Marcin Nicoś

Mechanisms of resistance to reversible inhibitors of EGFR tyrosine kinase in non-small cell lung cancer

Abnormalities of epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) patients consist of EGFR overexpression and EGFR (HER1) gene mutations. Structural dysfunction of the tyrosine kinase domain of EGFR is associated with the clinical response to tyrosine kinase inhibitors (TKI) in patients with NSCLC. The most common EGFR gene mutations occur as either deletions in exon 19 or as substitution L858R in exon 21 and cause a clinically beneficial response to gefinitib or erlotinib treatment. Unfortunately, the majority of patients finally develop resistance to these drugs. Acquired resistance is linked to secondary mutations localised in the EGFR gene, mainly substitution T790M in exon 20. Through intense research a few different mechanisms of resistance to reversible tyrosine kinase inhibitors have been identified: amplification of MET or IGF-1R genes, abnormalities of PTEN and mTOR proteins as well as rare mutations in EGFR and HER2 genes. Extensively investigated new drugs could be of significant efficiency in NSCLC patients with secondary resistance to reversible EGFR TKI.

Prevalence of rare EGFR gene mutations in nonsmall-cell lung cancer: a multicenter study on 3856 Polish Caucasian patients

Department of Pneumonology, Oncology and Allergology, Medical University of Lublin, Poland GENIM Ltd. Institute of Genetics and Immunology, Lublin, Poland Department of Clinical Oncology, Medical University of Poznan, Poland Department of Pathology, Specialist Pulmonary Hospital of Sokolowski, Zakopane, Poland Regional Centre of Oncology M. Kopernik Hospital, Lodz, Poland Mazovian Centre for Treatment of Lung Diseases and Tuberculosis, Otwock, Poland Department of Neurosurgery and Pediatric Neurosurgery, Medical University of Lublin, Poland E.J. Zeyland Wielkopolska Center of Pulmonology and Thoracic Surgery, Poznan, Poland Regional Oncology Centre in Gdansk, Poland Department of Clinical Patomorphology, Medical University of Lublin, Poland

Screening for ALK abnormalities in central nervous system metastases of non-small-cell lung cancer: ALK abnormalities in CNS metastases of NSCLC.

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%–7% of primary non‐small‐cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non‐smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ2 = 16.783) in former‐smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.

Screening for gene mutations in central nervous system metastases of non-small-cell lung cancer: Letter to the Editor

