Zoltan Lohinai

Subtype-specific KRAS mutations in advanced lung adenocarcinoma: A retrospective study of patients treated with platinum-based chemotherapy

BACKGROUND Platinum-based chemotherapy is the most common treatment in advanced-stage lung adenocarcinoma. Because the clinical significance of KRAS mutational status in this setting has not yet been clearly determined, a mutation subtype-specific analysis was performed in the so far largest cohort of Caucasian patients with KRAS mutant advanced-stage lung adenocarcinoma treated with platinum-based chemotherapy. METHODS 505 Caucasian stage III-IV lung adenocarcinoma patients with known amino acid substitution-specific KRAS mutational status and treated with platinum-based chemotherapy were included. The correlations of subtype-specific KRAS mutations with smoking status, progression-free and overall survival (PFS and OS, respectively) and therapeutic response were analysed. RESULTS Among 338 KRAS wild-type, 147 codon 12 mutant and 20 codon 13 mutant patients, there were no mutation-related significant differences in PFS or OS (P values were 0.534 and 0.917, respectively). Eastern Cooperative Oncology Group (ECOG) status and clinical stage were significant independent prognostic factors. KRAS mutation showed a significant correlation with smoking status (P=0.018). Importantly, however, G12V KRAS mutant patients were significantly more frequent among never-smokers than all other codon 12 KRAS mutant (G12x) subtypes (P=0.016). Furthermore, this subgroup tended to have a higher response rate (66% versus 47%; P=0.077). A modestly longer median PFS was also found in the G12V mutant cohort (233days; versus 175days in the G12x group; P=0.145). CONCLUSIONS While KRAS mutation status per se is neither prognostic nor predictive in stage III-IV lung adenocarcinoma, subtype-specific analysis may indeed identify clinically relevant subgroups of patients that may ultimately influence treatment decisions.

Distinct Epidemiology and Clinical Consequence of Classic Versus Rare EGFR Mutations in Lung Adenocarcinoma.

Introduction: Although classic sensitizing mutations of epidermal growth factor receptor (EGFR) are positive predictive markers for EGFR tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma, there are rare EGFR mutations with unknown epidemiology and influence on prognosis and TKI response. Methods: Eight hundred and fourteen lung adenocarcinoma patients with KRAS and/or EGFR mutation analyses for TKI therapy indication were identified. Six hundred and forty-five patients were included in the epidemiological analysis. The clinical outcome was analyzed in 419 advanced-stage patients with follow-up data. Results: Four hundred and eighty (59%) KRAS/EGFR double wild-type, 216 (27%) KRAS mutant, 42 (5%) classic, 49 (6%) rare, and 27 (3%) synonymous EGFR mutant cases were identified. Twenty previously unpublished non-synonymous mutations were found. Rare EGFR mutations were significantly associated with smoking (vs. classic EGFR mutations; p = 0.0062). Classic EGFR mutations but not rare ones were independent predictors of increased overall survival (hazard ratios, 0.45; 95% confidence intervals, 0.25–0.82; p = 0.009). TKI therapy response rate of patients harboring classic EGFR mutations was significantly higher (vs. rare EGFR mutations; 71% vs. 37%; p = 0.039). Patients with classic or sensitizing rare (G719x and L861Q) EGFR mutations had significantly longer progression-free survival when compared with the remaining rare mutation cases (12 vs. 6.2 months; p = 0.048). Conclusions: The majority of rare EGFR mutations was associated with smoking, shorter overall survival, and decreased TKI response when compared with classic EGFR mutations. However, studies characterizing the TKI sensitizing effect of individual rare mutations are indispensable to prevent the exclusion of patients with sensitizing rare EGFR mutations who may benefit from anti-EGFR therapy.

