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J. Botling
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MTE03 - Basics of Molecular Biology for the Clinician (Ticketed Session) (ID 297)
- Event: WCLC 2016
- Type: Meet the Expert Session (Ticketed Session)
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 07:30 - 08:30, Schubert 4
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MTE03.01 - Basics of Molecular Biology for the Clinician (ID 6542)
07:30 - 08:30 | Author(s): J. Botling
- Abstract
Abstract:
The rapid development of molecular biology in recent years has allowed us to understand the main molecular steps involved in the development and progression of lung cancer. The identification of molecular alterations in specific tumor genes that function as key drivers for neoplastic growth has laid the foundations for new therapeutic approaches with targeted agents. An accurate detection of target mutation is mandatory for an efficient treatment. The main limitation of targeted therapies is the occurrence of acquired resistance that makes cancer unresponsive to treatment. In many cases, through the acquisition of additional (secondary) mutations the tumor is able to acquire the heterogeneity which may enable it to adapt to various conditions of the microenvironment, including those determined by the effect of treatment with specific drugs. New generation drugs are constantly under development to overcome tumor resistance and increase survival of lung cancer patients. In this process, a constant monitoring of the mutational status of the tumor is required. Different types of genetic alterations are involved in tumor development, progression, and induction of resistance, including single nucleotide variants, indels, amplifications, fusions etc. Mutation detection before first line treatment is usually performed on tissue or cytological samples. Resected tumor samples, biopsies and cytological specimens are available in about 25%, 35% and 40% of NSCLC patients, respectively. At progression, a re-byopsy should be obtained to detect the emergence of resistance-inducing mutations. Transbronchial tissue biopsy is the most common sampling method used for re-biopsy. However, several factors limit the success rate of re-biopsy, such as the performance status of the patient, the difficulty of accessing some tumor sites, and the invasiveness of sampling methods. When the amount and/or quality of the biological material is insufficient for molecular analysis, circulating free DNA (cfDNA) can represent a valid alternative in selected patients. Liquid biopsies have several advantages over tissue or cells: they are less invasive, can be repeated over time, and have a more rapid turnaround time. However, there are some critical issues that must be considered: 1) the possibility to detect a mutation in cfDNA is dependent on several clinicopathological parameters, including tumor type, tumor burden, and particularly tumor stage (a locally advanced tumor has a significantly lower probability to spread mutant DNA in the blood than a metastatic tumor); 2) a large amount of wild-type DNA circulates in the plasma with only trace amounts of the mutant allele; therefore, the analysis of genetic aberrations in cfDNA is challenging, requiring well standardized pre-analytical/analytical protocols and dedicated techniques with high sensitivity and specificity. Different technologies/protocols are required for the detection of these genetic aberrations. Robust and sensitive molecular biology techniques are nowadays available to detect mutations in driver genes before initiating a targeted treatment or to identify the emergence of secondary mutations at disease progression. The use of multimarker assays, and in particular next generation sequencing, is progressively becoming popular, allowing on one hand to reduce the working time, costs per single assay, and the amount of nucleic acids required for testing and increasing, in the other hand, throughput and overall quality. Recently, semi-quantitative or quantitative detection methods for the assessment of genetic aberrations in cfDNA have been developed with a number of potential clinical implications. An accurate quantification of mutated alleles in cfDNA during the first days of treatment could: a) complement or replace more expensive and invasive methods to assess response in treated patients; b) represent a new way to compare the effectiveness of different drug; c) be an additional tool to evaluate the best treatment regimen for patients. In addition, a periodic quantification of the mutation burden during all the treatment time could allow an early detection of resistance-inducing mutations for possible changes to therapy.
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-022 - Establishing Reflex NGS Testing in NSCLC in a Regional Network of County Hospitals in Central Sweden (ID 4759)
14:30 - 15:45 | Author(s): J. Botling
- Abstract
Background:
Extended genetic testing of NSCLC tumor samples provides a foundation for personalized cancer treatment and use of new targeted medication. Testing with Next Generation Sequencing (NGS), mostly performed at university hospitals, has not been available for all patients due to geographic and economic reasons. Many lung cancer patients carry a heavy burden of disease and extensive travelling can negatively impact quality of life. The ability to perform a modern state-of the art work-up at local hospitals, without compromising on diagnostic quality, will enable equal access to personalized treatment for lung cancer patients.
Methods:
In Gävle County hospital routine diagnostic immunohistochemistry (IHC) on biopsies is performed at the local pathology lab. In the case of NSCLC the formalin-fixed, paraffin embedded (FFPE) tissue samples are sent to Uppsala University hospital for further molecular pathology and NGS testing. A targeted NGS test (18 gene panel) was established for mutation screening of small biopsies and cytology specimens (Moens et al., J Mol Diagn, 2015). Fusion genes - ALK, ROS1 and RET - are analysed by IHC, FISH and nanoString. Structured biobanking of surplus biopsies and blood samples during treatment, for explorative biomarker testing and research, was set up as a regional extension of the UCAN infrastructure, including detailed registration of clinical baseline and real-time follow-up data in a dedicated database.
Results:
Inclusion of patients in the biobanking cohort started gradually during 2015 in Uppsala, and in February 2016 in Gävle. The cumulative inclusion in the UCAN biobank is updated at www.u-can.uu.se (see Statistics). To date (July 2016) 70 patients have been included at Gävle County hospital covering 95% of the newly diagnosed NSCLC patients. So, far 242 patients from the region were tested by NGS yielding 23 EGFR+ (9.5%), 75 KRAS+ (31%), 5 BRAF+ (2.1%, codon 600), 2 MET (0.8%, exon 14 skipping), 1 ERBB2 (0.4%, exon 20 insertion), and 6 PIK3CA (2.5%, exon 9/20) cases. Fusion gene analysis resulted in 5 ALK+ (2.1%), 1 ROS1 and 1 RET patients.