Non-small-cell lung cancer (NSCLC) is characterized by aggressive clinical course including frequent occurrence of distant metastases. Central nervous system (CNS) metastases are diagnosed in 20%– 40% of NSCLC patients and they are considered as a pharmacological sanctuary lesions for most cytotoxic agents. Molecularly targeted therapies have shown relatively high activity in CNS metastases in patients harboring “drugable” abnormalities in EGFR or ALK genes. Especially promising activity of the secondgeneration ALK inhibitors (alectinib, ceritinib) and sequential or concurrent application of EGFR/ALK TKIs and WBRT/stereotactic radiotherapy has been postulated (4, 6, 8). To date the knowledge on the effectiveness of molecularly targeted therapies in NSCLC patients with CNS metastases is relatively scarce. The patients with untreated CNS metastases are excluded from recent clinical trials investigating new therapies in lung cancer (4, 8). Therefore, we retrospectively assessed the spectrum of “drugable” abnormalities in 10 genes, in CNS metastases of NSCLC (145 FFPE tissue samples—45 females and 100 males; median age 60 6 8.8 years; PS 5 0 or 1; all patients chemotherapy and TKI na€ıve), and determined the relationship between molecular status and clinical characteristics of our patients. The studied group was heterogeneous in terms of histopathology (80 adenocarcinoma, 29 squamous cell carcinoma, 22 large cell cancer and 14 nototherwise-specified NSCLC patients) and smoking status (73 current-smokers, 21 former smokers, 36 non-smokers). The molecular profile of selected mutations was assessed using different molecular methods. Mutations in EGFR (exons 18–21), KRAS (codons: 12; 13; 61), NRAS (codons: 12; 61), BRAF (codon: 600), PTEN (codon: 233) and AKT1 (codon: 17) genes were analyzed with commercially available kits certified for in vitro diagnostic (Entrogen, Woodland Hills, California, USA) or TaqMan probes for research use only (Applied Biosystem, Carlsbad, California, USA). To analysis mutation in, PIK3CA (codons: 542; 545; 1047), MEK1 (codons: 56; 57; 67), HER2 (exon 20) and DDR2 (codon: 768) genes we used originally designed methods which based on allele-specific PCR (ASP-PCR) and highresolution melting PCR (HRM-PCR). Moreover, direct sequencing and multi-temperature single strand conformation polymorphism (MSSCP) techniques were used to confirm results obtained by originally designed methods. ALK abnormal protein was determined using automated immunohistochemistry with Positive Rabbit Monoclonal Antibody D5F3 (Ventana, Tucson, Arizona, USA) according to manufacturer instructions (9). To confirm ALK gene rearrangement we used FISH technique with Vysis ALK Break Apart FISH Probe Kit (Abbot Molecular, Des Plaines, Illinois, USA). The criteria of FISH analysis were in accordance with FDA guidelines (10). In 30 patients, the material was simultaneously available from primary and metastatic NSCLC tumors. We identified at least one abnormality in 59 cases (41%): KRAS—21.4% (31/145), EGFR—6.2% (9/145), ALK—4.8% (7/145), DDR2—2.1% (3/145), PIK3CA—2.1% (3/145), NRAS— 1.4% (2/145) and HER2, AKT1, PTEN, MEK1 respectively in 0.7% of patients (1/145) (Figure 1A). Coexistence of two mutations (KRAS and DDR2) was found in one patient. Mutations were significantly more frequently observed in adenocarcinoma compared to other histologic types of NSCLC (P 5 0.001; v 5 15.9; Figure 1B,C) and in non-smokers compared to former/current smokers (P 5 0.034; v 5 11.39, Figure 1D,E). The mOS of patients with mutations was insignificantly longer than in patients with wild-type of analyzed genes (16 vs. 11.7 months; P 5 0.084; HR 5 1.36). Cox multivariate logistic regression demonstrated that the factors significantly prolonging patients’ survival were younger age (<60 years) and mutations’ presence (overall model: P 5 0.0459; v 5 6.161). In 30 matched primary NSCLC tumors, 23% had EGFR and 7% had KRAS mutations. Heterogeneity between primary tumor and CNS metastases concerned only KRAS mutations. In five cases KRAS gene mutations were identified in both specimens, in one case only in primary tumor and in one case only in CNS metastases. Most of our observations are in concordance with large epidemiological data from studies focusing on primary tumors of NSCLC. Barlesi et al indicated the presence of driver mutations in six examined genes in 50% out of 17 664 European and Caucasian patients. Prevalence of analyzed mutations was as follows: KRAS mutations in 29% (4894/17 001) of cases, EGFR mutations in 11% (1947/17 706) of cases, ALK rearrangement in 5% (388/8134) of cases, BRAF mutations in 2% (262/13 906) of cases, PIK3CA mutations in 2% (252/ 10 678) of cases and HER2 mutations in 1% of patients (98/11 723). The presence of a genetic alterations was significantly associated with longer duration of response to both firstand second-line of treatment, as well as with longer first-line PFS and with longer mOS. Cox multivariate analysis confirmed that the presence of ALK rearrangements and EGFR and HER2 genes mutations had a favorable effect on prognosis (1). Kris et al reported the driver mutations in 64% (466/733) of American patients with adenocarcinoma. The analysis included ten genes and spectrum of particular disorders was fallowing: KRAS mutations in 25% (182/733) of cases, EGFR activation mutations in 17% (122/733) of cases, ALK rearrangements in 8% (57/733) of cases, HER2 mutations in 3% (19/733) of cases, BRAF mutations in 2% (16/733) of cases; PIK3CA mutations in <1% (6/733) cases; MET amplification in <1% (5/733) of cases; NRAS mutations in <1% (5/733) of cases; MEK1 mutation in <1% (1/733) of cases. The authors have not identified a mutation in AKT1 gene. Moreover, they noted the co-existence of two or more mutations in 3% (24/733) of cases. PIK3CA gene mutations commonly overlapped with others genetic abnormalities. Kris et al observed significantly longer mOS in patients with molecular abnormalities who received targeted therapies

Immunohistochemical assays incorporating SP142 and 22C3 monoclonal antibodies for detection of PD-L1 expression in NSCLC patients with known status of EGFR and ALK genes

