Discordant ALK Status in Non-Small Cell Lung Carcinoma: A Detailed Reevaluation Comparing IHC, FISH, and NGS Analyses.

ALK detection was performed on 2813 EGFR-unmutated NSCLC cases by simultaneous use of immunohistochemistry (VENTANA® anti-ALK D5F3, Roche Molecular Systems, Inc., Rotkreuz, Switzerland) and fluorescence in situ hybridization with the ALK break apart and the ALK/EML4 fusion probe (ZytoVision, Bremerhaven, Germany). A total of 33 cases were positive discordant (FISH-positive, IHC-negative) and 17 cases were negative discordant (FISH-negative, IHC-positive). This study's aim was to reevaluate the methods used and compare discordant samples to positive concordant samples in order to ellucidate the differences. FISH signal variants were examined and compared. Positive discordant cases featured one pattern of ALK rearrangement in 41.4%, two patterns in 48.3%, and three patterns in 10.3% of analysed samples, with a higher variability of detected patterns and a higher number of ALK copy gains. Positive concordant cases displayed one pattern of rearrangement in 82%, two patterns in 17.8%, and three patterns in 0.6% of analysed samples. The association between number of patterns and concordance/discordance was statistically significant (p < 0.05). Eleven positive discordant and two negative concordant cases underwent NGS analysis, which resulted in identification of ALK fusion in one positive discordant and two negative discordant cases. Positive protein expression regardless of FISH result correlated more with a positive NGS result compared to samples with a positive FISH result with negative protein expression. FISH analysis was able to detect atypical or heterogenous patterns of rearrangement in a proportion of cases with negative protein expression, which may be associated with more extensive genetic alterations rather than true ALK rearrangement.

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements: Biopsy analysis of 70 cases from the Slovak Lymphoma Registry.

We performed a twelve-year retrospective analysis of diffuse large B-cell lymphoma (DLBCL) patients' biopsies with rearrangements of genes MYC, BCL2, and/or BCL6, commonly referred to as double-hit and triple-hit high-grade B-cell lymphomas (DH/TH HGBL). Our aim was to present complex characteristics of the DH/TH HGBL group of patients diagnosed in the Slovak National Lymphoma Register together with the evaluation of the relationship between immunohistochemical (IHC) protein expressions of c-myc, bcl2, bcl6, and cyclin D1 in tissue specimens and the presence of rearrangements of their protein-coding genes by FISH analysis in order to find a clinically relevant diagnostic algorithm that would be the most time- and cost-efficient. For this study, a standard panel of histomorphological, IHC, and FISH methods was used to analyze the characteristics of 70 DH/TH HGBL patients' biopsies. Our study showed a predominance of the immunohistochemical GCB subtype over the non-GCB subtype (59:10 cases) in DH/TH lymphomas. The centroblastic morphology was the most commonly observed (30/70 cases; 43%). Furthermore, our study showed a high predominance of DH lymphoma cases with simultaneous MYC and BCL2 genes rearrangements (40/70; 57%), followed by an almost equal incidence of DH lymphomas with rearrangements of MYC and BCL6 genes (16/70; 23%) and of TH lymphomas (14/70; 20%). 15 of 16 FISH-examined DLBCL cases were negative for CCND1 rearrangement. A great majority of DH/TH cases showed also immunohistochemical overexpression of corresponding proteins (62/70; 89%), mostly in a form of triple expressor of c-myc/bcl2/bcl6 proteins (36/70; 51%), followed by c-myc/bcl2 and c-myc/bcl6 double expressor proteins positivity (20/70 and 6/70, respectively). Comparing preferential FISH testing of DE/TE and GCB DLBCL cases for genetic rearrangements we would be able to detect 89% and 84% of our HGBL-DH, TH group of patients, respectively. None of the examined methods for economically rational FISH testing showed enough concordance with IHC analysis results. We might, therefore, advocate the complex testing of all DLBCL patients' biopsies including FISH analyses.