The Daily Sign-Out

SNAI1 functional variant associated with decreased risk for lung cancer

This is an abstract from the January 15, 2014 issue of American Journal of Respiratory and Critical Care Medicino that I just re-discovered this week and relates to one of my ongoing (and unrequited) obsession with epithelial-mesenchymal transition in lung cancer.

It appears that there are five major EMT regulatory genes (SNAI1, SLUG, ZEB1, ZEB2, and TWIST1) involved in EMT.  But--the relative contribution and importance of each of these genes in the development and progression of non-small cell lung cancer is not clear.

 This article adds another layer of complexity and I post it (rather belatedly) now because I find in it such an intriguing finding that a variant protein could have functional consequences that are observable as a clinical outcome with regard to COPD and NSCLC.  Moreover, this variant was discovered to attenuate Snai1’s ability to specifically up-regulate mesenchymal biomarkers (i.e., fibronectin and vimentin) expression, and to promote EMT-like changes, including morphologic changes, cell migration, and invasion.

Major Driver Mutations in Adenosquamous Lung Carcinomas

The June 2014 issue of Journal of Thoracic Oncology (abstract) features a thorough study of major known driver mutations (EGFR, KRAS, ERBB2, BRAF, PIK3CA, AKT1, RET, and ALK) in a series of 76 patients from Fudan University Shanghai Cancer Center with resected adenosquamous lung carcinoma (AdSqLC) by Wang et al. and compared this group with a group of 646 patients with resected adenocarcinoma (ADC) during the same study period.  This is a nifty paper that will serve well as a useful contemporary reference when you next encounter a patient with adenosquamous lung carcinoma.

From their "Table 1" data, it is of note that they found significantly higher smokers in AdSqLC versus ADC.

Known mutant kinases were demonstrated in 57% (43/76) patients.  EGFR mutations were the most common found (32%, 24/76) but were significantly lower compared to the ADC "control" group.  EGFR mutation was significantly higher in never smokers compared to smokers as expected, but it is notable that the frequency of EGFR mutation was higher in glandular dominant AdSqLC than squamous dominant AdSqLC.  Other gene mutations were similar to those found in ADC.  While the frequency of driver mutations was similar between "classical" AdSqLC and poorly-differentiated ADC, the frequency of RET and ALK fusion genes was higher in solid growth glandular component-predominant AdSqLC versus classical AdSqLC.

Microdissection of 13 tumors showed the same mutation in both glandular and squamous components in 9 tumors but 4 of 13 cases showed mutations (KRAS-2, HER2, EGFR) only in the glandular component.

Survival data including 58 AdSqLC and 246 ADC patients showed no significant difference in relapse-free survival between classical AdSqLC, poorly-differentiated ADC, and solid-predominant AdSqLC.  Both classical- and solid-types AdSqLC showed worse overall survival compared to well- and moderately-differentiated ADC.

 

Mutations in Lung Adenosquamous Carcinomas

Journal of Thoracic Oncology just issued a published ahead-of-print article that examined mutations of most frequent driver mutations seen in pulmonary adenocarcinomas in a series of 76 resected adenosquamous carcinomas of the lung.

This is an important study to note because it supports the recommendation by IASLC/ERS/ATS to send these tumors for molecular testing.  Another interesting finding for pathologists is the finding of excess ALK mutations associated with those tumors with a solid pattern histologic subtype in the adenocarcinoma component.

 

Analysis of Major Known Driver Mutations and Prognosis in Resected Adenosquamous Lung Carcinomas

Wang, Rui MD; Pan, Yunjian MD; Li, Chenguang MD,PhD; Zhang, Huibiao MD; Garfield, David MD; Li, Yuan MD; Ye, Ting MD; Hu, Haichuan MD; Luo, Xiaoyang MD; Li, Hang MD; Zhang, Yang MD; Zhang, Jie MD; Zhou, Xiaoyan MD; Shen, Lei MD; Pao, William MD,PhD; Sun, Yihua MD; Chen, Haiquan MD

Published Ahead-of-Print

 

Abstract

 

Introduction: Genotyping for driver mutations is now routinely used to guide clinical care of patients with lung cancer. Adenosquamous lung carcinoma (AdSqLC) is a subtype of cancer that contains both adenocarcinoma and squamous cell carcinoma. However, the incidence, clinicopathologic characteristics, and prognostic implications of major driver mutations in AdSqLCs are not well established.

