Mauro Ajaj Saieg

First-Line Erlotinib Followed by Second-Line Cisplatin-Gemcitabine Chemotherapy in Advanced Non–Small-Cell Lung Cancer: The TORCH Randomized Trial

PURPOSE Erlotinib prolonged survival of unselected patients with advanced non-small-cell lung cancer (NSCLC) who were not eligible for further chemotherapy, and two phase II studies suggested it might be an alternative to first-line chemotherapy. A randomized phase III trial was designed to test whether first-line erlotinib followed at progression by cisplatin-gemcitabine was not inferior in terms of survival to the standard inverse sequence. PATIENTS AND METHODS Patients with stage IIIB (with pleural effusion or supraclavicular nodes) to IV NSCLC and performance status of 0 to 1 were eligible. With a 95% CI upper limit of 1.25 for the hazard ratio (HR) for death, 80% power, a one-sided α = .025, and two interim analyses, a sample size of 900 patients was planned. RESULTS At the first planned interim analysis with half the events, the inferiority boundary was crossed, and the Independent Data Monitoring Committee recommended early termination of the study. Seven hundred sixty patients (median age, 62 years; range, 27 to 81 years) had been randomly assigned. Baseline characteristics were balanced between study arms. As of June 1, 2011, median follow-up was 24.3 months, and 536 deaths were recorded (263 in the standard treatment arm and 273 in the experimental arm). Median survival was 11.6 months (95% CI, 10.2 to 13.3 months) in the standard arm and 8.7 months (95% CI, 7.4 to 10.5 months) in the experimental arm. Adjusted HR of death in the experimental arm was 1.24 (95% CI, 1.04 to 1.47). There was no heterogeneity across sex, smoking habit, histotype, and epidermal growth factor receptor (EGFR) mutation. CONCLUSION In unselected patients with advanced NSCLC, first-line erlotinib followed at progression by cisplatin-gemcitabine was significantly inferior in terms of overall survival compared with the standard sequence of first-line chemotherapy followed by erlotinib.

EGFR gene status in cytological samples of nonsmall cell lung carcinoma: controversies and opportunities.

In nonsmall cell lung cancer (NSCLC), the development and clinical application of tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) has required the investigation of EGFR status by gene copy number and/or mutation analysis. This review aimed to present the current knowledge of the use of cytological specimens for EGFR testing in lung cancer.

“The petals and thorns” of ROSE (rapid on‐site evaluation)

The small samples obtained by these procedures can be used for diagnosis based on morphologic criteria alone and also, in many instances, for special ancillary studies, including molecular analysis. The inherent challenges involved in handling and processing limited samples in such a way as to allow multiple studies have triggered changes in the preanalytical phase and generated new protocols to maximize tissue/cell retrieval. 5 ROSE has been advocated as an effective way to ensure that samples are handled properly for morphological analysis, and that they meet all preanalytical requirements for specific diagnostic tests. Emerging novel molecular diagnostic technologies in current pathology practice have reinforced the essential role of ROSE in allowing pathologists to accurately divide diagnostic cytological material into small aliquots. This procedure ensures that sufficient quantities of cells of adequate quality are obtained to permit a complete diagnostic workup. Ultimately, this will translate into an appropriate treatment plan. For CT-guided and endoscopicguided procedures, ROSE has become increasingly more relevant because of the higher (although still low) rate of complications and the intrinsic risks of these procedures when compared with FNA of superficial lesions. A recent trial has concluded that ROSE of transbronchial aspirates from hilar and mediastinal lymph nodes enables clinicians to avoid additional biopsy without a loss in the diagnostic yield and reduces the complication rate of bronchoscopy. 6

Cyto-histologic agreement in pathologic subtyping of non small cell lung carcinoma: review of 602 fine needle aspirates with follow-up surgical specimens over a nine year period and analysis of factors underlying failure to subtype.

