•Liquid biopsy alone could underestimate coexistence of resistance mechanisms.•T790M plasma levels should be taken into account for clinical decisions.•Low EGFR-T790M/activating mutation ratio may ...suggest tissue re-biopsy.•p53 and Rb1 expression on diagnostic biopsy may predict SCLC transformation.
EGFR T790M mutation is the most common mechanism of resistance to first-/second-generation EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) and could be overcome by third-generation EGFR-TKIs, such as osimertinib. Liquid biopsy, a non-invasive technique used to test the presence of the resistant mutation, may help avoiding tissue re-biopsy. However, analysing only circulating-free DNA, information about other less frequent and coexisting resistance mechanisms may remain unrevealed.
All patients reported in this series participated in the ASTRIS trial, a real world treatment study testing the efficacy of osimertinib (80mg os die) in advanced T790M-positive NSCLC progressed to prior EGFR-TKI. Patients were considered eligible to osimertinib if T790M positive on tissue or plasma samples. In our patients, EGFR molecular testing on blood sample was conducted with digital droplet PCR (ddPCR).
We report our experience of five patients treated with osimertinib after T790M detection on liquid biopsy that presented a disease progression at first tumor assessment mediated by SCLC transformation, as evidenced at tissue re-biopsies. All patients showed low ratio T790M/activating mutation in the blood before osimertinib (lower than 0.03). For three patients, EGFR mutational analysis was T790M-negative when re-assessed by using a less sensitive method (therascreen®) on the same liquid biopsy sample analysed by ddPCR before osimertinib therapy.
Although liquid biopsy is a relevant tool to diagnose T790M presence in NSCLC patients resistant to EGFR-TKI, in case of a low ratio T790M/activating mutation, tissue biopsy should be considered to exclude the presence of SCLC transformation and/or other concomitant resistance mechanisms.
Trastuzumab has recently shown efficacy in the treatment of HER2-positive advanced gastric adenocarcinoma. Although antibody-based therapies target the metastatic disease, HER2 status is usually ...evaluated in the primary tumour because metastatic sites are rarely biopsied. The aim of this study was to compare HER2 status in primary and paired metastatic sites of gastric adenocarcinoma.
The HER2 status was assessed by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in 72 secondary lesions of gastric adenocarcinoma and in the corresponding primary tumours.
Concordance of FISH results, evaluable in 68 primary and matched metastatic sites, was 98.5%. Concordance of IHC results, available in 39 of the 72 paired cases, was 94.9%. Only one case showed discordance between primary tumour and metastasis, being negative by both IHC and FISH in the primary and showing HER2 overexpression and amplification in the corresponding pancreatic lymph node metastasis.
The high concordance observed between HER2 results obtained by both IHC and FISH on primary tumours and corresponding metastases suggests that in gastric cancer HER2 status is maintained in most cases unchanged during the metastatic process.
Resistance to osimertinib in advanced EGFR-mutated non-small cell lung cancer (NSCLC) constitutes a significant challenge for clinicians either in terms of molecular diagnosis and subsequent ...therapeutic implications.
This is a prospective single-centre study with the primary objective of characterising resistance mechanisms to osimertinib in advanced EGFR-mutated NSCLC patients treated both in first- and in second-line. Next-Generation Sequencing analysis was conducted on paired tissue biopsies and plasma samples. A concordance analysis between tissue and plasma was performed.
Sixty-five advanced EGFR-mutated NSCLC patients treated with osimertinib in first- (n = 56) or in second-line (n = 9) were included. We managed to perform tissue and liquid biopsies in 65.5% and 89.7% of patients who experienced osimertinib progression, respectively. Acquired resistance mechanisms were identified in 80% of 25 patients with post-progression samples, with MET amplification (n = 8), EGFR C797S (n = 3), and SCLC transformation (n = 2) the most frequently identified. The mean concordance rates between tissue and plasma for the EGFR activating mutation and for the molecular resistance mechanisms were 87.5% and 22.7%, respectively.
Resistance to osimertinib demonstrated to be highly heterogeneous, with MET amplification the main mechanism. Plasma genotyping is a relevant complementary tool which might integrate tissue analysis for the study of resistance mechanisms.
Ž. P. Marinšek, N. Nolde, I. Kardum‐Skelin, R. Nizzoli, B. Önal, T. Rezanko, E. Tani, K. T. Ostović, P. Vielh, F. Schmitt and G. Kocjan Multinational study of oestrogen and progesterone receptor ...immunocytochemistry on breast carcinoma fine needle aspirates
Objectives: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result.
Methods: Ten laboratories from eight countries participated in a two‐part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in‐house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator.
Results: There was great variability in the preparation of slides for in‐house controls and ICC methodology. The outcome of ICC staining of in‐house control slides was excellent in two laboratories, adequate in three, sub‐optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in‐house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep®) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process.
Conclusions: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.