Methods: Seventy-six resected AdSqLCs and 646 lung adenocarcinomas were screened for known genetic alterations involving EGFR, ERBB2, KRAS, BRAF, PIK3CA, AKT1, RET, and ALK. Tumors showing acinar, lepidic, micropapillary, or papillary growth in glandular component were classified as classical AdSqLC.

Results: Of the 76 AdSqLCs, 43 (56.6%) harbored known mutant kinases, including 24 (31.6%) with EGFR mutations, eight (10.5%) with KRAS mutations, two (2.6%) with AKT1 (2.6%) mutations, one (1.3%) with ERBB2 insertion mutation, one (1.3%) with PIK3CA mutation, four (5.3%) with ALK fusions, and three (4%) with KIF5B-RET fusions. No mutation was found in BRAF. The mutational profiles and clinicopathologic characteristics of classical AdSqLC were strikingly similar to that of poorly differentiated adenocarcinoma. However, AdSqLCs with solid growth pattern in glandular component had high frequency of ALK or RET fusions and low EGFR mutation rate.

Conclusions: To our knowledge, this is the first comprehensive study investigating major oncogenic driver mutations in a large cohort of AdSqLC patients in a Chinese population. The findings suggest that it will be clinically valuable to investigate the growth pattern of glandular component in AdSqLCs.

ETS2 mediates tumor suppressor function and MET inhibition in lung adenocarcinoma

There was a very intriguing paper published online by Clinical Cancer Research on May 9 last week that should be of interest for those involved in lung cancer research.  The authors are Mohamed Kabbout, Melinda Garcia, Junya Fujimoto, et al. from M.D. Ancerson Cancer Center in Houston and the title of the article is "ETS2 mediated tumor suppressive function and MET oncogene inhibition in
human non-small cell lung cancer." 

In brief, the authors find that there is significant downregulation of the ETS2 transcription factor in lung adenocarcinomas compared to normal lung tissues.  ETS2 expression is linked to various canonical cancer-associated pathways in lung cancer cells, in particular the HGF pathway.  Moreover, knockdown of ETS2 is associated with up-regulation of the ZEB1, SNAI2, RAB3B, SERPINB5 and SPDEF genes
with concomitant with elevated cell migration and invasion.  Last, low ETS2 protein is predictive of shorter time-to-recurrence in NSCLC and lung adenocarcinoma.  These data suggest that ETS2 functions as a tumor-suppressor in lung cancer cells by, at least in part, regulation of the migration- and invasion-promoting transcriptome.

More details. . .

Gene expression profiling showed significant decreased expression of ETS2 transcription factor transcripts in lung adenocarcinoma (ADC) compared to normal controls and quantitative RT-PCR confirmed decreased ETS2 expression.  Subset analysis should significantly decreased ETS2 expression in those ADCs from smokers compared to those from never-smokers.

They analyzed ETS2 expression by IHC in a microarray set of 201 NSCLC (135 ADC, 66 squamous cell carcinoma-SQC) tumors.  Low ETS2 expression was associated with shorter time-to-recurrence in stage I and all stages of patients.  Multivariate Cox proportional hazards analysis showed that, after adjusting for stage, low ETS2 expression was an independent predictor of shorter time-to-recurrence in NSCLC and ADC.

H441 human lung cancer cell lines transfected with small interfering RNA (siRNA) specific to ETS2 showed significantly reduced ETS2 transcript compared to controls.  ETS2 knockdown significantly increased cell growth, migration, and invasion.  Conversely, ETS2 overexpression in H1299 lung cancer cells showed significantly decreased cell migration and invasion.  Of interest, the magnitude of these changes following knockdown or overexpression of ETS2 was greater in measures of migration and invasion compared to cell growth--suggesting a tumor suppressor function for ETS2 at least in vitro.

Gene expression profiling of H441 lung cancer cell lines transfected with scrambled siRNA (control) versus ETS2-specific siRNA showed significant differential expression in 1,816 transcripts.  Functional analysis by Ingenuity Pathways Analysis (IPA) showed ETS2 knockdown modulated key oncogenic pathways, including HGF, integrin, tissue factor, semaphorin, and MAPK signaling.  Notably, HGF was the top modulated canonical pathway identified following knowdown, and further, an HGF-mediated gene-interaction network was among the among the top significant gene networks following topological arrangement of the genes found to be differentially expressed by IPA.