BACKGROUND The differential therapeutic efficacy and toxicity of targeted therapies has made subtyping of non-small lung cancer (NSCLC) mandatory. This study aimed to review the accuracy of NSCLC subtyping using lung fine needle aspirates (FNAs) in two periods (before and after the introduction of targeted therapy), checking the reasons for failure and the impact of the use of immunohistochemistry (IHC). METHODS An electronic search retrieved all NSCLC FNAs with a corresponding surgical specimen from 2001 to 2009. NSCLC, NOS (not otherwise specified) cases from 2005 to 2009 (after targeted therapy) were reviewed to determine reasons for failure in subtyping and to further subtype based solely on cytomorphology. The number of cases in which IHC was performed and the antibodies used were also recorded. RESULTS Cytohistological agreement of 602 lung FNAs (341 adenocarcinomas, 93 squamous cell carcinomas and 168 NSCLC, NOS) was achieved in 93.80%. There was a significant decrease in the percentage of cases not subtyped in the period after the introduction of targeted therapy (35.07% versus 24.57%). Final percentage of cases not subtyped after morphological review was 17.03%. IHC was performed in 157 cases, with an increased use in recent years. The number of antibodies did not influence the overall success in subtyping and an average of 3 markers was used. Most frequent antibodies used were TTF-1, CK7, high molecular weight keratin and p63. More than half of cases not subtyped even after IHC corresponded to poorly or undifferentiated neoplasms in the surgical specimens. For the NSCLC, NOS which IHC was not performed, a cell block was produced in 106 cases (75.71%). Review of the cell block slides from 2005 to 2009 showed that the majority (70.7%) had rare, few or no tumor cells. CONCLUSIONS Specific subtyping can be achieved in a high proportion of lung FNAs with high accuracy. The percentage of NSCLC, NOS has significantly decreased in recent years together with a trend for an increased use of IHC as well as increased number of cell blocks produced. An average of 3 IHC markers was used for subtyping and the number of markers did not influence the overall subtyping.

The use of FTA cards for preserving unfixed cytological material for high-throughput molecular analysis

Novel high‐throughput molecular technologies have made the collection and storage of cells and small tissue specimens a critical issue. The FTA card provides an alternative to cryopreservation for biobanking fresh unfixed cells. The current study compared the quality and integrity of the DNA obtained from 2 types of FTA cards (Classic and Elute) using 2 different extraction protocols (“Classic” and “Elute”) and assessed the feasibility of performing multiplex mutational screening using fine‐needle aspiration (FNA) biopsy samples.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples

Numerous genomic abnormalities in B‐cell non‐Hodgkin lymphomas (NHLs) have been revealed by novel high‐throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex‐associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B‐cell NHLs, through use of a customized multiplex mutation assay.

Preanalytic parameters in epidermal growth factor receptor mutation testing for non–small cell lung carcinoma: A review of cytologic series

The results from molecular assays can be affected significantly by the preanalytic condition of cytologic samples. The authors review current knowledge on the use of cytologic samples for epidermal growth factor receptor (EGFR) mutation testing in non–small cell lung cancer with a focus on preanalytic parameters. A systematic electronic search of the MEDLINE database was performed to identify original articles that reported the use of cytologic samples for EGFR molecular analysis and included a minimum of 100 samples. The information collected included author(s), journal, and year of publication; number of patients and samples; sampling method; type of preparation; type of fixative; staining techniques; mutation analysis techniques; tumor cellularity; the percentage of tumor cells; data on DNA quantity, quality, and concentration; failed assays; and the mutation rate. EGFR mutation analysis was conducted on 4999 cytologic samples from 22 studies that fulfilled the inclusion criteria. Fine‐needle aspirates and pleural effusions were the most common types of specimens used. DNA was mainly extracted from cell blocks and smears, and the most commonly reported fixatives included formalin, ethanol, and CytoLyt. Cellularity assessments and DNA yields were available from 5 studies each. The average success rate for the assays that used cytologic specimens was 95.87% (range, 85.2%‐100%). The mutation rate ranged from 6% to 50.46%, and a higher mutation detection rate and lower numbers of insufficient cases were reported for pleural effusions and lymph node samples from endobronchial ultrasound‐guided transbronchial needle aspiration compared with histologic specimens. Low cellularity and a low percentage of tumor cells were associated with higher test failure rates. Future guidelines should consider the current data for specific recommendations regarding cytologic samples. Cancer (Cancer Cytopathol) 2015;123:633–643.

