The Daily Sign-Out

The new IASLC/ATS/ERS Lung Adencarcinoma Classification is a Stage-Independent Predictor of Survival

 I blogged on this new classification over a year ago when it was first published, but now papers are rolling out showing its utility.  My take on this recent paper below is that it confirms the idea of histologically grading lung ADC according to the predominant different subtypes (lepidic predominant grade 1 "well-differentiated" versus solid predominant being grade 3 "poorly-differentiated".  Moreover, it asserts that it is sufficient to regard only the predominant histologic type with regard to survival--which is a relief from going through the tedious task of estimating percentages of different minor subtypes after complete histologic examination.  Get this article--good for discussion with surgeons, oncologists, tumor conference, etc. 

The Novel Histologic International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification System of Lung Adenocarcinoma Is a Stage-Independent Predictor of Survival.

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.

Is Notch signaling involved in lung carcinogenesis?

This is the question asked by Joyce Osanyingbemi-Obidi and colleagues from Johns Hopkins in an interesting recent paper published in Molecular Cancer Research.  The cancer stem cell hypothesis proposes that there is a small proportion of tumor cells with stem cell-like characteristics, including self-renewal capability, as well as a much larger proportion of "differentiated" cells with limited proliferative capability.  While stem cell-like cells have been characterized in a variety of malignancies, such a population for NSCLC has not been reported.  I previously blogged on a murine model for evidence of lung stem/progenitor cells. The authors cite the emerging body of data (perhaps not surprisingly) suggesting that the same key signaling pathways in embryonic and fetal development (e.g. Notch, Wnt, and Hedgehog) are also involved in the regulation of cancer stem cells.  The intracellular domain of Notch receptors (NICD) is released upon ligand binding and translocates to the nucleus where it converts the CSL complex from a transciptional repressor into an activator.  However, the NICD-CSL complex requires mastermind-like (MAML) proteins to recruit and bind coactivators that drive expression of downstream targets that promote cell growth and proliferation.  Aberrant Notch signaling has been described in a variety of malignancies (albeit with both activating and inactivating Notch mutations) and, indeed, elevated expression of Notch family members and a key downstream target, "hairy and enhancer of split 1" (Hes1), have been reported in NSCLC.

Using NSCLC cell lines showing overexpression of Notch3 and Hes1, the authors demonstrated that pharmacologic inhibition of Notch was associated with suppression of Hes1 expression and inhibition of clonogenic growth.  The authors used a clever LSL-KRAS^G12D mouse model in which an oncogenic KRAS allele is silenced by a transcription stop site flanked by lox P sites; inhalation of an adenovirus vector containing the Cre recombinase promotes rearrangment of the lox sites and deletion of the transcriptional stop and selective overexpression of mutant KRAS in bronchial and alveolar cells.  The authors showed a transient induction of Hes1 expression by immunohistochemistry in airway epithelial cells 1 week postinfection with adenovirus and gradually diminshing Hes1-positive cells in tumors over time.  They then used an novel inducible double-negative MAML knock-in murine model to show that inhibition of Notch signaling in vivo was not sufficient to suppress carcinogenesis or tumor progression in the context of mutant KRAS-driven carcinogenesis.

So--these data suggest that Notch signaling may have a role in early lung adenocarcinogenesis through the transformation of a tumorigenic stem cell-like cell but is not likely required for adenocarcinogenesis.  The authors provide several notable caveats and qualifications of this conclusion but, I think the key consideration is that Notch signaling is considerably more complex that we are given to understand.  Notch signaling in development can maintain stem cell characteristics in certain cell populations or drive cell differentiation in other.  Likewise, Notch pathway activation in neoplasia appears to have tumor-promoting and tumor-suppressing effects.  Also, not all Notch family members appear to equally contribute to lung neoplasia.  Finally, individual Notch isoforms may have opposing effects on carcinogenesis.

This paper may not be of general interest, especially for the busy practicing pathologist, but is worthwhile to reflect upon and ruminate over.  Intriguing stuff.