Western blot analysis and ELISA both showed increased phosphorylation MET following ETS2 knockdown.  Importantly, co-transfection of H441 lung cancer cells with MET-specific siRNA abrogated cell migration, invasion, and, to a lesser effect, cell growth mediated by knockdown of ETS2 expression alone.  Furthermore, co-transfection of cells with MET-specific siRNA significantly attenuated the induction of SNAI2, RAB3B, SPDEF and SERPINB5 mediated by knockdown of ETS2 alone as well as reduced expression of ZEB1. These data demonstrate that ETS2 suppresses key features of the lung cell malignant phenotype, at least in part, through inhibition of the MET oncogene. 

The authors also find that ETS2 suppresses EGF-mediated signaling in lung cancer cells.  HGF treatment increased ETS2 protein expression and phosphorylation of MET and ERK1/2 MAPK in H441 cells.  Conversely, expression of ETS2, phospho-MET, and phospho-ERK1/2 was attenuated by co-treatment of H441 cells with the MET TKI PHA-665752.  HGF treatment also significantly increased the expression of SNAI1, RAB3B, SPDEF, ZEB1, and SERPINB5 genes.  Moreover, the expression of these genes was significantly higher in HGF-treated cells transfected with ETS2-specific siRNA compared to similarly treated control cells.  Conversely, overexpression of ETS2 in H1299 cells decreased HGF-induced phosphorylated MET levels and decreased HGF-induced cell migration in the wound healing assay. 

Finally, NSCLC (p=0.01) or ADC (p=0.02) patients with relatively low ETS2 protein expression and higher phospho-MET membrane immunoreactivity showed shortest tiime-to-recurrence compared to other patient groups, specifically those with higher ETS2 and higher phospho-MET
expression.

NKX2-1/TTF-1 drives pulmonary-specific differentiation in lung adenocarcinoma

Snyder et al. recently published an intriguing report in Molecular Cell (21 March 2013) (featured free article) in which they convincingly demonstrate how tumors in which Nkx2-1 was deleted show striking different morphology from those in which Nkx2-1 is expressed.

Nkx2-1/TTF-1 is a highly conserved homeodomain-containing transcription factor that is expressed at the onset of lung and thyroid development and has been shown from murine knockout experiments to be required for branching morphogenesis in very early lung development. Nkx2-1 is expressed in about 80% of human lung adenocarcinomas (ADCs). Recent studies have shown that Nkx2-1-negative ADCs have a worse prognosis and have a higher histologic grade (more poorly differentiated) compared to Nkx2-1-positive tumors. These data suggest that Nkx2-1 may coordinate a lineage-specific differentiation program on lung adenocarcinomas that restrains their malignant potential. Further data have shown that the Nkx2-1 gene is amplified in 10%–15% of human lung ADCs, suggesting that it can also act as a lineage-survival oncogene in a subset of tumors. The authors' previous work using a mouse model for lung ADC showed that Nkx2-1 restrained the ability of Kras-driven lung tumors to evolve to a poorly differentiated, Hmga2-positive state. 

The authors cleverly designed a mouse model in which Cre recombinase can activate a conditional allele of Kras ^G12D and delete both alleles of Nkx2-1 from the lungs of mice.  Simultaneous activation of Kras ^G12D and deletion of Nkx2-1 yielded peripheral lung ADCs that showed a distinct glandular growth pattern of malignant cells with abundant MUC5A-positive mucin resembling human lung mucinous ADC. In contrast, control Nkx2-1-positive mice developed tumors showing a predominantly papillary non-mucinous pattern. They also showed in a different model that in established tumors, Nkx2-1 deletion induced a glandular pattern and mucin production similar to de novo tumors and promoted long-term tumor growth. In addition, this study demonstrated that Nkx2-1 deletion induced diffuse alveolar epithelial hyperplasia in the alveolar space.  The hyperplastic cells lacked expression of canonical Nkx2-1 targets and exhibited increasing mucinous accumulation and morphology over time.

