The Daily Sign-Out

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.

ALK -Positive Lung Adenocarcinoma: NEJM Case Records

The July 14, 2011 issue of The New England Journal of Medicine features a nice case report as part of the weekly "Case Records of the MGH" series.  I thought those interested in lung cancer would benefit from reading this, especially in light of recent advances recognizing ALK translocations as driver mutations in about 5% of lung ADC--and the drug crizotinib to treat these particular tumors (my previous post on ALK inhibition in lung cancer).

Incidentalomas revisited

Dr. Bruce Friedman posted a thoughtful blog on Lab Soft News recently concerning "incidentalomas" ("How to Avoid the Risks of a CT Incidentaloma").  This post happened to coincide with a new working committee at my hospital commissioned with developing a virtual clinic for following patients who have incidental lung nodules discovered.

Perhaps unlike other sites, there are widely disseminated radiographic recommendations for following lung nodules (Fleishner criteria).  Of interest, a recent article found that despite widespread awareness of these criteria, actual incorporation of these criteria into practice has been found wanting--only around 50% of radiology practices actually used these recommendations in reports.  One issue that we will be challenged with is how to track these patients across multiple different providers for appropriate follow-up.  One model we are considering is already in place: using a breast "navigator" to follow-up patients with abnormal mammograms.  This issue arose recently because of a series of patients presented at our Tumor Board who had incidental lung nodules identified but were not followed appropriately and then were re-discovered with inoperable advanced stage disease months later.

This idea also came up just today in a conversation I was having with a urologist colleague regarding renal incidentalomas treated with cryoablation.  I recently diagnosed an incidentally discovered 2.5-cm mass in an 80-year-old woman on CT-guided needle biopsy as an muscle-predominant angiomyolipoma (mini-case report on its way!).  For better or worse, the tumor was ablated probable before it was even grossed!  But, to return to Dr. Friedman's post, what if the patient was simply observed with serial imaging?  The urologist was unaware of any recommendations similar to the Fleishner criteria for lung nodules.  If you, dear reader, know of any please post them in a comment.

Finally, today's email brought a "Published Ahead of Print" article from Journal of Thoracic Oncology on-- mediastinal incidentalomas.  This is a prospective study of 88 patients in which mediastinal lymphadenopathy was found as an incidental finding on CT scans for indications other than staging.  The title is a bit deceptive but the scenario is common.  The finding of incidental mediastinal lymph nodes on CT were characterized by multiple Naruke stations involved, relative small sizes of nodes (median ranging from 6 to 14 mm for all nodes), and coexistence with hilar adenopathy.  In this study, sarcoidosis was diagnosed by EBUS-FNA in 22% of patients and 2 patients developed lung cancer 2 years after initial finding.

While I concur with the basic premise of the post (i.e. that one shouldn't jump into a full-blown diagnostic work-up of an incidental finding), I think we should also insist on developing evidence-based criteria for managing these types of findings such as, Fleishner criteria for lung nodules.

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.

ASCO issues Provisional Clinical Opinion on EGFR testing in Non-Small-Cell Lung Cancer

Hot off the electronic press is the PCO issued by ASCO recommending that patients with advanced stage NSCLC should have their tumors tested for EGFR mutation if they are being considered candidates for EGFR-TKI therapy.  Read here  for the abstract or Download ASCO EGFR testing in NSCLC for more deets.

Provisional Clinical Opinion
On the basis of the results of five phase III randomized controlled trials, patients with
NSCLC who are being considered for first-line therapy with an EGFR TKI (patients who have
not previously received chemotherapy or an EGFR TKI) should have their tumor tested for
EGFR mutations to determine whether an EGFR TKI or chemotherapy is the appropriate
first-line therapy.

If you haven't been following this topic, the paper nicely summarizes the clinical trials forming the basis for this recommendation.

Of especial interest to pathologists is the issue of adequate tissue for testing in patients with advanced stage disease:

the ad hoc panel commented that there is often a limited amount of tissue for mutation testing and, therefore, endorses a relevant recommendation in ASCO’s stage IV NSCLC guideline update: “In order to obtain tissue formore accurate histologic classification or for investigational purposes, theUpdate Committee supports reasonable efforts to obtain more tissue than what is contained in a routine cytology specimen.”  Properly fixed material from cytology cell block preparations is generally required for analysis, as opposed to cytology smear preparations.

Finally, note that this PCO only addresses mutation analysis for activating mutations.  Giving recent literature showing concommitant EGFR T790M resistance mutation in 30%-35% of patients at the time of diagnosis, I think we should take this into consideration when looking at testing pathways.  This PCO specifically does not recommend EGFR testing by FISH or IHC at this time.  By the way, testing issues are discussed in the companion journal, Journal of Oncology Practice.  I'll hunt down the link and post later.

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.