The Daily Sign-Out

Possible false positive interpretation of Napsin A staining in evaluating lung non-small cell carcinomas

Dr. Nelson Ordonez (MD Anderson Cancer Center, Houston) published a brief and very helpful article in the March 2012 American Journal of Surgical Pathology (abstract) that addresses a very practical issue of using IHC staining for Napsin A in evaluating primary lung NSCLC for confirmation of adenocarcinoma type.

Napsin A is normally expressed in type II alveolar pneumocytes but may also be localized immunohistochemically in intraalveolar macrophages presumably from phagocytosis.  The expression of both Napsin A and pro-surfactant protein B (ProSP-B) are regulated by thyroid transcription factor-1 (TTF-1), which is one of the common IHC stains initially performed on NSCLC not diagnostic for adenocarcinoma on routine H&E.  Napsin A is localized to lamellar bodies with ProSP-B and is thought to be involved in post-translational processing of ProSP-B.

This paper must be understood in the context of new IASLC lung adenocarcinoma classification and the algorithm recommended to histological type NSCLC.  Napsin A has emerged as a useful adjunct IHC stain, such as, in those cases in which a tumor is negative or equivocal for initial screening markers for both adenocarcinoma (ADC) and squamous cell carcinoma (SQC).  Until recently, Napsin A was found to be highly specific for lung ADC.  But, as Dr. Ordonez points out, several recent publications have found Napsin A positivity in lung squamous cell carcinomas and, thus, cast doubt over the high degree of specificity of Napsin A for lung adenocarcinoma and utility of this marker in this context.

Dr. Ordonez studied Napsin A expression by IHC in 90 primary lung SQC, including whole section and biopsy specimens, and 64 non-pulmonary SQC.  None of the primary lung SQC or the non-pulmonary SQC showed Napsin A positive staining in the malignant cells.

The key observation is the identification of entrapped type 2 pneumocytes and/or intraalveolar macrophages in desmoplastic areas adjacent to the tumor and in small clusters within the tumor--which could be a source of false-positive interpretation in SQC.  Dr. Ordonez found 5 biopsies in which these entrapped cells were difficult to distinguish from malignant cells and CK 5/6 was used in 2 of these cases to rule out the possibility that these were Napsin A-positive malignant squamous cells.

This is a critical distinction and very welcome contribution to the lung cancer pathology literature.  The reader should keep point this in mind, especially when evaluating biopsy material.  The illustrations are excellent in demonstrating these points and should also be kept close at hand.  Congratulations to Dr. Nelson Ordonez for an excellent paper.

WCLC 2011 Oral Presentations: Squamous Cell Carcinoma

There were several interesting presentations regarding squamous cell lung carcinoma that are worthy of attention.

O06.02 presented in the Biomarkers I session by Selinger et al. was a study of a possible prognostic marker for SQC called Special AT-rich Binding Protein-1 (SATB1).  SATB1 is a global genome organizer that recruits chromatin remodeling proteins for epigenetic regulation of multiple genes in a tissue-specific manner.  SATB1 is normally expressed in normal respiratory ciliated columnar epithelium in bronchi and terminal bronchioles but the authors observed loss of this protein by IHC expression in preinvasive squamous lesions and in NSCLC.

In their cohort of 243 NSCLC patients, the authors found that loss of SATB1 was an independent predictor of decreased cancer-specific survival in SQC patients (HR 2.06, P=0.016).  The authors suppose that loss of SATB1, which can act as a transcriptional activator or repressor, in early lung lesions results in widespread disruption of tissue-specific epigenetic modifications, leading to activation or repression of key cancer genes and promotion of lung tumorigenesis.

This provocative study raises more questions than it can answer but is worthy of notice because of its identification of a possible prognostic marker for SQC (as opposed to ADC) but also the interesting mechanistic questions it raises with regard to changes in tissue-specific epigenetic regulation for the development of or progression of cancer.

