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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.

 

More on Landmark Lung Screening Trial: What Does It Mean for Clinicians and Their Patients? - The ASCO Post

This month's ASCO Post has an interview with Dr. Christine Berg, one of the authors of the recently published trail in NEJM.

A Landmark Lung Screening Trial: What Does It Mean for Clinicians and Their Patients? - The ASCO Post.

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.

 

FDA draft guidelines on IVD "companion diagnostics"

Digital Pathology Insights blog posted a nice succinct comment on the FDA draft guidance on companion diagnostics that are of acute interest for anyone interested in digital pathology.

The issue is the development of drugs (i.e., so-called "targeted" drug therapies, such as, trastuzumab) that depend on the result of a predictive test (HER2 protein overexpression by IHC, amplification by FISH) to meet the claims of the drug's effectiveness as approved by the FDA.  As the FDA document states in the introduction,

When an appropriate scientific rationale supports such an approach, FDA encourages the development of therapeutic products that depend on the use of approved or cleared IVD companion diagnostic devices — several such IVD companion diagnostic devices for use with corresponding therapeutic products have already been approved or cleared.

As pointed out in the blog post, the implication is that the drug will not get approval unless the companion diagnostic test or methodology gets approval (and vice versa?).

You may not want to read the FDA document before operating heavy machinery or driving.  

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.