The authors compared Kras-mutant/Nkx2-1-deleted tumors and Nkx2-1-positive tumors by mRNA expression profiling and found 1,828 differentially expressed genes, including 1,137 upregulated and 691 downregulated genes. MetaCore analysis for disease-specific gene collections and networks identified genes associated with gastric diseases that were enriched in  ‘‘stomach neoplasms’’ and ‘‘stomach diseases’’ as categories that were enriched in Kras-mutant/Nkx2-1-deleted tumors.  Finally, they determined whether human mucinous lung adenocarcinomas also exhibit evidence of gastric differentiationby evaluating the expression of two stomach-restricted proteins (GKN1 and CTSE) in 37 human lung adenocarcinomas. In 11 NKX2-1-negative mucinous human lung adenocarcinomas (n = 11), they found that GKN1 was expressed in 6 out of 11 cases and that CTSE was strongly and diffusely expressed in all 11 tumors. In contrast, NKX2-1-positive lung adenocarcinomas (n = 26) were entirely negative for GKN1. Most NKX2-1-positive lung tumors were either negative (n = 14) or only focally positive (n = 10) for CTSE. 

The DL

There is much more to unpack from this article than I can concisely summarize in this post. The fascinating point for me is that lung alveolar epithelial cells have a latent gastric differentiation program that becomes activated by default when Nkx2-1/TTF-1 is deleted and this is most dramatically evinced in pulmonary mucinous adenocarcinomas. These particular tumors are nettlesome diagnostically because of the question raised of whether the tumor is primary or metastatic.

Wonderful paper and lots to think about!

 

Video: Dr. Jack West discusses changing views on molecular testing in NSCLC

The Lungevity blog features a video of Dr. Jack West from Swedish Cancer Institute in Seattle discussing his evolving views regarding molecular testing for actionable driver mutations in non-small-cell lung cancer (NSCLC). Dr. West is a thought-leader in lung oncology and I found this a tidy and brief summary of current issues and reasonable argument for expansion of such testing.

This is important for pathologists as we are often tasked with triaging testing as the time of diagnosis, managing results reporting, and driving testing. My own views as a pathologist with a concentration on lung cancer have changed over the last two years based on clinical practice, research meetings, recent publications, and listening to clinicians' views. Initially, it seemed to me that reflex testing of all advanced stage (stage 2B and up) lung adenocarcinomas and excluding tumors with squamous histology; since we generally are unaware of the smoking history, I ignored smoking history as a criterion for testing. I okayed this with the oncologists and off we went. Dr. West succinctly contrasts the two views concerning molecular testing in NSCLC: essentially testing all patients with advanced stage NSCLC versus testing focused on "enriched" populations, e.g. adenocarcinoma histology, never/light smokers.

My own views began to change as a result of one memorable lung cancer research meeting at Rush we had when one of the thoracic surgeons related how a patient grilled him on why he received an expensive bill for all this molecular testing without any discussion with the surgeon or oncologist and, moreover, when he had already expressed his preference to not receive any further treatment following biopsy and mediastinoscopy. These aren't cheap tests and someone (not the pathologist) must explain these results to the patient. I also reviewed our experience with molecular testing and found several instances where testing was sent on early-stage patients and squamous histology tumors, which I didn't think was appropriate. Based on another round of discussions with the oncologists, I decided that we should consult with the oncologist before ordering molecular testing.

But as Dr. West says, "Some patient's cancer don't read the books." He relates several recent experiences that have caused him to revise his views: squamous tumors that show driver mutations, including one tumor with both EGFR activating mutation and ALK rearrangement. He advocates broader, more expansive molecular testing as well as consideration of testing recurrences and re-biopsy of clinically stable lesions.

The bottom line is that this is still an evolving area--we must be aware of new data coming out and be prepared to modify our views and practices.

Epithelial-Mesenchymal Transition and Lung Cancer: Mechanism for Resistance to EGFR-TKIs?

Despite all of the encouraging clinical results with molecularly targeted therapies against non-small cell lung cancer, there are still important and significant questions that continue to vex.  The discovery of activating mutations in EGFR radically changed the treatment options for these patients, and, indeed, treatment of advanced stage patients with EGFR-mutated tumors with erlotinib (in the US) is now front-line therapy.  However, even patients who initially show dramatic responses invariably relapse due to the development of drug resistance.  Although such resistance can be explained in about 50% of these patients, what are the mechanisms for the other 50%?  Conversely, why do some, albeit rare, patients who are wild-type for EGFR respond to EGFR-TKIs?  Also, why don't conventional chemotherapy and molecularly targeted therapy produce a synergistic effect?  In fact, patients treated with combined therapy seem to actually do worse than if they get just one or the other--why?