Lung adenocarcinoma subtyping: practical issue

We have adopted subtyping adenocarcinoma routinely as part of the examination of lung resections along the lines suggested in the recent "comprehensive histologic subtyping" article by Motoi et al. (Am J Surg Pathol 2008;32:810-827).  A recent case exposed an unexpected issue with such subtyping.  The primary tumor measured 8.0 cm and appeared to show an exclusively solid pattern (6 blocks of tumor submitted):

But the lymph node metastases showed an almost exclusively papillary/acinar pattern:

Final stage was IIIA (T3N2).  The primary tumor is EGFR wild-type but showed the KRAS mutation Gly12Cys.  The case demonstrates a significant morphological discrepancy in histological subtypes between the primary tumor and lymph node metastases.  What is the significance of this?  What does this phenomenon suggest about the relationship between the primary tumor and its metastases?

A key question in lung adenocarcinoma currently is the association between histological subtypes and outcomes and mutational profiles. 

I call your attention to the timely and thoughtful editorial by Dr. Sanja Dacic from UPMC, Pittsburgh published in last month's Archives of Pathology and Laboratory Medicine which touches on the issues and problems concerning adenocarcinoma subtyping.  Dr. Dacic's own excellent study published in Modern Pathology last year showed a large morphologic overlap between tumors with/without KRAS and EGFR mutations.  I found this to be an especially provocative quote:

At this point, there are not enough data to prove an independent prognostic significance of detailed histologic subtyping, and it is uncertain whether histologic subtype of adenocarcinoma is an independent predictor of therapy response.

On the research side, I have been retrospectively annotating our cases in several large databases at Rush University Medical Center.  One critical issue in archival specimens is sampling--there is a wide range in the number of sections taken of tumor.  I think it reasonable (and practical) to entirely submit T1 tumors for histological exam.  But currently, there are no guidelines or protocols (or even suggestions!) for what constitutes adequate sampling to be "representative" of tumors in the T2-T3 categories.  The Motoi study only included 101 cases and included stage I to III cases.  Of note, cases were selected on the basis of available frozen tumor not as part of a consecutive series (!).  The range of H&E stained slides reviewed was 1 to 23 with a average of 6.8.

From the clinician's and patient's points-of-view, adenocarcinoma subtype is meaningful if it provides some prognostic and predictive information.  The data are just not out there right now that subtyping provides this.  So should we bother?  Long enough post for a Friday afternoon--I'll give my answer next week.

WCLC 2011 Oral Presentations: (More) Genomics

Continuing posts summarizing my highlights from the oral presentations from WCLC 2011.

The Biomarkers V presentation highlight (O39.01) was a retrospective analysis of the CALGB 30406 study looking at the impact of specific EGFR mutations on outcomes.  CALGB 30406 was a prospective trial in never/light smokers, previously untreated patients with advanced lung adenocarcinoma randomized to receive erlotinib (ERL) or ERL plus carboplatin and paclitaxel (C/P) as first-line therapy.  Patients with EGFR mutations had similar outcomes (RR, PFS, OS) in both arms of the study vs. EGFR-wild type  patients but no significant difference was seen in outcomes in exon 19 deletion vs. L858R mutation, irregardless of treatment arm.  Good evidence to address a certainly relevant question.

Recent studies have shown that NSCLC can be differentiated into "epithelial-like" and "mesenchymal-like" tumors based on gene expression and EMT biomarkers have been associated with erlotinib activity in both in vitro and clinical specimens.  Increasing evidence also suggests that changes in DNA methylation also are associated with the phenotypic EMT transition in lung cancer cells.  MO16.02, presented in the Biomarkers VII session, is a proof-of-principle study by Genentech researchers using an integrated genomics approach combining gene expression profiling and methylated DNA immunopecipitation (meDIP) to define phenotypic subsets of NSCLC by their DNA methylation patterns.  They further developed quantitative methylation-specific PCR assays for 14 markers identified by meDIP and pyrosequencing assays for another 5 markers.  They tested a panel of NSCLC cell lines with defined responses to erlotinib by these assays and showed good correlation with erlotinib activity or EL/ML status.  This study showed the potential for using DNA methylation profiles in targeted therapy profiles.  This seemed like a lot of fancy razzle-dazzle in this context and I'm unclear what it would really add in a practical sense; it seems to me most of the work on DNA methylation patterns in lung cancer has been focused on identifying markers for early diagnosis in serum.