Minimizing delays in DNA retrieval: The "freezer method" for glass coverslip removal. Letter to the editor regarding comparative study of epidermal growth factor receptor mutation analysis on cytology smears and surgical pathology specimens from primary and metastatic lung carcinomas.

Removing the glass coverslips for retrieving DNA from archival slides can be time-consuming, as pointed out by Khode et al. Traditionally, xylene has been used as the solvent for glass coverslip removal, which can take several days and thereby impose delays in molecular assays. We want to bring their attention to a method that applies “freezing” to old stained slides that can overcome this problem. The method was described for slides requiring restaining or destaining, but we have been using it in projects that involved molecular analysis, shortening the time required for getting the slides ready for DNA extraction. According to the “freezer method,” the slide should be placed flat in the freezer as the initial step. Although the original recommendation described a maximum exposure of 10 minutes to one hour, in our experience slides can rest in the freezer (at a temperature of 220 C) for only 1 to 2 minutes. After removing the slide from the freezer and wearing eye protection, immediately place the tip of a scalpel blade under one corner edge of the coverslip, lift up the coverslip, and remove it. The slide should be still frozen/cold when using the blade, otherwise the technique will not work. If the coverslip fails to lift off, return the slide to the freezer for an additional minute. If the coverslip still does not lift off during that period of time, it may indicate that the mounting medium was applied too recently. After the coverslip has been removed, allow the now uncoverslipped slide to return to room temperature before soaking it in xylene for 1 or 2 minutes until the remaining mounting media has been removed. The slide can be then sent for manual or laser capture microdissection for collecting cells for DNA extraction. Similar approaches have described how to remove coverslips from Araldite-mounted preparations using an ice block on the surface of the coverslip and liquid nitrogen. Apparently the basic principle consists of the differences in freezing between the glass slide and the mounting media. Archival stained smears and cytospin preparations are a precious source of DNA for molecular analysis and in some cases might be the only resource for detecting molecular abnormalities such as mutations in epidermal growth factor receptor (EGFR), BRAF, and KRAS genes to direct targeted therapies. We anticipate the broad use of archival slides in studies involving cases with exhausted histological material or those with only cytological specimens as a source for DNA. The “freezer method” for coverslip removal can facilitate the preparation of these samples.

Subclassification of lymphoproliferative disorders in serous effusions: a 10-year experience.

Rare studies have reported the application of multiple ancillary tests to the diagnosis of lymphoproliferative disorder in serous effusions. In the current study, the authors evaluated the effectiveness of using an algorithm for the triage of serous effusions and the contribution of ancillary studies to achieve a specific subtype of lymphoproliferative disorder.

A proposal for cellularity assessment for EGFR mutational analysis with a correlation with DNA yield and evaluation of the number of sections obtained from cell blocks for immunohistochemistry in non-small cell lung carcinoma

Aims Different approaches have been described for reporting specimen adequacy for epidermal growth factor receptor (EGFR) mutation analysis. We aimed: (1) to conduct cellularity assessment and to investigate its association with DNA yield, (2) to compare the H&E slides taken before and after the thick sections (curls) obtained for EGFR testing and (3) to evaluate the number of ancillary studies performed. Methods Cell block (CB) slides of 110 non-small cell lung carcinoma cases submitted to EGFR analysis from 2010 to 2012 were reviewed for total cellularity (ranges 1–100, 100–250, 250–500, 500–750, 750–1000 and >1000 cells), tumour cellularity (ranges 1–50, 50–100, 100–300 and >300 cells) and the percentage of tumour cells. Precurl and postcurl H&E slides were compared using the three criteria. The number of immunohistochemistry (IHC) markers and special stains and DNA yield were recorded. Results DNA yield was significantly associated with the total cellularity, number and percentage of tumour cells. For 46 cases with precurl and postcurl slides, only three (6.5%) were classified as being different and in two of them the postcurl slide had greater cellularity than the precurl. IHC was performed in 83 cases, with a minimum of 1 and a maximum of 11 markers (median of 3) per case. Conclusions An association between the total cellularity and the tumour cellularity with the DNA yield was demonstrated using the ranges described. Evaluation of a postcurl slide is an unnecessary practice. The majority of the CB had sufficient material for ancillary studies (up to 11 markers) and mutation testing.