Background
: Pre-operative cytology in thyroid disease remains the most appropriate diagnostic test for defining the nature of a thyroid nodule before surgical excision.
Materials and methods
: We ...selected the most recent 825 surgical thyroid procedures performed in our institution from January 2004 to June 2007; 776 were total thyroidectomies, 23 were lobe-isthmectomies, and 26 were radical neck dissections. We distributed the data based on pre-operative cytology. Each cytological diagnosis was compared to results obtained by definitive histology. Tumors were called incidentalomas if they consisted of a neoplastic focus with a low grade of aggressiveness, as demonstrated by dimension <5 mm, non-aggressive histological subtype.
Results
: Of the 541 cases of benign disease, 417 were confirmed as benign. The other 124 cases are listed as follows: 29 follicular adenoma; 76 papillary carcinoma (35 found as incidentalomas), and 19 follicular carcinoma (3 incidentalomas). Cytology suggestive of papillary carcinoma was correct in 95.2% of cases (119/125). The 135 tumors termed “follicular neoplasm” were staged on pathology thus: 56 adenoma (41.4%), 26 carcinoma (19.2%), 13 (9.6%) absence of follicular proliferation, 38 (28.1%) papillary follicular variant, 2 (1.4%) undifferentiated cells. Medullary carcinomas were both confirmed. The “suspicious group” exhibited no malignancy on fine needle aspiration cytology (12 of 21; 57%).
Conclusions
: Cytology has good reliability in malignant lesions. Incidental tumors occurring in benign disease have little impact on clinical and surgical management; “follicular neoplasm” posed two problems — the impossibility of identifying the nature of the tumor, as well as the newer difficulty in distinguishing papillary follicular subtype.
BACKGROUNDPre-operative cytology in thyroid disease remains the most appropriate diagnostic test for defining the nature of a thyroid nodule before surgical excision. MATERIALS AND METHODSWe selected ...the most recent 825 surgical thyroid procedures performed in our institution from January 2004 to June 2007; 776 were total thyroidectomies, 23 were lobe-isthmectomies, and 26 were radical neck dissections. We distributed the data based on pre-operative cytology. Each cytological diagnosis was compared to results obtained by definitive histology. Tumors were called incidentalomas if they consisted of a neoplastic focus with a low grade of aggressiveness, as demonstrated by dimension <5 mm, non-aggressive histological subtype. RESULTSOf the 541 cases of benign disease, 417 were confirmed as benign. The other 124 cases are listed as follows: 29 follicular adenoma; 76 papillary carcinoma (35 found as incidentalomas), and 19 follicular carcinoma (3 incidentalomas). Cytology suggestive of papillary carcinoma was correct in 95.2% of cases (119/125). The 135 tumors termed "follicular neoplasm" were staged on pathology thus: 56 adenoma (41.4%), 26 carcinoma (19.2%), 13 (9.6%) absence of follicular proliferation, 38 (28.1%) papillary follicular variant, 2 (1.4%) undifferentiated cells. Medullary carcinomas were both confirmed. The "suspicious group" exhibited no malignancy on fine needle aspiration cytology (12 of 21; 57%). CONCLUSIONSCytology has good reliability in malignant lesions. Incidental tumors occurring in benign disease have little impact on clinical and surgical management; "follicular neoplasm" posed two problems - the impossibility of identifying the nature of the tumor, as well as the newer difficulty in distinguishing papillary follicular subtype.
Background: The HER-2/neu gene is amplified in 20–30% of human breast cancers and has been shown to have prognostic and predictive value for treatment with chemotherapy, hormone therapy and ...antibodies against the HER-2/neu domain (trastuzumab). The aim of our study was to evaluate the reliability of HER-2/neu determination by fluorescence in situ hybridization (FISH) on fine-needle aspirates (FNAs) from primary breast cancer patients by comparison with the results obtained by FISH and immunohistochemistry (IHC) on the corresponding histological sections. Materials and methods: HER-2/neu amplification was determined by FISH on 66 breast cancer FNAs. Twenty-three and 36 corresponding formalin-fixed, paraffin-embedded sections were assayed by FISH and by IHC, respectively, in order to detect HER-2/neu amplification and HER-2/neu protein expression. Results: Twenty-seven per cent (18/66) of breast cancer FNAs showed amplification of HER-2/neu by FISH. Paired results by FISH cytology and FISH histology were available in 22 cases. Concordance was 91% (20/22). Paired results by FISH cytology and IHC were available in 36 cases. Concordance was 92% (33/36). Eighteen of 66 breast cancer FNAs were also submitted to flow cytometric DNA analysis. None of the diploid cases showed HER-2/neu amplification by FISH. Six out of the eight aneuploid cases were amplified and two were polysomic. Conclusions: HER-2/neu gene amplification can be reliably estimated by FISH on breast cancer FNAs and a good correlation has been found between FISH and IHC results from the corresponding histological sections.
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