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: Low-dose Spiral CT Screening for Lung Cancer

Of course, the recently published study of the National Lung Cancer Screening Trial in NEJM on low-dose spiral CT for lung cancer screening in high-risk individuals received the spotlight at the commencement of the WCLC meeting.  Dr. John Field, Chair of IASLC CT Screening Task Force, hailed the results as transformative for the field of lung cancer and framed the discussion in terms of making a transition to implementation.  One of the study’s co-authors, Dr. Denise Aberle, noted that while the trial recorded 24% positive screens compared with 6.9% for chest radiographs, it also had a 95% false positive rate.  A panel discussing the study concurred that the opportunity for early detection outweighs the potential physical and psychological burden of a false result.

 

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.

 

WCLC 2011 Oral Presentations: Genomics

There were 3 presentations on genomics from the Biomarkers II session that I thought noteworthy for extending the understanding of genotype driver mutations in lung ADC.

O016.01 is an update from the U.S. Lung Cancer Mutation Consortium (LCMC) on 911 patients registered to date who have undergone comprehensive testing for currently known driver mutations in NSCLC.  KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, and NRAS have been testing using multiplexed assays, whilst ALK rearrangements and MET amplification have been assessed using FISH (which are reported in a separate abstract from the conference).

Multiplexed assays have been completed in 420 patients and a genetic alteration has been identified in >50%.  All patients had advanced stage (3B or 4) ADC.  The median age of this population is 61 years and 59% are women.  There was no significant difference in the age between those patients with tumors associated with identifiable mutations and those having tumors without mutations.  Neither did gender or smoking history show any significant association with mutation status.

This is very interesting early data from this project that contrasts the clinical observations of driver mutations being associated with female gender and never (or light) smoking history.  This data also supports mutation testing for all patients with lung ADC, at least in advanced stage patients who might be considering erlotinib therapy or enrollment into a clinical trial testing other “targeted” agents.

O016.05 was a presentation of another high profile project, the Lung Cancer Mutation Analysis Project (LC-MAP) from Memorial Sloan Kettering Cancer Center.  The authors presented their 2-year results of comprehesive mutation analysis of 1231 NSCLC (ADC=896, other non-SQC=49).  Evidence of driver mutations was found in 56% of tumors as follows: KRAS 30%, EGFR 20%, ALK-EML4 7%, HER2 5%, PIK3CA 1%, BRAF 1%, MEK1 0.2%, AKT1 0.1%.

This study provides some preliminary benchmarking data to discuss with clinicians, although I would provide a caveat that there is likely to be a referral bias (since this is a single-institutional study from one of the world’s most reknown cancer centers) that overestimates the frequency of driver mutations in ADC.

Finally, O016.06 is another presentation from the MSKCC that examined the prognostic impact of driver mutations in lung ADC with respect to smoking history.  There is important background that puts this study in context, namely, the demonstration of an independent dose-dependent relationship between smoking history and survival in patients with advanced stage NSCLC with never-smokers living 50% longer than smokers (Janjigian et al., Cancer 2010).  The authors reviewed 301 consecutive never smokers and 373 consecutive smokers with lung ADC who underwent testing for EGFR and KRAS mutations and ALK rearrangements between May 2009 and May 2010.

They found statistically significant differences in the frequency of these mutations between never smokers and smokers: KRAS mutations in 4% of never smokers vs. 43% smokers (P<0.0001); EGFR mutations in 37% of never smokers vs. 14% smokers (P<0.0001); ALK rearrangements in 12% of never smokers vs. 2% smokers (P<0.0001).  By multivariate analysis, there were significant survival differences between never smokers with EGFR mutations vs. KRAS mutations (HR for death 4.2, favoring EGFR mutations) and between never smokers with EGFR mutations vs. ALK rearrangements (HR for death 0.31, favoring ALK rearrangements).  But, within a given genotype, there were no significant survival differences between never smokers vs. smokers.

