The Daily Sign-Out

Medscape Interview with NIH Director

Medscape recently produced a fascinating interview with Dr. Francis Collins, Director of the National Intitutes of Health that focuses on genomics but ranges broadly over several areas, including medical education and preventative medicine.  It's not that long and there is also a text format if you don't want to watch the interview.  Highly recommended.  This a thought-provoking interview.

I think Dr. Collins is overly optimistic about the impact of genomics on day-to-day patient care.  Pharmacogenomics, for one example he uses, has not made any impact thus far in my experience.  While it is a tool, perhaps, for demonstrating why a patient may not be responding to a particular drug, the use of pharmacogenomic testing before initiating treatment has not been prospectively studied to my knowledge in any trials, for any drug.  This leaves aside the further studies regarding cost-benefit analyses as well as the issue of payment/reimbursement.  Right now, it seems cheaper and tried to put a patient on a standard dose and see what happens.

Another example is the idea of genomic sequencing of cancers.  It sounds good because it provides a "personalized" profile of a cancer.  What about tumor heterogeneity?  Do we sequence the primary tumor or metastasis?  Which part of the primary tumor?  Which metastasis?  Finally, how does one compare "unique" tumor genotypes and develop groups to statistically compare treatment responses?  At some point, patients have to be lumped together (and thus muddying up personalized differences) in order to find appropriate guidelines for treatment and provide some prognostic information.

Lots more to think about!

Review of microsatellite instability (MSI) in colorectal cancer

Clinical Cancer Review just published an "OnlineFirst" article on February 2, 2012 (link to abstract) that reviews microsatellite instability (MSI) in colorectal cancer.  There have been several recent reviews on this topic in various journals, including Archives of Pathology and Laboratory Medicine in October 2011.  The review by Drs Geiersbach and Samowitz in Archives is excellent (and recommended too) but has a decidedly technical, lab-oriented focus. I recommend this one to pathologists looking for a more clinically-oriented review that especially addresses current and future studies looking at how the molecular heterogeneity of "MSI" tumors may have different chemosensitivity responses.  It is very readable and certainly is the most current.  If you're looking for an article that makes you look good in tumor board, this is it!

A couple of highlights:

• MSI as prognostic marker in CRC.  Observational studies, retrospective data from randomized clinical trials and meta-analyses have consistently shown that tumors that show MSI or deficient mismatch repair (MMR) are independently associated with improved survival and reduced recurrence rates compared with individuals with MSS tumors.
• ECOG 5202 is a ongoing trial of stage II CRC patients stratifying them into low-risk versus high-risk based on MSI and allelic loss of 18q.  Low-risk patients (MSI and intact 18q) are assigned to an observational arm post-op while high-risk patients (MSS and allelic loss of 18q) are assigned to standard post-op chemo for stage III patients (FOLFOX).  Unfortunately, this study will not address the use of MSI as a predictive marker for response to 5-FU therapy.
• MSI as predictive marker in CRC.  Mutliple studies, including RCTs, retrospective case series, in vitro cell line data, and a meta-analysis, have consistently shown that MSI is a negative predictive marker for response to 5-FU.  It has been recently recommended that patients with stage II colon cancer showing MSI not receive 5-FU as adjuvant therapy given their favorable prognosis and lack of benefit from 5-FU.  Incidentally, in discussing whether to routinely test for MSI in stage II and II patients with the oncologists, it was evident that there is a lot of "catch-up" and education that needs to be done here. 
• Currently, there are only limited data in MSI tumors from patients treated with oxaliplatin combined with 5-FU and predictive information is lacking.  This is an important confounding problem interpreting the literature since standard first-line treatment for CRC is 5-FU and oxaliplatin.  It appears, however, from in vitro studies that oxaliplatin sensitivity is independent of the MMR system.
• Since KRAS testing is becoming more routine for CRC, especially in high stage III/stage IV cancers, it is important also to note that sporadic MSI CRCs (with epigenetic inactivation of hMLH1) show frequent (~50%) co-occurrence of BRAFV600E mutations (compared to an overall BRAF mutation frequency of about 10% among all CRCs).  Note: The presence of a BRAF mutation indicates a sporadic MSI CRC and essentially excludes a diagnosis of Lynch syndrome.
• There's a lot of interesting stuff on possible additional targets for therapy you should check out.

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 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).

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.

A Consensus Statement of EGFR Mutation Testing in NSCLC

The International Association for the Study of Lung Cancer (IASLC) and European Thoracic Oncology Platform recently published a consensus statement regarding EGFR mutation testng in NSCLC.  There was agreement that the decision to request EGFR mutation testing should be made by the treating physician.  The worskshop group underscored that the most important issue is cooperation and communication between the various disciplines with regard to tumor specimen handling.  The group did not reach a consensus on a preferred methodology but did stress the need for good laboratory practice with regard to validation and standardization of whatever methodology is used.  You can read more in the October 2010 Journal of Thoracic Oncology.

This is interesting because of the fact that a number of institutions already are routinely referring NSCLC tumors for molecular testing, usually incorporating a panel that includes EGFR, KRAS, and ALK-EML4.  Moreover, routine EGFR mutation testing for NSCLC is being aggessively marketing by various companies.  I suppose that these "consensus" recommendations do not truly reflect what is already happening in practice, at least at "academic" medical centers in the U.S.  Perhaps someone in the U.K. or Europe might comment.