KRAS-mutation incidence and prognostic value are metastatic site-specific in lung adenocarcinoma: Poor prognosis in patients with KRAS mutation and bone metastasis

Current guidelines lack comprehensive information on the metastatic site-specific role of KRAS mutation in lung adenocarcinoma (LADC). We investigated the effect of KRAS mutation on overall survival (OS) in this setting. In our retrospective study, 500 consecutive Caucasian metastatic LADC patients with known KRAS mutational status were analyzed after excluding 32 patients with EGFR mutations. KRAS mutation incidence was 28.6%. The most frequent metastatic sites were lung (45.6%), bone (26.2%), adrenal gland (17.4%), brain (16.8%), pleura (15.6%) and liver (11%). Patients with intrapulmonary metastasis had significantly increased KRAS mutation frequency compared to those with extrapulmonary metastases (35% vs 26.5%, p = 0.0125). In contrast, pleural dissemination and liver involvement were associated with significantly decreased KRAS mutation incidence (vs all other metastatic sites; 17% (p < 0.001) and 16% (p = 0.02) vs 33%, respectively). Strikingly, we found a significant prognostic effect of KRAS status only in the bone metastatic subcohort (KRAS-wild-type vs KRAS-mutant; median OS 9.7 v 3.7 months; HR, 0.49; 95% CI, 0.31 to 0.79; p  = 0.003). Our study suggests that KRAS mutation frequency in LADC patients shows a metastatic site dependent variation and, moreover, that the presence of KRAS mutation is associated with significantly worse outcome in bone metastatic cases.

High circulating activin A level is associated with tumor progression and predicts poor prognosis in lung adenocarcinoma

Activin A (ActA)/follistatin (FST) signaling has been shown to be deregulated in different tumor types including lung adenocarcinoma (LADC). Here, we report that serum ActA protein levels are significantly elevated in LADC patients (n=64) as compared to controls (n=46, p=0.015). ActA levels also correlated with more advanced disease stage (p<0.0001) and T (p=0.0035) and N (p=0.0002) factors. M1 patients had significantly higher ActA levels than M0 patients (p<0.001). High serum ActA level was associated with poor overall survival (p<0.0001) and was confirmed as an independent prognostic factor (p=0.004). Serum FST levels were increased only in female LADC patients (vs. female controls, p=0.031). Two out of five LADC cell lines secreted biologically active ActA, while FST was produced in all of them. Transcripts of both type I and II ActA receptors were detected in all five LADC cell lines. In conclusion, our study does not only suggest that measuring blood ActA levels in LADC patients might improve the prediction of prognosis, but also indicates that this parameter might be a novel non-invasive biomarker for identifying LADC patients with organ metastases.

From bench to bedside: Attempt to evaluate repositioning of drugs in the treatment of metastatic small cell lung cancer (SCLC)

Backgrounds Based on in vitro data and results of a recent drug repositioning study, some medications approved by the FDA for the treatment of various non-malignant disorders were demonstrated to have anti-SCLC activity in preclinical models. The aim of our study is to confirm whether use of these medications is associated with survival benefit. Methods Consecutive patients with pathologically confirmed, stage 4 SCLC were analyzed in this retrospective study. Patients that were prescribed statins, aspirin, clomipramine (tricyclic antidepressant; TCA), selective serotonin reuptake inhibitors (SSRIs), doxazosin or prazosin (α1-adrenergic receptor antagonists; ADRA1) were identified. Results There were a total of 876 patients. Aspirin, statins, SSRIs, ADRA1, and TCA were administered in 138, 72, 20, 28, and 5 cases, respectively. A statistically significant increase in median OS was observed only in statin-treated patients when compared to those not receiving any of the aforementioned medications (OS, 8.4 vs. 6.1 months, respectively; p = 0.002). The administration of SSRIs, aspirin, and ADRA1 did not result in a statistically significant OS benefit (median OS, 8.5, 6.8, and 6.0 months, respectively). The multivariate Cox model showed that, besides age and ECOG PS, radiotherapy was an independent survival predictor (Hazard Ratio, 2.151; 95% confidence interval, 1.828–2.525; p <0.001). Conclusions Results of drug repositioning studies using only preclinical data or small numbers of patients should be treated with caution before application in the clinic. Our data demonstrated that radiotherapy appears to be an independent survival predictor in stage 4 SCLC, therefore confirming the results of other prospective and retrospective studies.

