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.

Atomic force microscopy! EMT and idiopathic pulmonary fibrosis

Here is a very intesting article I recently found with some very cool technology:

Abstract:

Epithelial-mesenchymal transition (EMT) is closely implicated in the pathogenesis of idiopathic pulmonary fibrosis. Associated with this phenotypic transition is the acquisition of an elongated cell morphology and establishment of stress fibers. The extent to which these EMT-associated changes influence cellular mechanics is unclear. We assessed the biomechanical properties of alveolar epithelial cells (A549) following exposure to TGF-β1. Using atomic force microscopy, changes in cell stiffness and surface membrane features were determined. Stimulation with TGF-β1 gave rise to a significant increase in stiffness, which was augmented by a collagen I matrix. Additionally, TGF-β1-treated cells exhibited a rougher surface profile with notable protrusions. Simultaneous quantitative examination of the morphological attributes of stimulated cells using an image-based high-content analysis system revealed dramatic alterations in cell shape, F-actin content and distribution. Together, these investigations point to a strong correlation between the cytoskeletal-associated cellular architecture and the mechanical dynamics of alveolar epithelial cells undergoing EMT.

http://dx.doi.org/10.1016/j.nano.2011.06.021

I know this is a little out there for the pedestrian pathologist (ahem, like me) but I thought this was so cool--definitely worth at least scanning for the images of atomic force microscopy alone! But this article also provides data at the cellular level to explain some of the mechanical forces behind the changes that occur in IPF.

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. 

Evidence for tissue-specific differences in EMT

Remodeling of the extracellular matrix is a key process in cancer invasion.  Matrix metalloproteases (MMPs) have been shown to be important in ECM remodeling but identifying individual MMPs with specific roles in various cancers has been difficult due to functional overlap with other proteases.  The current Diseases Models and Mechanisms has an interesting article by Hald et al. in which they demonstrate that double-knockout mice lacking both plasmin and MMP-9 develop inflammatory colonic mass lesions that resemble mucosal prolapse lesions in humans.  In contrast, wound healing in skin in these mice is normal.  The findings indicate tissue-specific differences in ECM remodeling--and thus potential therapeutic targets. Further, this study demonstrates the potential utility of other mouse models for studying ECM remodeling in other tumors.  Provocative stuff and more of the EMT story unravels.

P53 in tumor-associated fibroblasts in invasive ductal carcinoma

Hasebe et al. have authored an interesting article in a recent Modern Pathology (2010;23:662-672) which builds on their earlier work that showed P53 expression in tumor-stromal fibroblasts (TSF) not forming fibrotic foci to be an important predictor of outcome in patients with invasive ductal carcinoma.

The fibrotic focus has been shown to be a prognostic factor in breast cancer.  Moreover, criteria for fibrotic foci proposed by Van den Eynden et al. in 2007 (Dr. Hasebe was a co-author) appear to both clinically significant and reproducible in daily practice.  I am surprised that the use of this feature has not been adopted for routine use since presenting breast cancer cases with prominent fibrotic foci never fails to elicit curiosity and questions in Tumor Board.  Further, a fibrotic focus has been proposed as a marker for tumor hypoxia and angiogenesis.

The purpose of the current study was to determine whether the combined assessment of P53 expression in tumor-stromal fibroblasts both forming and not forming fibrotic foci serves as a significant predictor of outcome in patients with invasive ductal carcinoma using multivariate analysis with well-established prognostic factors.

The authors studied 1,039 consecutive patients at a single Japanese institution who received surgery including axillary dissection; 873 also received conventional chemotherapy, endocrine therapy, or both.  Using FFPE serial sections of each tumor, the authors evaluated 8 different histological factors, including measuring the fibrotic focus using 8 mm as the cutoff per the Van den Eynden criteria.  Immunohistochemical staining for P53 was evaluated using the Dako mouse monoclonal antibody clone DO7 and staining of tumor-stromal fibroblasts was scored according to modified Allred scoring.  Furthermore, the authors stratified P53 staining in tumor-stromal fibroblasts into 3 groups each for those tumors with and without fibrotic foci.

373 out of 1,039 cases had fibrotic foci.  Notably, in 97 of these 373 (26%) P53 staining could not be evaluated because the tumor tissue sections examined by P53 IHC did not contain a fibrotic focus identified initially at routine examination.

The authors found significantly higher P53 Allred scores in TSFs not forming compared with TSFs forming fibrotic foci.  But P53 Allred scores >4 in TSFs forming fibrotic foci were also associated with fibrotic focus diameter > 8 mm: the size previously established as a cut-off for predicting adverse outcome independent of stage.

Based on Allred scores, the authors segregated P53 expression for TSFs forming and not forming fibrotic foci into risk classes.  Patients in pTNM stages I-III with intermediate to high risk classes of P53 expression showed a significantly higher tumor recurrence rate than patients in the low risk classes.  In addition, stage II and III patients with intermediate to high risk classes of P53 expression showed a significantly higher disease-related mortality rate.

By multivariate analysis, for stage I patients, intermediate to high risk classes of P53 expression in TSFs forming and not forming fibrotic foci and histological grade 3 were significantly associated with tumor recurrence (but not disease-specific death).  For stage II patients, intermediate to high risk classes of P53 expression in TSFs forming and not forming fibrotic foci were associated with increased tumor recurrence and tumor-related death.  Lymphovascular invasion and blood vessel invasion were significantly associated with increased tumor recurrence, whilst pT1 and fibrotic focus > 8 mm were significantly associated with increased tumor-related death.

(Next to) Bottom Line: P53-expression TSFs located both in the inner fibrotic foci an outer invasive front increase the malignant potential of invasive ductal carcinomas across stages I to III.

SO--should we start doing P53 IHC on all invasive ductal carcinomas?  Perhaps not ready yet for prime-time, but this study provides evidence that may, at least in part, explain the prognostic value of fibrotic focus diameter.  Measuring the fibrotic focus, however, may just be ready for prime-time and you may want to consider reviewing the original paper by Van den Eynden et al published in Histopathology 2007;51:440-451.

BUT I do have a few points to consider.  First, this is a single institution paper, so the methodology needs to be independently validated and a larger, multi-insitutional patient cohort needs to be studied to confirm the association between patient outcomes and P53 staining.  Second, other P53 antibodies should be evaluated to optimize the assay.  Third, the number of invasive ductal carcinomas with fibrotic foci that were excluded from the study is disquieting and may be a major source of observational bias.  Finally, the role of image analysis in providing quantitative P53 cut-offs may provide better discrimination between prognostically different groups.