Different immunohistochemical (IHC) assays were approved for PD-L1 expression examination on tumor cells in qualification to immune-checkpoint inhibitors therapy in NSCLC patients. These assays have some similarities, but also very serious differences. We assessed 2 IHC tests for PD-L1 expression evaluation in NSCLC tumors with different pathological diagnoses and genetic abnormalities. We enrolled 48 NSCLC patients (median age: 65 years) with known status of EGFR and ALK genes. We compared the effectiveness of PD-L1 expression examination of two IHC assays with 22C3 (Dako) and SP142 antibodies (Ventana). IHC tests were performed in resected tissue samples and in cellblocks from bronchoscopy biopsies (formalin-fixed paraffin-embedded). IHC staining was carried out on Dako Autostainer Link 48 and Ventana Benchmark GX. The percentage of tumors with PD-L1 expression of ≥5% and ≥50% on tumor cells was significantly (p<0.05) higher in assay with 22C3 (66.7% and 45.8%) than with SP142 antibody (39.6% and 22.9%). The median percentage of tumor cells with PD-L1 expression was significantly (p<0.0001) higher in test with 22C3 than with SP142 antibody. Percentage of squamous cell carcinoma (SCC) patients with PD-L1 expression was significantly higher than of non-SCC patients. Large group of patients without PD-L1 expression on tumor cells was identified among patients with common EGFR mutations and ALK rearrangement. Our results support that the highest PD-L1 expression on tumor cells occurs in SCC patients and in adenocarcinoma patients without common, druggable genetic abnormalities. The above mentioned results were clearly visible in IHC assay with 22C3 (strong cell staining).

Discrepancies between ALK protein disruption and occurrence of ALK gene rearrangement in Polish NSCLC patients

Background Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) rearrangement are predisposed to molecularly targeted therapies. Proper diagnostic is crucial for quick and correct patients qualification to optimal treatment method. Genetic tests to detect predictive factors could be performed sequentially. After excluding EGFR mutations, abnormal ALK protein expression should be tested using immunohistochemistry (IHC) method. In patients with disrupted ALK expression, the rearrangement of the ALK gene should be confirmed by FISH method. Despite few years of experience in analysis of these predictive factors, there are still problems in interpretation of diagnostic tests results. Especially, some recommendations for ALK IHC diagnosis are not precise. Methods Mutations in EGFR gene were examined using real-time PCR technique in 1,108 formalin-fixed paraffin-embedded (FFPE) tissues, 398 FFPE cell-blocks and 470 cytological specimens of NSCLC. The disrupted ALK protein expression was analysed in 1,100 samples including 782 histological and 306 cytological (cell-blocks) samples using IHC. Twelve materials (1.1%) were non-diagnostic in IHC. ALK gene rearrangement using FISH method was analysed in IHC positive cases. Results The frequency of EGFR mutations was 8.6%. EGFR mutations occurred significantly more often in females (P=0.00001, χ2=62.732) and in adenocarcinoma cases (P=0.0002, χ2=14.222). The exon 19 deletions (49%) and exon 21 Leu858Arg substitution (38%) were the most common, rare EGFR mutations occurred in 13% of patients. Any expression of abnormal ALK protein was detected in 202 cases (18.57%). ALK gene rearrangement was confirmed in 49 cases (4.5%). ALK gene rearrangement is significantly more common in female than in male (P=0.0105, χ2=6.541). In patients with ALK gene rearrangement, the median percentage of nuclei with ALK rearrangement was only 25.5%. The polysomy (≥4 gene copy number per nuclei) of ALK gene was observed in 39 cases (21.4% of patients with diagnostic result of FISH examination). Median number of ALK gene copy per nuclei was 2.9±0.77. Significant positive correlation between percentage of cells with abnormal ALK expression in IHC test and percentage of nuclei with ALK rearrangement in FISH method was detected (R=0.617, P<0.00001). Significant negative correlation between the number of copies of ALK gene and the percentage of cells with expression of abnormal ALK was observed (R=-0.2004, P<0.05). ALK gene rearrangement was significantly more frequently observed in the material with coarse-grained cytoplasmic and membranous IHC staining than in materials with light cytoplasmic stippling. The occurrence of cytoplasmic stippling correlated with the increase of ALK gene copy number. Conclusions We indicated that diagnosis of ALK disruption in NSCLC patients should be notably careful using IHC and FISH methods. Recommendations for ALK diagnosis should include the way of interpretation of cases with low percentage of cells with abnormal ALK protein expression in IHC test, character of IHC reaction, and cases with ALK gene polysomy in FISH method.