Conclusion:
Decentralised local patient care, tissue/blood sampling and biobanking in combination with centralised molecular testing allows advanced lung cancer diagnostics and clinical research in networks of county hospitals. Survival benefits from modern targeted drugs, for national lung cancer cohorts, can only be achieved and evaluated in population-based settings without bias related to selective referral to major cancer centers.
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P1.02-063 - Mutation Profiling by Targeted Next-Generation Sequencing of an Unselected NSCLC Cohort (ID 4147)
14:30 - 15:45 | Author(s): J. Botling
- Abstract
Background:
Non-small cell lung cancer (NSCLC) is a heterogeneous disease, with a wide diversity when it comes to molecular variations. In the non-squamous subset a large variety of altered driver genes have been identified.
Methods:
The mutational status was evaluated in a consecutive Swedish NSCLC cohort consisting of 354 patients, who underwent surgical resection between 2006 and 2010. DNA was prepared from either fresh frozen or formalin fixed paraffin embedded tissue (FFPE) and used for library preparation using a Haloplex gene panel and subsequently sequenced on an Illumina Hiseq instrument. The gene panel covers all exons of 82 genes, previously identified in NSCLC. The panel design utilizes two strand capture and reduced target fragment length compatible with degraded FFPE samples (Moens et al., J Mol Diagn, 2015).
Results:
All previously known hotspot alterations in the driver genes KRAS, EGFR, HER2 (exon 20 insertions), NRAS, BRAF, MET (exon 14-skipping) and PIK3CA (exon 9 and 20) were analyzed in the 252 non-squamous cases, see figure. KRAS mutations were found in 98 patients (39%) whereas EGFR alterations were present in 33 (13%). The prevalence of KRAS mutations is higher than normally reported and could be due to the large fraction of smokers included in this cohort. The EGFR prevalence is a bit higher than previously demonstrated (Sandelin et al. Anitcancer Res, 2015). Mutations in the other driver genes were detected at low frequencies (HER2(3%), BRAF(2%), NRAS(1%), MET(1%) and PIK3CA(1%)). Figure 1
Conclusion:
The preliminary analysis of mutational status in this large unselected Swedish NSCLC cohort reveals mutation frequencies in the common driver genes resembling previous reports on western populations with a high smoking rate. Ongoing analysis of the remaining genes will be used for pathway analysis and could provide a more complete picture of the lung cancer pathogenesis.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-021 - Reproducibility of Comprehensive Histologic Assessment and Refining Histologic Criteria in P Staging of Multiple Tumour Nodules (ID 5365)
14:30 - 15:45 | Author(s): J. Botling
- Abstract
Background:
Multiple tumor nodules (MTNs) are being encountered, with increasing frequency with the 8[th] TNM staging system recommending classification as separate primary lung cancers (SPLC) or intrapulmonary metastases (IM). Pathological staging requires assessment of morphological features, with criteria of Martini and Melamed supplanted by comprehensive histologic assessment of tumour type, predominant pattern, other histologic patterns and cytologic features. With publication of the 2015 WHO classification of lung tumours, we assessed the reproducibility of comprehensive histologic assessment and also sought to identify the most useful histological features.
Methods:
We conducted an online survey in which pathologists reviewed a sequential cohort of resected multifocal tumours to determine whether they were SPLC, IM, or a combination. Specific histological features for each nodule were entered into the database by the observing pathologist (tumour type, predominant adenocarcinoma pattern, and histological features including presence of lepidic growth, intra-alveolar cell clusters, cell size, mitotic rate, nuclear pleomorphism, nucleolar size and pleomorphism, nuclear inclusions, necrosis pattern, vascular invasion, mucin content, keratinization, clear cell change, cytoplasmic granules¸ lymphocytosis, macrophage response, acute inflammation and emperipolesis). Results were statistically analyzed for concordance with submitting diagnosis (gold standard) and among pathologists. Consistency of each feature was correlated with final determination of SPLC vs. IM status (p staging) by chi square analysis and Fisher exact test.
Results:
Seventeen pathologists evaluated 126 tumors from 48 patients. Kappa score on overall assessment of primary v. metastatic status was 0.60. There was good agreement as measured by Cohen’s Kappa (0.64, p<0.0001) between WHO histological patterns in individual cases with SPLC or IM status but proportions for histology and SPT or IM status were not identical (McNemar's test, p<0.0001) and additional histological features were assessed. There was marked variation in p values among the specific histological features. The strongest correlations (<0.05) between p staging status and histological features were with nuclear pleomorphism, cell size, acinus formation, nucleolar size, mitotic rate, nuclear inclusions, intra-alveolar clusters and necrosis pattern. Correlation between lymphocytosis, mucin content, lepidic growth, vascular invasion, macrophage response, clear cell change, acute inflammation keratinization and emperipolesis did not reach a p value of 0.05.
Conclusion:
Comprehensive histologic assessment shows good reproducibility between practicing lung pathologists. In addition to main tumour type and predominant patterns, nuclear pleomorphism, cell size, acinus formation, nucleolar size, and mitotic rate appear to be useful in distinguishing between SPLC and IM.
