Our results confirm the difficulty in managing an IHC test without amplification in the absence of confirmatory FISH analysis, as well as the possibility of performing a direct diagnosis in approximately 90% of patients by the VENTANA ALK (D5F3) CDx Assay. On the basis of the recent regulatory changes, the data that have emerged from the literature, and the results of the present study, a new algorithm for ALK assessment in non-small cell lung cancer has been devised.

Purpose: The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells.Experimental Design: We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 AE 2,370 sequences per sample were obtained by NGS.Results: In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS.Conclusions: The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies.

NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed by using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

The success of TYS will depend on the acceptance of TYS by all the relevant pathology and gynecologic oncology communities who, by their joint efforts, will adopt, critically evaluate, and optimize this method with the only aim of further improving the impact of endometrial cytology on patients' care.

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