Sabato 27 ottobre 2012 - Pacini Editore

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340 ALK (A)) and E6;A20, which also referred to as variants 1 and 3, respectively. These have been respectively detected in 33% and 29% of NSCLC patients with EML4-ALK rearrangement 3 4 . Consequent to the EML4-ALK rearrangement, the encoded protein contains the N-terminal part of EML4 and the intracellular catalytic domain of ALK. Replacement of the extracellular and transmembrane domain of ALK with a region of EML4 results in constitutive dimerization of the kinase domain and thereby a consequent increase in its catalytic activity 3 . Detection of ALK gene fusion is currently indicated as a predictive marker of treatment response to small molecule inibitors of ALK in NSCLC: crizotinib. Crizotinib, a new and selective TKI targeting ALK and MET has been approved on August 26, 2011, by the FDA to treat locally advanced or metastatic NSCLC that harbor the abnormal fusion gene. In Phase I and Phase II trials, crizotinib was shown to be highly active in patients with advanced ALKpositive NSCLC, with overall response rates of 50–60%. EML4- ALK is also associated with anti-EGFR TKI resistance. The gold standard method to detect ALK rearrangement is fluorescent in situ hybridization (FISH). However many published studies using reverse transcritase-polymerase chain reaction analysis to characterize EML4-ALK. FISH detects any rearrangement involving ALK, including potentially rare uncharacterized rearrangement. PCR identifies specific fusion variants. Although both/these techniques are sensitive and specific for detecting EML4-ALK, we want to compare the data obtain through the two different approaches. We aimed to estabilish if PCR can be used to take additional advantage of genetic analyses, especially in case were the FISH was not applicable. Matherials and method. This study comprised a series of 46 patients with NSCLC (39 adenocarcinoma, 5 metastasis of lung adenocarcinoma, and 2 Squamous cell lung cancer) who underwent surgery in the Department of Surgery at the University of Pisa from 2007 to 2011. From each paraffin block, 4 consecutive 10µm sections were cut to extract the DNA (for EGFR and KRAS mutation analysis) and the RNA (for EML4-ALK analysis). An adjacent 5µm section was used for FISH (for EML4-ALK analysis). All samples were analyzed using both FISH and PCR approach in parallel. Nucleic acid was isolated using a commercially kit specific to formalin-fixed and paraffin-embedded (FFPE) sample. The quality and quantitation of RNA and DNA were verified by Agilent 2100 bioanalyzer. RNA (500ng) from each sample was reverse transcribed using random hexamer primers to produce cDNA from all RNAs. For detection of EML4- ALK fusion cDNAs, we have carried out three independent PCR. The first one, was performed using a set of primers to identify the variant 1; forward primer is located at exon 13 of EML4, whereas the reverse primer is located at exon 20 of ALK, resulting in a 170-base pair PCR product. To search for other variants of rearrangement, as described in the study of Soda et al. 3 , we used primers that target fusion variant 1 (247-bp). These primers could also detect variant 2 which would have yielded a considerebly longer amplication (≈1Kb); the forward primer Fusion-RT-S is located in exon 13 of EML4, while the reverse primer Fusion-RT-AS, in exon 20 of ALK. The latter variant may have been expressen in the fusion positive cases but missed in our assay because of the difficulties in amplifying long amplicons from RNA extracted from FFPE. To analyze the shorter variant of EML4-ALK transcript (155/188-bp), the variant 3, the ALK Fusion- RT-AS primer was combined with a forward primer located in exon 6 of EML4: EML4-ex6F. PCR primers GAPDH-S and GAPDH-AS for glyceraldehyde-3phosphate dehydrogenase cDNA (452bp) were used as control for cDNA integrity. Polymerase chain reaction products were assessed and visualized by 1,5% agarose gel electrophoresis. PCRs were performed in triplicate, each one in 25µl reactions containing 100 ng cDNA, 12,5µl of Taq PCR Master Mix and 0.5 µl of each primer (20µM). CONGRESSO aNNualE di aNatOmia patOlOGiCa SiapEC – iap • fiRENzE, 25-27 OttOBRE 2012 To asses for rearrangement of the ALK locus on 2p23 using commercially available ALK (Vysis ALK Break Apart FISH Probe Kit, Abbott Laboratories) probes we performed on 4µm paraffinembedded thin sections. The test employs two differently labeled probes on opposite sides of the breakpoint of the ALK gene. When hybridized with the Vysis ALK Break Apart FISH Probes, the 2p23 ALK region in its native state will be seen as two immediately adjacent or fused orange/green (yellow) signals. However, if a chromosome rearrangement at the 2p23 ALK breakpoint region has occurred, one orange and one green signal separated by at least two signal diameters will be seen. Alternatively, a single orange