Numerous research groups are trying to identify more effective biomarkers to identify predictive markers for response or resistance to EGFR-TKIs in order to optimize therapy for individual patients.  Our group at Rush University Medical Center has been focused on the role of epithelial-mesenchymal transition as a prognostic marker for early-stage NSCLC but also as a predictive marker for response to EGFR-TKIs.  So I read with great interest the article by Byers et al. recently published online in Clinical Cancer Research on December 20, 2012 (abstract here).

Some important notes:

 

• The EMT first principal component correlated better with E-cadherin protein level than did even the best CDH1 mRNA probe set.
• Mesenchymal cell lines were highly resistant to erlotinib as compared to epithelial cell lines, which agrees with previous work.  It is interesting, however, that while cell lines with EGFR-activating mutations were the most sensitive to erlotinib, in a subset of 40 cell lines WT for both EGFR and KRAS, the correlation between EMT signature and erlotinib resistance was still observed with significantly greater resistance in mesenchymal-like cell lines.  An important corollary is that the EMT signature was a better predictor of erlotinib response than mRNA probe sets for either CDH1 (E-cadherin) or VIM genes individually--a finding that agrees with the work we've done in our lab.
• EMT does not appear to be a general marker of resistance.  Indeed, mesenchymal cells were not more resistant to other targeted agents, such as sorafenib or to commonly used cytotoxic chemotherapies, including pemetrexed, docetaxel, paclitaxel, and platinum-doublets. Instead, a trend toward greater relative sensitivity was seen in mesenchymal cells as compared with epithelial for cisplatin. 
• An EMT signature may be a predictive marker in EGFR wild-type/KRAS wild-type patients with advanced stage NSCLC.  The authors analyzed the association between EMT signature and clinical outcome in patients enrolled in the BATTLE-1 trial but limited their analysis to patients whose tumors were WT for EGFR and KRAS.  Although the N is only 20, patients with disease control at 8 weeks (the primary study endpoint) showed a more epithelial-like signature as compared with those without disease control, although the difference is of borderline significance (P=0.05, by t test).  However, among the full group of 101 of 139 clinically evaluable patients (all treatment arms), expression of EMT signature genes was not prognostic of 8-week disease control or progression-free survival (PFS) in the overall group (all treatment arms).

This is a dense article but well-worth reading through in detail if you are interested in either EMT in NSCLC or mechanisms of EGFR-TKI drug resistance.  I offer a few further comments.

First, note that this is a study of NSCLC cell lines, so the findings have to be taken as hypothesis-generating for confirmation in clinical tumor specimens.  The limitations of studying cell lines ought to be well-known to you all.  But one point that has to be appreciated is that the phenomenon of EMT in vivo is context-dependent and critically depends on interaction with the tumor microenvironment--and this crucial fact is not and cannot be studied in this type of model.  Second, although it seems obvious to state this, histological subtype matters greatly with regard to EGFR mutations, EMT, and response to erlotinib.  Why do so many recently published studies still lump together non-small-cell lung cancer?  We recently focused exclusively on a group of patients with resected lung adenocarcinomas who received erlotinib upon disease progression in a study before the advent of EGFR mutation testing.  We identified two separate "epithelial" (high E-cadherin/low vimentin) and "mesenchymal" (low E-cad/high VIM) as well as intermediate/transitional tumors and found a trend toward worse DFS that did not quite reach statistical significance.  Unfortunately, our study was small (N=45) and I think that much larger numbers will be needed to show any importance difference.  In this study, out of 35 cell lines with adenocarcinoma histology, 29 (83%) had epithelial signatures, while 8 (23%) had mesenchymal signatures. Of 4 cell lines with squamous histology, there was an even distribution between epithelial and mesenchymal subsets.  Thus, the adenocarcinomas more commonly expressed an epithelial signature (P<0.0016, Chi-square test).  Finally, I have observed in our studies that EMT in vivo is a heterogenous process throughout a tumor.  Indeed, even if you focus on the invasive edge of a tumor, where one expects EMT to be occurring (as we did in a recently published abstract at AACR 2012), there is frequently significant variation in the expression and distribution of E-cadherin and vimentin.  What is the significance of this variation?  Can it be quantified and does the amount of variation in EMT have any prognostic or predictive significance?  Don't know--image analysis and quantitative immunohistochemical studies could possibly answer these questions. 

Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer

Thought this was an intriguing abstract. Signaling crosstalk between EGFR and integrins was a hot topic at AACC meeting earlier this year.