 

O037.07 by Savic et al. was presented in the Biomarkers IV session and adds to the growing number of studies on Sex Determining Region Y-box 2 protein (SOX2) in NSCLC.  SOX2 is located on the long arm of chromosome 3, an area that has been previously identified as being associated with loss of 3q in early stages of SCQ carcinogenesis.  This study involved a tissue microarray constructed from 568 patients with stage 1 NSCLC with detailed histopathological and clinical data.  SOX2 gene data was determined by FISH and SOX2 protein expression was assessed by IHC.  This data was correlated then with clinicopathological findings and OS and cancer-specific survival.

Increased SOX2 gene amplification or polysomy was found in 4% of (17/429) evaluable samples but 16/17 (94%) were associated with non-ADC histology.  SOX2 protein expression was found in 48% of NSCLC and was associated with non-ADC histology (P<0.001): SOX2 expression was identified in 78% of SQC specimens (178/227) but only in 15% of ADC (29/191).  Further, SOX2 protein expression was significantly associated with high grade tumors, patients >50 years, and male gender.  Finally, they noted a significant association between increased SOX2 gene number and protein expression (P<0.001).

While these are some intriguing findings, this presentation doesn’t quite deliver. Unfortunately, the authors did not report any correlation data with survival (and this is a glaring deficiency) nor do they offer any speculation on the discrepancy between the few cases that show increased SOX2 gene copy number and the frequent observation of SOX2 protein expression in SQC.  Further work on SOX2 definitely seems warrented, especially because of this association with SQC histology and its location in a chromosome region frequently lost in early stages of squamous carcinogenesis.

 

WCLC 2011 Summary from Plenary Session

There were three themes from the Monday, July 3 plenery session at the 2011 14th World Conference on Lung Cancer.  First, the effect of smoking cessation on lung cancer incidence was discussed in relation to a recent study showing that women who smoke 20 cigarettes a day have a risk of developing lung cancer 30 times higher than non-smokers.  However, the study also showed that quitting before age 50 reduced the risk to sic times higher and quitting before age 40 reduced it to 1.2 times higher almost completely eliminating the risk from smoking.

The second major theme discussed the shift from squamous cell carcinoma (SQC) to adenocarcinoma (ADC) as the most prevalent histologic type of lung cancer.  The development of cigarette filters was discussed as one reason for this shift.  It was proposed that cigarette filters causes smokers to inhale more deeply which delivers smoke to areas of the lung predisposed to adenocarcinogenesis.  Further, low tar/low nicotine formulations cause smokers to inhale more often, resulting in an increased volume per puff, to achieve the same nicotine dose.

Finally, the third theme explored recent work describing the association between variants in chromosome 15q25, which encodes nicotinic acetylcholine receptors, and smoking behaviors that not only make it harder for people to quit smoking but also are associated with increased smoking intensity.

 

DDR2 Kinase Mutations in Lung Squamous Cell Carcinoma

Yes--you read that right!

The journal Cancer Discovery reports in a publish ahead-of-print article (abstract)the discovery of mutations in the discoidin domain receptor 2 tyrosine kinase (DDR2) gene in a series of lung squamous cell carcinomas (SQC) that may identify a  potential therapeutic target similar to those described for lung adenocarcinomas.  Interestingly, DDR2 is a receptor kinase that normally binds collagen as its ligand and has been shown to promote cell migration, proliferation, and survival when activated by ligand binding and phosphorylation.

While subtypes of lung adenocarcinoma (ADC) have been increasingly better defined and characterized on multiple levels with respect to identifying therapeutic targets/responses, minimal progress has been made with squamous cell carcinoma.