signal(deletion of green signal) in addition to a fused or broken apart signal may be seen. A minimum of 50 tumor cells must be scored and a specimen is considered positive for ALK rearrangement when > 15% of the cells show split signals. All experiments were done without knowledge of the RT-PCR results for EML4-ALK. Mutation analysis of EGFR (exons 18-21) and KRAS (codons 12, 13 and 61) were performed using the HotStarTaq Master Mix Kit, as per manufacturer’s instructions. The presence of an appropriate PCR product was confirmed by resolving the PCR products on a 1.5% agarose gel. PCR products were purified using the PCR Purification Kit and sequenced using fluorescent dye-terminator chemistry. Mutations were identified by visual analysis of the sequence chromatograms using SeqScape. Results. Among 46 NSCLC analyzed 6 (13%) showed EML4- ALK rearrangement by FISH analysis; while, EML4-ALK transcript, detected by RT-PCR, were found in 9 cases of 46 (21.7%). Of 9 positive-PCR cases 6 (13%) showed EML4-ALK fusion variant 1 and 3 (6.5%) showed EML4-ALK fusion variant 3. We identified only one (2.17%) in-frame deletion in EGFR exon 19 (E746-A750del); while, single amino acid substitution involving codons 12 and 13 of KRAS, were identified in 7 of 46 NSCLC (15%). In particular, we found that 3 patients had G12C, 3 patients had G12D and 1 patient had G12S. Discussion. An overall concordance of FISH and RT-PCR (being either negative or positive in both tests) was found in 39 cases (84.7%) and a discrepancy was identified in 7 cases (15.3%). In detail, 4 samples resulted positive in both FISH and RT-PCR analysis, 2 FISH positive samples were RT-PCR negative, 5 samples showing transcript expression were FISH negative (or below the cut-off value) and 35 samples were negative for EML4- ALK rearrangement detection. ALK fusion and EGFR and KRAS mutations appears to be mutually exclusive. Although, we identified one patient who had both EML4-ALK rearrangement and KRAS mutation (G12D); while, all positive cases for ALK rearrangement did not show EGFR mutations. In conclusion, the usefulness of RT-PCR analysis for EML4- ALK rearrangement detection is still not fully satisfactory due to the fact that either alternative EML4 exons were involved or that EML4 was not the ALK fusion partner in all the FISH positive patients. As a consequence a multiplex RT-PCR reaction should be performed to fully cover the fusion variants but this approach is difficult to apply in routine condition poor quality samples. Finally FISH analysis remains the gold standard methods for EML4-ALK rearrangement detection. references 1 Chiarle R, et al. The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer 2008;8:11-23. 2 Solomon B, Varella-Garcia M, Camidge DR. ALK gene rearrangements: a new therapeutic target in a molecularly defined subset of non-small cell lung cancer. J Thorac Oncol 2009;4:1450-4. 3 Soda M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561-6. 4 Martelli MP, Sozzi G, Hernandez L, et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. The American Journal of Pathology 2009;174:661-670.
COmuNiCaziONi ORali True 3q chromosomal amplification in squamous cell lung carcinoma by FISH and aCGH molecular analysis: impact on targeted drugs M. Brunelli1 , E. Bria2 , A. Nottegar1 , S. Cingarlini2 , A. Caliò1 , A. Eccher3 , C. Parolini1 , A. Iannucci2 , E. Gilioli3 , S. Pedron1 , F. Massari2 , G. Tortora2 , I. Borze4 , S. Knuutila4 , S. Gobbo1 , A. Santo5 , L. Tondulli5 , F. Calabrò6 , G. Martignoni1 , M. Chilosi1 1 University of Verona, Department of Pathology and Diagnostic, Verona, Italy; 2 University of Verona, Department of Medical Oncology, Verona, Italy; 3 Azienda Ospedaliera Universitaria Integrata di Verona, Ospedale Civile Maggiore, Pathology Unit, Verona, Italy; 4 University of Helsinki, Haartman Insitute, Department of Pathology, Helsinki, Finland; 5 Azienda Ospedaliera Universitaria Integrata di Verona, Oncology Unit, Ospedale Civile Maggiore, Verona, Italy; 6 Azienda Ospedaliera Universitaria Integrata di Verona, Ospedale Civile Maggiore, Thoracic Surgery Unit, Verona, Italy Introduction. Squamous lung carcinoma lacks specific “ad hoc” therapies. Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics 1,2,3 . There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs anti-PI3K and -SOX2 mapping on 3q 4 . Materials and method. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Results. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20%) showed ≥ 8 3q signals. Twenty out of 40 (50%) showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%). When corrected by whole chromosome 3 signals, only cases with ≥8 signals maintained a LSI 3q/CEP3 ratio > 2. Only the cases showing 3q amplification by aCGH (+3q25.3-3q27.3) showed ≥8 fluorescent signals at FISH evidencing a 3q/3 ratio > 2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. Conclusion. We concluded that: 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2) a case results truly “amplified for chromosome 3q” when showing ≥8 fluorescent 3q signals; 3) trials involving anti-PI3CA and –SOX2 drugs for squamous lung carcinoma therapy have to consider false versus true 3q chromosomal amplification. references 1 Kettunen E, el-Rifai W, Bjorkqvist AM, et al. A broad amplification pattern at 3q in squamous cell lung cancer--a fluorescence in situ hybridization study. Cancer Genet Cytogenet 2000:117:66-70. 