From Evernote:




Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer
Clipped from: http://clincancerres.aacrjournals.org/content/early/2012/10/19/1078-0432.CCR-11-2162.abstract?papetoc



Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer

Shunsuke Kondo1,Satoshi Iwata2, Taketo Yamada4, Yusuke Inoue7, Hiromi Ichihara3, Yoshiko Kichikawa2, Tomoki Katayose2, Akiko Souta-Kuribara2, Hiroto Yamazaki2, Osamu Hosono2, Hiroshi Kawasaki2, Hirotoshi Tanaka2, Yuichiro Hayashi4, Michiie Sakamoto4, Kazunori Kamiya5, Nam H. Dang8, and Chikao Morimoto6+
Authors' Affiliations: 1Hepatobiliary and Pancreatic Oncology Division, National Cancer Center Hospital; 2Department of Rheumatology and Allergy, Research Hospital, 3Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo; 4Department of Pathology, School of Medicine, 5Department of Pathology and Department of Surgery, Division of General Thoracic Surgery, School of Medicine, Keio University;
6Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University,
Tokyo; 7Department of Diagnostic Radiology, Kitasato University Hospital, Kanagawa, Japan; and 8Division of Hematology and Oncology, University of Florida Shands Cancer Center, Gainesville, Florida


Corresponding Author:
Chikao Morimoto, Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine,
Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81-3-3868-2310; Fax: 81-3-3868-2310; E-mail: [email protected]


Abstract

Purpose: In a substantial population of non–small cell lung cancer (NSCLC), expression and activation of EGF receptor (EGFR) have been reported and is regarded as a novel molecular target. A growing body of evidence has shown the signaling crosstalk between EGFR and integrins in cellular migration and invasion. NEDD9 is an integrin signaling adaptor protein composed of multiple domains serving as substrate for a variety of tyrosine kinases. In the present study, we aimed at elucidating a role of NEDD9 in the signaling crosstalk between EGFR and integrins.


Experimental Design: Using NSCLC cell lines, we conducted immunoblotting and cellular migration/invasion assay in vitro. Next, we analyzed metastasis assays in vivo by the use of xenograft transplantation model. Finally, we retrospectively evaluated clinical samples and records of patients
with NSCLCs.


Results: We showed that tyrosine phosphorylation of NEDD9 was reduced by the inhibition of EGFR in NSCLC cell lines. Overexpression of constitutively active EGFR caused tyrosine phosphorylation of NEDD9 in the absence of integrin stimulation. By gene transfer and gene knockdown, we showed that NEDD9 plays a pivotal role in cell migration and invasion of those cells in vitro. Furthermore, overexpression of NEDD9 promoted lung metastasis of an NSCLC cell line in NOD/Shi-scid, IL-2Rγnull mice (NOG) mice. Finally, univariate and multivariate Cox model analysis of NSCLC clinical specimens revealed a strong correlation
between NEDD9 expression and recurrence-free survival as well as overall survival.


Conclusion: Our data thus suggest that NEDD9 is a promising biomarker for the prognosis of NSCLCs and its expression can promote NSCLC
metastasis. Clin Cancer Res; 1–13. ©2012 AACR.



Footnotes

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).



Received August 23, 2011.
Revision received August 20, 2012.
Accepted August 28, 2012.

©2012 American Association for Cancer Research.

Lack of NKX2-1 (TTF-1) expression in lung adenocarcinoma and EGFR mutations

My last post discussed one of the two papers I thought were notably in the October 2012 Journal of Thoracic Oncology. Here's my take on the other paper by Vincentin et al (J Thorac Oncol 2012;7:1522-1527). NK2 homeobox 1 is the "proper" name that we pathologists more affectionately call "TTF-1." NKX2-1 is normally expressed in type 2 pneumocytes and non-ciliated bronchiolar epithelial cells and has been shown to play an active role in sustaining lung adenocarcinoma. Since NKX2-1 is found in about 95% of lung adenocarcinomas with EGFR mutations, the authors studied whether NKX2-1 IHC staining is a useful provisional marker for guiding EGFR-TKI therapy in advanced stage lung adenocarcinoma while EGFR mutation analysis is being completed.

This is a large study including 810 consecutive NSCLC tumor specimens referred for routine diagnostic EGFR mutation testing. While one may debate the ultimate practicality of this study with respect to it's purpose, what recommends it to me is that the study population more closely mirrors what a general pathologist encounters in routine practice. The study includes biopsies (N=594), resections (N=162), and cytology specimens (N=54) and the overall incidence of EGFR mutations was 14%. It is important to note that the authors used the TTF-1 8G7G3/1 antibody clone for IHC testing. There are substantial differences between the different antibody clones for TTF-1 that have been recently noted in the literature and it is important to take note of this detail when analyzing articles that test NKX2-1 or determining the best antibody to use in your lab.

