Rearrangements of anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) define a molecular subgroup of tumors characterized clinically by sensitivity to ALK tyrosine kinase inhibitors such as crizotinib. Although ALK rearrangements may be detected by reverse transcriptase-PCR, immunohistochemistry or fluorescence in situ hybridization (FISH), the optimal clinical strategy for identifying ALK rearrangements in clinical samples remains to be determined. We evaluated immunohistochemistry using three different antibodies (ALK1, 5A4 and D5F3 clones) to detect ALK rearrangements and compared those with FISH. We report the frequency and clinicopathologic features of lung cancers harboring ALK translocations in 594 resected NSCLCs (470 adenocarcinomas; 83 squamous carcinomas, 26 large cell carcinomas and 15 other histological subtypes) using a tissue microarray approach. We identified an ALK gene rearrangement in 7/594 cases (1%) by FISH and all anti-ALK antibodies correctly identified the seven ALK-positive cases (100% sensitivity), although the intensity of staining was weak in some cases. These data indicate that the use of antibodies with high sensitivity and avidity to ALK may provide an effective pre-screening technique to complement the more expensive and labor-intensive approach of ALK FISH testing.

The function of the tumor suppressor p53 is universally compromised in cancers. It is the most frequently mutated gene in human cancers (reviewed). In cases where p53 is not mutated, alternative regulatory pathways inactivate its tumor suppressive functions. This is primarily achieved through elevation in the expression of the key inhibitors of p53: Mdm2 or Mdmx (also called Mdm4) (reviewed). In breast cancer (BrCa), the frequency of p53 mutations varies markedly between the different subtypes, with basal-like BrCas bearing a high frequency of p53 mutations, whereas luminal BrCas generally express wild-type (wt) p53. Here we show that Mdmx is unexpectedly highly expressed in normal breast epithelial cells and its expression is further elevated in most luminal BrCas, whereas p53 expression is generally low, consistent with wt p53 status. Inducible knockdown (KD) of Mdmx in luminal BrCa MCF-7 cells impedes the growth of these cells in culture, in a p53-dependent manner. Importantly, KD of Mdmx in orthotopic xenograft transplants resulted in growth inhibition associated with prolonged survival, both in a preventative model and also in a treatment model. Growth impediment in response to Mdmx KD was associated with cellular senescence. The growth inhibitory capacity of Mdmx KD was recapitulated in an additional luminal BrCa cell line MPE600, which expresses wt p53. Further, the growth inhibitory capacity of Mdmx KD was also demonstrated in the wt p53 basal-like cell line SKBR7 line. These results identify Mdmx growth dependency in wt p53 expressing BrCas, across a range of subtypes. Based on our findings, we propose that Mdmx targeting is an attractive strategy for treating BrCas harboring wt p53.

Although fine‐needle aspiration (FNA) is accepted as the method of choice for the initial evaluation of lymph nodes for metastatic carcinomas, its utility as the initial diagnostic procedure for hematopoietic processes is less established. We review our experience over a 3‐year period with 127 FNA cases accompanied by flow cytometric (FC) analysis from 117 patients. Fifty cases had subsequent histologic examination. A hematopoietic process was identified in 85 cases, a reactive process in 27 cases, and a nonhematopoietic process in 15 cases. All non‐Hodgkin lymphomas (NHL) were B‐cell processes except for one T‐cell lymphoma. By FNA/FC, 44 NHL had sufficient findings to be subtyped; of these, 27 had subsequent histologic examination. The correlation between the FNA/FC and histologic classification in these cases of NHL was 100%. One case was insufficient for diagnosis by FNA and six cases were inadequate for FC. We conclude that FNA in conjunction with FC can be used as the initial diagnostic approach for both primary and recurrent hematopoietic processes. Diagn. Cytopathol. 2001;24:1–10. © 2001 Wiley‐Liss, Inc.

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in highthroughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86-96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof-of-principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting.Lung cancer remains one of the major causes of cancer mortality in both men and women worldwide 1 . Genomic alterations identified in lung cancer have significant predictive value in the treatment of the disease including EGFR mutations found commonly in advanced non-small cell lung cancer (NSCLC) that are associated with a favourable response to tyrosine kinase inhibitors 2,3 .More recently, structural genomic rearrangements involving the anaplastic lymphoma kinase (ALK) gene have been identified in NSCLCs. The most common rearrangement, occurring between ALK and echinoderm microtubule-associated protein-like 4 (EML4), is an inversion event resulting in the fusion of the 5′ end of EML4 to the 3′ end of ALK, leading to constitutive kinase activity and malignant growth 4 . Multiple EML4-ALK variants have subsequently been described, all occurring in the same region of the ALK gene but involving different breakpoints within the EML4 gene. Variants 1 and 3a/b account for the highest proportions, at 33% and 29% respectively 5 . Other fusion partners have also been described such as kinesin family member 5B (KIF5B), TRK-fused gene (TFG) and kinesin light chain 1 (KLC1) [5][6][7][8] . Subsequent studies have reported ALK rearrangements occurring in 3-5% of NSCLCs, where they are associated with younger patients who have a light or no smoking history 9 .The development of kinase inhibitors such as crizotinib has led to a breakthrough in the treatment of NSCLC patients carrying ALK fusions, who gain significant survival benefit following treatment 10 . Crizotinib has recently shown therapeutic efficacy in NSCLC patients carrying other rearrangements and alterations, including those involving ...

Our findings show good correlation between FISH versus CISH in the detection of ALK and ROS1 rearrangements. FISH versus IHC showed good correlation in the detection of ALK rearrangements but showed weak correlation in the detection of ROS1 rearrangements. These results suggest CISH and IHC could be complimentary detection methods to FISH in the detection of ALK and ROS1 rearrangements.

We characterized genome alterations in 1255 clinically annotated lung tumors of all histological subgroups to identify genetically defined and clinically relevant subtypes. More than 55% of all cases had at least one oncogenic genome alteration potentially amenable to specific therapeutic intervention, including several personalized treatment approaches that are already in clinical evaluation. Marked differences in the pattern of genomic alterations existed between and within histological subtypes, thus challenging the original histomorphological diagnosis. Immunohistochemical studies confirmed many of these reassigned subtypes. The reassignment eliminated almost all cases of large cell carcinomas, some of which had therapeutically relevant alterations. Prospective testing of our genomics-based diagnostic algorithm in 5145 lung cancer patients enabled a genome-based diagnosis in 3863 (75%) patients, confirmed the feasibility of rational reassignments of large cell lung cancer, and led to improvement in overall survival in patients with EGFR-mutant or ALK-rearranged cancers. Thus, our findings provide support for broad implementation of genome-based diagnosis of lung cancer.

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This case of an unusual DFSP demonstrates that genomic interrogation provides additional potential targets such as a therapeutic avenue with anti-EGFR therapies and shows the power of molecular characterisation of unusual tumours for a personalised medicine approach.