This is very interesting data suggesting that the prognostic impact of smoking is driven by the type of mutation (at least in those patients in whose tumor has a detectable driver mutation).  Integration of smoking history with the type of mutation detected should refine estimation of the overall prognosis of patients with lung ADC.

 

WCLC 2011 Oral Presentations: Tumor microenvironment

So I'm finally finished sorting through the abstracts from the 14th World Conference on Lung Cancer that I recently attended and this begins a series of posts of what I found of interest from the oral abstract presentations.

There were two presentations that focused on the immune cell response in the tumor microenvironment that were of particular interest to me in that I have a current project looking at CD3+ lymphocyte responses in resected NSCLC through my ongoing work with the Thoracic Oncology Research Group at Rush University Medical Center.

MO 22.01 from session VIII examined tissue microarrays constructed from 479 patients with stage 1 adenocarcinoma and a median follow-up of 4.3 years.  They looked at a wide variety of different immune markers by immunohistochemistry and assessed localization (tumor vs. stromal) of immune cell infiltration, the grade of that immune cell infiltrate (low vs. high), and expression (sum of intensity of staining plus semi-quantitative assessment of distribution).

Results: The 5-yr relapse free survival (RFS) was 82% for the entire cohort.  There was no independent prognostic significance for any subpopulation of immune cells.  However, the ratio between stromal FOXP3+ (Treg cells) and CD3+ (total T cells) and tumor expression of IL7R and IL12Rbeta2 were found to be significant factors in predicting recurrence by univariate analysis.  Tumor expression of IL12Rbeta2 and stromal FOXP3:CD3 were prognostic even in tumors less than 2 cm.  By multivariate analysis, gender, stage (1A/1B), lymphatic invasion, tumor IL12Rbeta2 and stromal FOXP3:CD3 were independent predictors associated with recurrence.

This study highlights the importance of localization of immune cell response but also underscores the challenge for future studies to use more refined quantitative assessments of immune cell subpopulations and relative expression of IHC staining intesity (such as utilizing digital imaging and image analysis). 

MO 22.04, also from session VIII, presented interesting complementary data to the above presentation. This study examined 196 resected stage 1-3A NSCLC using IHC for infiltrating CD8+ and FOXP3+ cells.  They counted cells expressing these markers in tumor and stromal regions in five "randomly selected" high-power-fields and reported their data as the number of cells per mm² for each region.  They used median cell count as the cutoff to define patient subgroups.

Results:  Increased tumor islet CD8+ and stromal FOXP3+ infiltration were associated with better prognosis, whilst increased stromal CD8+ and tumor islet FOXP3+ infiltration were associated with worse prognosis.  Further, high CD8+ tumor islet/stromal infiltration was associated with better prognosis, whilst high FOXP3+ tumor islet/stromal infiltration was associated with worse prognosis.

Again, this study reinforces the crucial importance of identifying the microlocalization of immune cell subpopulations.  But I note several critical issues with methodology in this study that are worth considering for future studies.  First, the investigators did not clarify their methodology as to how high-power-fields were "randomly selected."  Further, their use of the term "tumor islet" suggests a non-random selection of such foci (tumor budding?) for assessment.  As a pathologist, I can safely say that this is quite unlikely "random"--which would undoubtedly lead to bias towards areas of the tumor that show higher concentrations of positively staining cells.  In fact, our Rush group recently began re-evaluating EMT markers assessed by IHC in a group of NSCLC patients who underwent surgical resection to specifically assess and grade distribution and extent of staining at the tumor-stromal interface rather than the global expression of such staining.  The data are being analyzed currently.  Moreover, I have been assessing the lymphocytic response just by H&E and note an association between foci of tumor budding and intensity of lymphocytic response at the tumor invasive front.  Second, manual counting of "events" just doesn't make it for this type of study and (again!) I must reiterate the need to use image analysis for this type of study.