The authors of this study used conventional Sanger sequencing of 201 genes, including the entire tyrosine kinome, in an initial set of 20 primary lung SQC and matched normal controls and identified 2 samples with DDR2 mutations; a secondary screen of 48 lung SQC samples identified as additional 4 DDR2 mutations.  Following this, the authors sequenced DDR2 in a validation cohort of 222 primary lung SQC samples and identified 5 additional samples with mutations.  Excluding samples from lung SQC cell lines, there was an overall frequency of 3.2% of DDR2 mutations in primary lung SQC samples--about the same frequency reported for ALK fusion mutations in lung ADC.

The authors analyzed the TKIs imatinib and dasatinib, previously reported to inhibit DDR2, to evaluate whether targeting DDR2 mutated in SQC might be a potential therapeutic strategy.  Dasatinib showed particular efficacy in inhibiting proliferation and promoting cell death through apoptosis in SQC cell ines with DDR2 mutations.  In addition, they report one of 7 subjects from an early-phase trial of dasatinib or combination dasatinib and erlotinib with advanced stage lung SQC cancer who exhibited significant shrinkage in tumor size while receiving the dasatinib/erlotinib combination.  The authors were able to perform direct sequencing of DDR2 in a pretreatment tumor specimen from this individual and identified a novel DDR2 kinase mutation.

This is an intriguing paper that you might well keep in mind.  Hopefully, this may stimulate other investigators to not only look for other potential genomic alterations that either drive SQC and/or represent targetable proteins but also go back and scrutinize histological and other clinicopathological characteristics of lung SQC that may have therapeutic or prognostic importance.

Aberrant Wnt1 and beta-catenin expression in NSCLC

This month's April 2011 Journal of Thoracic Oncology has an interesting paper by Xu and colleagues (abstract) that examines Wnt1 expression in non-small cell lung cancer (NSCLC) in relation to downstream Wnt signaling molecules, including beta-catenin, and correlates different marker expression with traditional clinicopathological parameters.

This is an immunohistochemical study of a tissue microarray composed of 262 NSCLC resected specimens.    The authors define aberrant beta-catenin expression as: 1) decreased membranous pattern in less than 70% of tumor cells, 2) cytoplasmic pattern of expression, or 3) nuclear pattern of expression.  As expected, the majority of patients are stage 1 or 2 but nearly 36% of the study population are never-smokers--over 50% in the adenocarcinoma histology group (!).  The authors found that Wnt1 overexpression was significantly associated with never-smoker status and ADC histology; aberrant beta-catenin expression was associated with SQC histology.  While each Wnt1 overexpression and aberrant beta-catenin expression were independently associated with decreased OS regardless of TNM stage, patients that demonstrated both had worse OS than other combinations of Wnt1/beta-catenin expression.  Further, Wnt1/beta-catenin expression was also an independent factor adjusted by stage, pleural invasion, lymphatic invasion, ECOG PS or stage, age, gender, histology, and p53 expression.

A few comments about this paper.  One significant caveat to the findings is the over-representation of never smokers (35%) in this Asian study population, especially nearly 54% of never smokers in the ADC group, compared to a more typical American population in a community hospital setting (about 10%).  Overexpression of Wnt1 was significantly associated with never-smoking and as well as ADC histology, but, somewhat paradoxically, Wnt1 overexpression was associated with significantly shorter overall survival.  Unfortunately, the authors do not break this further down into Wnt1 overexpression and OS with respect to histological type--perhaps some SQCs showing Wnt1 overexpression and dismal OS are skewing the data here since one would expect never-smokers with ADC to have a better prognosis in general.  Another is the usual issues with using TMAs to represent complex heterogenous tumors.  The authors do not sufficiently describe from where they target obtaining cores for constructing the TMA--are they targeting the invasive margin, the central tumor, wherever?  what about mixed histology tumors?  These are critical methodological issues--especially when extrapolating the data concerning marker expression.  One admirable feature of this paper is the clear definition, supported by appropriate photomicrographs, of "aberrant" beta-catenin expression.  As I am currently working on projects that are similarly assessing beta-catenin expression in NSCLC, I found this very helpful in assessing this paper's findings.