Results and Authors' Conclusions

NKX2-1 was evaluable in 98% of cases and was positive in 68%, negative in 32%. NKX2-1 positive immunostaining showed a strong correlation with EGFR mutations: in the EGFR-mutated cases, NKX2-1 was positive in 92% and negative in 8%. The probability of absent of EGFR mutation in all adenocarcinomas is 80%; in NKX2-1-negative adenocarcinomas that probability rises to 96%. The authors found a negative predictive value of negative NKX2-1 IHC for EGFR mutation to be >95%.

The authors conclude that if treatment is clinically urgent, the NPV of NKX2-1 expression is appropriate to use as a surrogate marker to choose conventional chemotherapy as starting treatment pending EGFR mutation analysis. The PPV of NKX2-1 is not sufficient to warrent adding an EGFR-TKI. Also, the result of NKX2-1 is not specific enough to exclude a specimen from EGFR-mutation analysis.

My take-aways

While the clinical situation in which NKX2-1 testing would influence an urgent treatment decision may uncommon, I think it is important to note this paper because when it does occur, the pathologist can play a vital role in assisting the clinician determine the management for such a patient. If one is following the diagnostic algorithm from the 2011 IASLC guidelines, one would not routinely test either histologically diagnostic adenocarcinomas or squamous cell carcinomas for NKX2-1 (TTF-1). Another point is that the SPT24 clone for TTF-1 is a more sensitive antibody for TTF-1/NKX2-1 (a point that the authors make as well) and, therefore, one would expect the percentage of NKX2-1-negative cases to be fewer and the NPV to be higher. Finally, the authors used direct sequencing of EGFR exons 19-21, followed by HRM screening, and PCR-sequencing for identifying EGFR mutations; another methodology could influence the denominator of EGFR-mutated cases and, thus, the predictive value of NKX2-1 IHC testing.

Loss of PTEN as a poor prognostic marker for lung adenocarcinoma

The October 2012 Journal of Thoracic Oncology presents two papers of interest to pathologists.

The first study is a paper by Yanagawa et al. that correlates PTEN expression with gene copy number, P53 expression, EGFR and KRAS mutations in surgically resected NSCLCs and examines the relationship between PTEN status, clinicopathologic characteristics and survival.

Recall that PTEN functions as a lipid phosphatase that negatively regulates the PI3K/AKT anti-apoptotic and proliferation pathway, which is a key downstream component of the EGFR pathway. Loss of PTEN is most commonly due to promoter hypermethylation, while homozygous deletion and nonsense mutations with LOH seem to be uncommon.

PTEN expression was examined by immunohistochemistry in a tissue microarray constructed from 152 patients with a median follow-up time of 2.3 years. The study consisted of 62% adenocarcinomas, 29% squamous cell carcinomas and 9% of tumors called “other.” Since this is a study of surgically resected patients, there is obviously an overrepresentation of stage one tumors compared to all patients typically diagnosed with lung cancer. Smokers comprised 79% of the study population. EGFR mutations were found in 15% and KRAS mutations and 22%. The authors defined loss of PTEN as complete absence of cytoplasmic staining in tumor cells.

Loss of PTEN was found in 63 out of 152 cases (41.4%). No significant differences were found between loss of PTEN and smoking, EGFR mutations, KRAS mutations, or p53 expression.

Patients with loss of PTEN expression had significantly worse disease-free survival compared with patients with retained PTEN expression (HR 1.76, log-rank p=0.038) but the prognostic effect was related directly to tumor histology: loss of PTEN was significantly associated with shorter DFS in adenocarcinoma only (HR 2.72, log-rank p=0.002). This association was also significant in multivariate analysis controlling for sex, age, histology and stage.

Recent data suggest that loss of PTEN may contribute to EGFR TKI resistance in NSCLC. PIK3CA mutations only account for 2% to 4% of NSCLC but loss of PTEN would result in constitutive activation of the PI3K pathway. This study suggests that lung adenocarcinomas selected for PTEN status may be susceptible to PI3KCA inhibitors that are currently under investigation.

My next post will discuss the other interesting paper in JTO this month.