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Implications of clonality in multifocal non small cell lung cancer

About 5% to 8% of NSCLC patients present with 2 or more anatomically separate lung tumors at diagnosis. The distinction between synchronous primary tumors and intrapulmonary metastases has important implications for staging and, thus, treatment strategies.

Arne Warth and colleagues from University Hospital Heidelberg published a single institution study in the June 1, 2012 European Respiratory Journal (abstract) analyzing the clonal relationship between multifocal NSCLC with indistinguishable morphology in a series of 78 patients.  Their analysis included LOH analysis of 14 polymorphic microsatellite markers and EGFR and KRAS mutation screening using single-strand conformation polymorphism, followed by direct sequencing of abnormally migrating bands. 

This study included 58 adenocarcinomas and 20 squamous cell carcinomas; 59 pT3, 18 pT4, and 1 pM1a. 97.4% of all specimens could be classified as clonal, likely clonal, or non-clonal; only 2 cases were non-informative.

Some results--

1. Tumor nodules of 22 patients (28%) showed divergent LOH status--indicating non-clonal origin. This is a surprising finding--remember this study only included multifocal tumors with indistinguishable morphology.
2. Activating EGFR mutations were found in both synchronous tumors in 4/58 ADC but none of the SQC.
3. KRAS mutations were found in 20 patients (17 ADC, 3 SQC) but in 6 patients the mutation was identified in only 1 of the tumors and in 1 patient there were divergent EGFR and KRAS mutations.
4. With combined allelotyping of LOH and mutational status of EGFR and KRAS, about 36% of cases (28/78 cases) showed evidence that they were not clonally related, including:
5.  14/18 ADC and 6/10 SQC located in the same lobe.
6. Kaplan-Meier curves illustrating the impact of clonality of multifocal NSCLC on overall survival did not identify a statistically significant difference but did show a tantalizing trend that patients with 2 clonally unrelated tumors have a better OS; notably, this difference did reach statistical significance by univariate analysis.

Practical "take-home" points.

There is no specific anatomical or morphological criteria that reliably indicate a clonal or nonclonal origin of multifocal NSCLC tumors. The authors reasonably recommend performing predictive molecular testing (EGFR and KRAS mutational analysis) or allelotyping separately in all cases with synchronous tumors to identify clonally related tumors. This study presents some intriguing data regarding the prognostic value of identifying nonclonalty, but the prognostic impact of clonality needs to be further analyzed in larger cohorts of NSCLC patients with multifocal synchronous tumors.

Establishing clonality in multifocal nonsmall cell lung cancer

Up to 8% of nonsmall cell lung cancer (NSCLC) patients present with 2 or more anatomically separate lung tumors.  Distinguishing between synchronous primary tumors and intrapulmonary metastases is an important clinical question because this determination significantly changes tumor staging and subsequent therapeutic decisions.

Arne Warth et al. have published a single institution study from University Hospital Heidelberg in the current issue (1 June 2012) of European Respiratory Journal (abstract) that examines the clonal relationship in a series of patients with morphologically indistinguishable multifocal NSCLC.

The authors screened their archives for synchronous multifocal NSCLC in the same lobe (pT3), different ipsilateral lobes (pT4), and bilateral lobes (pM1a).  An important part of this study is that the authors only included those cases in which morphology between the tumors was indistinguishable using published criteria from the literature (Girard et al, Am J Surg Pathol 2009--actually a great reference).  They included 78 patients in this study.

The authors performed EGFR and KRAS mutation screening by using single-strand conformation polymorphism sensitive gel elctrophoresis and then subjected aberrently migrating bands to direct DNA Sanger sequencing.  The authors analyzed 14 polymorphic microsatellite markers for loss of heterozygosity. A clever part of the analysis of clonality in this study was the utilization of individual clonality scores for each tumor pair by multiplying the likelihood of the observed findings for each informative variable.  Thus, the clonality score defines the statistical error probability of a clonal or non-clonal relationship.

Results

1. The study included 58 adenocarcinomas (ADC) and 20 squamous cell carcinomas (SQC).  59 were pT3, 18 pT4, and 1 pM1a.  The mean follow up for all patients was 25.6 months. 97.4% of all specimens could be classified as clonal, likely clonal or nonclonal.
2. Tumors from 22 patients (28%) showed divergent LOH status indicative of non-clonal origin (14/58 ADC, 8/20 SQC).
3. Activating EGFR mutations were found in both synchronous tumors in 4/58 ADC and none of the SQC showed EGFR mutation.
4. KRAS mutations were found in 20 cases (17 ADC, 3 SQC), including in both synchronous tumors in 13 cases but in only 1 tumor in 6 cases. 1 patient had divergent EGFR and KRAS mutations in separate tumors.
5. Combined allelotyping of LOH and EGFR and KRAS mutational status showed that 36% of synchronous primary tumors were not clonally related.  Remember these were tumors that were determined to be morphologically indistinguishable.
6. Impact of clonality of multifocal NSCLC on overall survival: Kaplan-Meier curves show a trend that patients with 2 clonally unrelated tumors have a better outcome but this did not reach statistical significance by univariate analysis.

Conculsions

There is no objective, specific anatomical or morphological criteria that reliably indicate a clonal or non-clonal origin of multiple NSCLC tumors.  Therefore, the authors recommend performing predictive molecular testing (EGFR and KRAS) separately in all cases with synchronous tumor nodules or allelotyping to identifiy clonally related tumors.

This paper will influence the way I approach these situations.  First, I will specifically incorporate the Girard criteria to identify morphologically indistinguishable cases.  Second, I will send out separate tumor specimens for EGFR and KRAS mutation analysis before assigning my pathologic staging to the case.  A couple practical questions are not addressed by this study.  Given the remarkable morphological heterogeneity of lung ADC--and the high percentage of "mixed" subtype tumors, isn't it possible that morphological disparate tumors could still be clonally related?  The authors don't specifically address the issue of mixed subtype in relation to "morphologically indistinguishable" tumors--but this would be a major practical issue.  Should we simply assume that morphologically different-ish tumors are non-clonal or do the testing anyway?  A great complimentary study would test all synchronous tumors regardless of histology since this study showed that morphological criteria could not predict clonal origin even in a narrow series of synchronous tumors that looked alike in pairs.  An intriguing point of this study is the possible relationship between clonal relationship and OS.  The authors rightly conclude that this should be tested in larger cohorts of NSCLC patients to identify any statistically significance to the trend that they identified.  I am perplexed as to why clonally unrelated tumors should have better outcomes.  If any one has any thoughts, I'd love to hear them--please post a comment with your thoughts. 

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.