Optimal panel for differentiating NSCLC in small specimens

There has been a plethora of papers, abstracts, and marketing materials in the last 12 to 18 months regarding the "optimal" panel for differentiating adenocarcinoma (ADC) from squamous cell carcinoma (SQC) in non-small cell lung cancer (NSCLC).  The December 2010 American Journal of Surgical Pathology features a paper from Terry, Leung, Laskin et al. that evaluates an optimal panel and, perhaps, more importantly, a testing algorithm for differentiating ADC from SQC in small biopsy specimens.

Caveat: one of the authors is the Medical Director/Chief Pathologist for a commercial pathology reference lab specializing in IHC diagnostics (PhenoPath Laboratories).

This study makes several practical points that can be nicely summarized as bullets:

• For ADC the most sensitive marker and best negative predictor is CK7: 93% sensitivity, 91% NPV.
• For ADC the most specific marker and best positive predictor is Napsin A: 94% specificity, 90% PPV.
• TTF-1 has similar specificity as Napsin A for ADC.
• For SQC the most sensitive marker and best negative predictor is p63: 84% sensitivity, 86% NPV.
• For SQC the most spepcific marker and best positive predictor is NTKR2 (a neurotrophic tyrosine kinase receptor, rarely used outside of research labs) but CK5/6 has a nearly identical specificity and PPV: about 95% specificity, about 95% PPV.
• No single marker is BOTH highly sensitive and specific for either ADC or SQC.

The authors identified a 6-marker panel that includes p63, TTF-1, CK5/6, CK7, Napsin A, and mucicarmine has demonstrating the best AUC in ROC analysis.  Since a 6-marker panel may not be feasible (or even possible) due to various factors with small specimens with limited diagnostic tumor material, the authors develped a binary scheme using a sequential addition of markers (Fig. 3, 1809).

The authors do point out, however, that the "best" sequential order may vary by laboratory according to the actual senstitivities and specificities acheived in individual labs and this is important to remember if your lab does not perform all of the stains.

I think one important caution, however, is to note that this study was performed using tissue microarrays derived from resected lung cancer specimens and did not study or validate the performance of these markers on "real" small biopsy specimens.  The authors claim that the results are "similar" to earlier studies using actual patient biopsy material.  Given their careful statement above regarding the "best" sequential order, I think that this is somewhat disingenuous elision over important details regarding significant differences between resected tumors and small biopsy specimens.  Notwithstanding this major issue, there are some good quantitative data here that warrants your consideration in evaluating your own panels at least for comparing your own sensitivity and specificity for individual markers.

Association between EGFR mutations and ERCC1 expression in NSCLC

Gandara and colleagues from UC Davis Cancer Center have published ahead-of-print on October 21, 2010 in Journal of Thoracic Oncology an interesting article examining the association between EGFR activating mutations and ERCC1 gene expression in NSCLC.

NSCLC tumors that show EGFR activating mutations are also more likely to show low expression of ERCC1 mRNA levels according to this study.  This might explain (at least in part) the clinical observation that suggest enhanced efficacy of platinum-based chemotherapy in patients with EGFR-mutant NSCLCs.

The authors performed microdissection of tumors from 1207 patients with NSCLC and analyzed EGFR mutation by allele-specific by PCR and ERCC1 mRNA expression by RT-PCR.

Median ERCC1 expression was histology-related: adenocarcinoma (ADC) showed a lower median expression (1.68 (0.22-11.31) versus squamous cell carcinoma (SQC) (2.42 (0.51-14.28) (p<0.001).  The presence of EGFR mutations was highly associated with low ERCC1 expression (p<0.001).

