it's not CK5/6 positive, TTF-1 negative it's not a squamous cell carcinoma of lung. Apmis 116:526–529, 2008.[CrossRef][Medline]

8. Kargi A, Gurel D, Tuna B: The diagnostic value of TTF-1, CK 5/6, and p63 immunostaining in classification of lung carcinomas. Appl Immunohistochem Mol Morphol 15:415–420, 2007.[CrossRef][Medline]

9. Khayyata S, Yun S, Pasha T, et al: Value of P63 and CK5/6 in distinguishing squamous cell carcinoma from adenocarcinoma in lung fine-needle aspiration specimens. Diagn Cytopathol 37:178–183, 2009.[CrossRef][Medline]

10. Pu RT, Pang Y, Michael CW: Utility of WT-1, p63, MOC31, mesothelin, and cytokeratin (K903 and CK5/6) immunostains in differentiating adenocarcinoma, squamous cell carcinoma, and malignant mesothelioma in effusions. Diagn Cytopathol 36:20–25, 2008.[CrossRef][Medline]

11. Rossi G, Marchioni A, Milani M, et al: TTF-1, cytokeratin 7, 34betaE12, and CD56/NCAM immunostaining in the subclassification of large cell carcinomas of the lung. Am J Clin Pathol 122:884–893, 2004.[Abstract/Free Full Text]

12. Brownson RC, Loy TS, Ingram E, et al: Lung cancer in nonsmoking women: Histology and survival patterns. Cancer 75:29–33, 1995.[CrossRef][Medline]

13. Feinstein AR, Gelfman NA, Yesner R: Observer variability in the histopathologic diagnosis of lung cancer. Am Rev Respir Dis 101:671–684, 1970.[Medline]

14. Field RW, Smith BJ, Platz CE, et al: Lung cancer histologic type in the Surveillance, Epidemiology, and End Results registry versus independent review. J Natl Cancer Inst 96:1105–1107, 2004.[Abstract/Free Full Text]

15. Greenberg ER, Korson R, Baker J, et al: Incidence of lung cancer by cell type: A population-based study in New Hampshire and Vermont. J Natl Cancer Inst 72:599–603, 1984.[Medline]

16. Stang A, Pohlabeln H, Muller KM, et al: Diagnostic agreement in the histopathological evaluation of lung cancer tissue in a population-based case-control study. Lung Cancer 52:29–36, 2006.[CrossRef][Medline]

17. Grilley-Olson J, Hayes D, Miller R, et al: Inter-observer reliability for the diagnosis of lung cancer in a clinical cohort using the WHO classification system, version 3. J Clin Oncol 26: 2008 (suppl; abstr 22020.

18. Grilley-Olson J, Hayes D, Qaqish B, et al. Diagnostic reproducibility of squamous cell carcinoma (SC) in the era of histology-directed non-small cell lung cancer (NSCLC) chemotherapy: A large prospective study Annual meeting of the American Society of Clinical Oncology, May 29-June 2, 2009, abstr 8008.

19. Travis WD, Brambilla E, Muller-Hermelink HK, and Harris CC. World Health Organisation Classification of Tumours: Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. Lyon, France: IARC Press, 2004.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

Related Article

• Morphology and a Limited Number of Immunohistochemical Markers May Efficiently Subtype Non–Small-Cell Lung Cancer
  Giulio Rossi, Mauro Papotti, Mattia Barbareschi, Paolo Graziano, and Giuseppe Pelosi
  JCO 2009 27: 141-142 [Full Text]

This Article Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Google Scholar Articles by Mansukhani, M. M. Articles by Rosenfeld, N. PubMed PubMed Citation Articles by Mansukhani, M. M. Articles by Rosenfeld, N. Related Articles Related Article Social Bookmarking                            What's this?