Preventing central nervous system metastases in non-small cell lung cancer

ABSTRACT Introduction: There are no effective central nervous system (CNS) metastases prevention methods in lung cancer patients. Prophylactic cranial irradiation has a limited effectiveness and relatively high toxicity. Systemic chemotherapy is not relevant in reducing the risk of CNS in lung cancer patients. The understanding of molecular background of brain metastases in non-small cell lung cancer (NSCLC) patients could contribute to the development of personalized treatments for such patients. Areas covered: This article summarizes the latest clinical trials concerning the use of radiotherapy, chemotherapy, molecularly targeted therapies, and immunotherapy in lung cancer patients, with particular consideration of brain lung cancer metastasis prevention. The literature search was undertaken via PubMed and EMBASE searches and relevant articles are included in this review. Expert commentary: The recent data supports that EGFR-TKIs and ALK inhibitors are clinically relevant for first-line treatment to prevent and treat CNS metastases in molecularly selected NSCLC patients. In the future, high hopes for the prevention of CNS metastases in NSCLC patients are associated with immunotherapy concerning immune check-points inhibitors.

Analysis of KRAS, NRAS, BRAF, and PIK3CA mutations could predictmetastases in colorectal cancer: A preliminary study

BACKGROUND Colorectal cancer (CRC) is usually diagnosed in the metastatic stage, when chemotherapy and molecularly-targeted therapies, instead of surgery, play the most important therapeutic role. Application of anti-epidermal growth factor receptor (EGFR) therapy requires the analysis of RAS mutation status and only RAS wild-type (wt) patients are qualified for the therapy. OBJECTIVES The objective of this study was to analyze driver mutations in KRAS, NRAS, BRAF, and PIK3CA genes in CRC patients. MATERIAL AND METHODS We assessed the KRAS, NRAS, BRAF, and PIK3CA genes in 102 inoperable, locally advanced and advanced CRC patients. Real-time polymerase chain reaction (RT-PCR) and high resolution melt PCR (HRM-PCR) techniques with DNA intercalating dye were applied in the study. RESULTS Forty-six patients demonstrated the presence of examined mutations (45.1%). No significant differences in driver mutation occurrence between men and women, as well as between younger (<65 years) and older (≥65 years) patients were found. The mutations were present significantly more frequently in metastatic than in primary tumors (p = 0.039) due to the high incidence of KRAS gene mutations in metastatic tissue. BRAF and PIK3CA mutations were found only in primary tumors. The incidence of PIK3CA mutations was significantly higher (11.77%) in early than in advanced stages of the disease (1.96%; p = 0.05); NRAS mutations were found only in metastatic cancer (7.85%; p = 0.041). Only a single mutation of the PIK3CA and no mutations of NRAS were found in rectal cancer. CONCLUSIONS Our results have shown low occurrence of driver mutations in Polish CRC patients, involving also mutations in rarely tested genes. The extent of the research panel of additional mutations could contribute to creating a better method of qualifying patients for molecularly targeted therapies and obtaining a better outcome for these therapeutic strategies.

Do we apply a personalised lung cancer therapy? Use of molecular tests in scheduling a multilineage treatment in a patient with lung adenocarcinoma

Molecularly targeted therapies, which can be used in genetically selected patients, play an increasing role in the multidisciplinary approach to the treatment of lung cancer. Treatment personalisation extends the scope of therapy, prolongs survival of patients and decreases the risk of life-threatening side effects. In this report, we present the diagnostic and therapeutic history of a 57-year-old male with lung adenocarcinoma and activating EGFR gene mutation. The whole therapeutic approach involved a diagnostic segmentectomy, cytoreductive surgery of the primary tumour, and a palliative hemipelvectomy of metastases in the right hip joint followed by adjuvant radiotherapy as well as six lines of systemic treatment based on standard cytostatics and novel personalised agents. Regardless of a patient’s good performance status and relatively good tolerance of the treatment, futile continuation of the therapy despite the lack of a longer stabilisation of the disease remains questionable.

Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique

PurposeRT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods.MaterialsWe evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status.Results21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results.ConclusionUtility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.