Reply to Rare Versus Artifactual EGFR Mutations

EGFR-tyrosine kinase inhibitor (TKI) sensitivity of theses rare mutations differ from classic EGFR mutant. As a matter of fact, they are similar to EGFR wildtype patients. Lohinai et al.’s results are in line with previous report. Rare EGFR mutations had a high incidence, almost the same as classic and synonymous (5%, 6%, and 4%; respectively). They were also associated with smoking and poor response to EGFR-TKI as opposed to classic mutations. In addition, of the 49 rare mutations, 20 were never described before (not registered in the Catalogue of Somatic Mutations in Cancer [COSMIC] database). We must be aware that the diagnostic of advanced non–small-cell lung cancer is often made by biopsy rather than resected specimen, and these rare mutations were all noted from samples of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. Artifacts can easily be observed when sequencing multiple polymerase chain reaction (PCR) amplification products of very small amounts of DNA, particularly if the DNA is isolated from paraffin-embedded tissue. Therefore, the hypothesis that several of these rare mutations might be actually PCR-amplification artifacts must be discussed. Akbari et al. performed direct sequencing of the PCR-amplified coding region of the uracil-DNA glycosylase gene using DNA isolated from FFPE tissue specimens from patients with gastric cancer (only one mutation in the uracil-DNA glycosylase gene in human cancer had been previously reported in a sporadic human glioblastoma). In nearly 35% of the samples, they detected base substitutions, which, after further investigation, proved to be PCR artifacts. They also demonstrated that very low concentration of DNA template in PCR mix can give rise to false base substitutions. Marchetti et al. identified 45 rare EGFR mutations in 70 samples of lung tumor DNA extracted from FFPE tissue, and they were all found to be artifacts. This was confirmed because they also found the same mutations in multiple amplifications of DNA extracted from FFPE of normal tissue (50 patients’ lymph nodes without neoplasm). In addition, series whose fresh-frozen tissue was used do not observed rare EGFR mutations. There are some hypotheses for the occurrence of artifactual mutations. For instance, base damages and largescale DNA fragmentation caused by the chemical preparation of FFPE samples might result in cytosine deamination. Thereby the taq DNA polymerase would insert an adenosine instead of a guanosine resulting in C → T and G → A transitions (so-called “a-rule”). Moreover, degraded PCR products allowed the taq DNA polymerase performs a “jump” from a damaged template to another to continue the extension. Some strategies were reported to prevent artifactual mutations. Routine application of microdissection and use of fresh-frozen tissue to enrich tumorcell DNA are one option. Also, if small amounts of DNA extract from FFPE were inevitable, addition of uracil-Nglycosylase to the DNA template before PCR amplification and the examination of multiple amplifications are imperative. Because analysis of EGFR gene is mandatory for decision regarding EGFR-TKI use, the correct interpretation of EGFR exons 18 to 21 sequencing and its genetic alterations is crucial to select patients whom would benefit from treatment. Therefore, these data preclude the indiscriminate use of EGFR-TKI in patients harboring EGFR rare mutations and drive us to a more careful molecular analysis to identify molecular artifacts.

Differences in the Epidemiology of Rare EGFR Mutations in Different Populations

In Response: We thank Elghissassi et al. for their comment on our study. This is a great opportunity to further discuss the differences in the epidemiology of rare epidermal growth factor receptor gene (EGFR) mutations in different patient populations. The complete coverage of exons 18 to 21 and the EGFR analysis in patients with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations—as we all agree— could very well be one reason for the increased rate of rare EGFR mutations. Importantly, it seems that smoking status can also have a great—and indeed patient population–dependent—impact on the rate of rare EGFR mutations. Unfortunately, in Hungary as well as in our patient cohort, the number of current smokers is very high, which could lead to enrichment for rare EGFR mutations. Interestingly, unlike in white cohorts, the rare EGFR mutations in Asian populations do not seem to be associated with smoking. Notably, the findings of Errihani et al. suggest that the epidemiology of such mutations in Morocco has greater resemblance to that in the Asian population than to that in white study cohorts. We cannot emphasize enough that the lack of identical methods makes side-by-side comparison of the different studies very difficult. A number of commercial mutation analysis methods provide very high sensitivity, but only for a preselected set of molecular alterations that might enrich for classical EGFR mutations. In contrast, direct (Sanger) sequencing has a lower sensitivity but can identify novel mutations. Indeed, a number of rare mutations described in our study were reported in lung adenocarcinoma for the first time. Of note, we cannot rule out that some of

O13. Subtype-specific KRAS mutations in advanced lung adenocarcinoma

Background So far there is no strong evidence for the predictive effects of KRAS mutations on the clinical outcome of platinum-based chemotherapy in advanced lung adenocarcinoma. However, most of the studies did not take into account the subtype-specific mutations in the KRAS gene.