2 Qian J, Massion PP. Role of chromosome 3q amplification in lung cancer. J Thorac Oncol 2008;3:212-5. 3 Bjorkqvist AM, Husgafvel-Pursiainen K, Anttila S, et al. DNA gains in 3q occur frequently in squamous cell carcinoma of the lung, but not in adenocarcinoma. Genes Chromosomes Cancer 1998;22:79-82. 4 McCaughan F, Pole JC, Bankier AT, et al. Progressive 3q amplification consistently targets SOX2 in preinvasive squamous lung cancer. Am J Respir Crit Care Med 2010;182:83-91. 341 InV(2)(p21p23) and t(2;2)(EMLA4;ALK) detection in pure squamous lung carcinoma. time to assess ALK translocation in squamous subtype of lung cancer? A. Caliò1 , M. Brunelli1 , A. Nottegar1 , S. Pedron1 , S. Knuutila4 , F. Simionato2 , S. Cingarlini2 , E. Bria2 , G. Tortora2 , F. Calabrò3 , G. Martignoni1 , M. Chilosi1 1 Università di Verona, Dipartimento di Patologia e Diagnostica, Sezione di Anatomia Patologica; 2 Università di Verona, Dipartimento di Medicina clinica e sperimentale, Sezione di Oncologia medica; 3 Azienda Ospedaliera Universitaria Integrata di Verona, Ospedale Civile Maggiore, Unità di Chirurgia Toracica, Verona, Italy Introduction. In 2007 ALK was shown to be involved in the oncogenesis of a subset of non small cell lung cancer (NSCLC) and 20% NSCLC from never smokers. The most common 5’ fusion partner in NSCLC is EML4 (echinoderm microtubule-associated protein like 4), but other, rarer 5’ fusion partner, notably, KIF5B (kinesin family member 5B) and TGF (TRK- fused gene) have also described. The EML4-ALK fusion results from a small inversion within the short arm of chromosome 2, which encodes a chimeric tyrosine kinase, leading to constitutive activation. The main clinico-pathologic features of these NSCLC, to date, include younger age at diagnosis, never or light smoking history, adenocarcinoma histology, and signet ring cells. However, tumors with squamous cell carcinoma or adenosquamous histologies, although with much lower frequency than adenocarcinoma, have been reported to harbor ALK translocation. Recently, it has been raised the question whether squamous cell lung cancer may be evaluated for ALK gene rearrangement due to single case report evidencing cases of adenosquamous and squamous lung cancer ALK translocated. Materials and methods. A consecutive series of 55 lung carcinomas with pure squamous morphology were analyzed. We studied the immunohistochemical expression of protein 63 (p63), cytokeratin CK5/6, thyroid transcrition factor 1 TTF-1 and Napsin. Moreover, we used two distinct FISH kit; the first one, a break apart dual color probe, approved by FDA, has the goal to detect the ALK rearrangement but not the partner of the fusion; the second one, a dual-color assay Kreatech FISH probes, has the specific goal to assess the specific ALK-EML4 t(2;2); inv(2). Results. All tumors stained positive, by immunohistochemistry, for p63 and CK5/6 and negative for TTF-1 and Napsin. One case with diffuse ALK rearrangement was found. Primarly, by using FISH technique with a break apart dual color probe, we observed the split signals in more than 40% of the nuclei (150 neoplastic nuclei scored). Secondarly, by the ALK/EML4 t(2;2); inv(2), the squamous carcinomatous tissue showed the ALK-EML4 fusion by visualing of 2 red-green fused, 1 red and 1 green fluorescent signals in > 70% of nuclei (150 neoplastic nuclei scored) Conclusions. We observed a case of lung carcinoma with pure squamous morphology with diffuse ALK rearrangement; we additionally reviewed the Literature focusing on squamous cell lung cancer and adenosquamous carcinoma characterized by ALK rearrangement by FISH technique and we found 14 cases and 12 cases respectively. Furthermore, we did not observed any other squamous lung cancer with ALK/EML4 t(2;2); inv(2). We think that the histotype per se do not preclude the presence of ALK rearrangement and we propose, in agreement with other Authors, to screen squamous cell lung carcinoma for ALK rearrangement. references 1 Travis WD, et al. United States lung carcinoma incidence trends: declining for most histologic types among males, increasing among females. Cancer 1996;77:2464-70. 2 Travis WD, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol;6:244-85.
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212 Key words: EGC, Prognosis, Trea
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COmuNiCaziONi ORali Immunohistochem
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