It would be interesting if future or additional work focused specifically on ADC as a subgroup and perhaps the authors might consider re-analyzing their data and presenting this data.  I suggest this as a practical concern since we are not routinely testing SQC for EGFR mutation.  In addition, the next step is to examine the relationship between EGFR mutation and low versus high ERCC1 expression and response to combined EGFR-TKI and platinum-based chemotherapy, especially in advanced stage disease.

mRNA subtypes in lung squamous cell carcinoma

With all the recent studies looking at various distinct histological and molecular subtypes in lung adenocarcinoma, lung squamous cell carcinoma has been somewhat neglected.  Pathologists have recognized variability in morphological appearances in squamous cell carcinomas and there is a wide range of clinical outcomes.  Although the recent WHO classification recognizes four uncommon histological variants, their biological and clinical significance is not clear.  Moreover, clinical differences between other less-defined morphological patterns are unknown.

A paper published online on July 19, 2010 on Clinical Cancer Research by Wilkerson et al. from University of North Carolina sheds a new light of lung squamous cell carcinoma (abstract).  They describe four novel reproducible mRNA expression subtypes different survival outcomes, histological features, functional biological processes or themes, and association with normal lung cell types.

The methodology of subtype discovery and validation is worth a separate blog itself (for someone else to tackle I'm afraid) but well-worth seeing for yourself because it is quite clever.  The four subtypes described in terms of their mRNA overexpression relative to other types are:

• Primitive: cellular proliferation theme.  Genes include minichromosome maintenance 10 (MCM10), E2F transcription factor 3 (E2F3), thymidylate synthase (TYMS), and polymerase alpha 1 (POLA1), and a previously published "proliferation signature."  PARP1 is also overexpressed--which may be a potential target for the new class of PARP inhibitors. Target genes for E2F transcription factor are also overexpressed.  Other functional themes of this subtype are RNA processing and DNA repair.
• Classical: xenobiotics metabolism theme.  Genes include GPX2, ALDH3A1, AKR1C3 which have been found in smokers' airway transcriptomes and lung cell lines exposed to smoke; TP63, a transcription factor essential to development of stratified squamous epithelium.  This subtype has the greatest percentage of smokers and the heaviest smokers compared to other subtypes.
• Secretory: immune response theme.  Genes include Rho GDP dissociation inhibitor beta (ARHGDIB) and tumor necrosis factor receptor 14 (TNFRSF14); also has a NF-kappaB regulation theme and expression of NF-kappaB target gene overexpression.  Also shows overexpression of lung secretory cell markers (MUC1 and pulmonary surfactant proteins).  TTF-1 (NKX2-1/TTF1)--which is highly expressed in adenocarcinomas--is overexpressed in this type relative to other subtypes.
• Basal: cell adhesion theme and epidermal development theme.  Genes relating to the cell adhesion theme include laminins, collagens, integrins, and claudin-1.  Genes relating to the epidermal development theme include keratin 5, psoriasin, and gap junction protein beta 5 (GJB5).  This subtype also shows overexpression of several S100 family genes.  Interestingly, several of the genes overexpressed in this subtype are also overexpressed in basal-like tumors from other organ sites: an head and neck SCC subtype identified by Chung et al (2004) and the basal-like breast cancer subtype. This suggests that these various "basal" subtypes may share some biological properties.

This is all interesting--but does it have any impact on clinical outcome?

The authors found that tumor subtype is significantly associated with grade: the primitive subtype has an overrepresentation of poorly-differentiated tumors, while the basaloid type has an overrepresentation of well-differentiated tumors.  However, subtype showed no association with tumor stage.  Nevertheless, overall and relapse-free survival was significantly different between different SqCC subtypes.  The primitive subtype was found to have the worst OS and RFS compared to other subtypes across all stages, whereas the classical, secretory and basal subtype all have similar outcomes.  In multivariate models, only subtype was significantly associated with OS and RFS.  The authors comment, however, that tumor stage was not have achieved significance because of an underrepresentation of higher stage tumors--which is the usual referral bias of tertiary-care single institution studies.

This paper is significant because one could use this data to evaluate an IHC panel to subtype SqCC not only for prognosis but also to identify patients who might respond to certain targeted therapies, like PARP inhibitors.