Issue 4 - August 2010 - Pacini Editore

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lectures Mutation analyses of KRAS in colorectal cancer and egfr in non-small cell lung cancer: a task for pathologists? M. Dietel Institute of Pathology, Charité Universitätsmedizin Berlin, Germany Background. Colorectal cancer (CRC) and non-small cell lung carcinoma (NSCLC) are the two most common malignancy in the western world with estimated 350,000 new cases reported for the United States in 2008 1 . Since both tumors are being predominantly diagnosed at advanced stage, the option of a curative therapy then does no longer exist in many instances and chemotherapy is often the treatment of choice. However, conventional anti-cancer drugs have been shown to be of limited value accompanied by strong side effects. This situation was the driving force to search for new more specific drugs. The membrane bound epidermal growth factor receptor (EGFR1) molecule was found to be of major importance in growth stimulation und thus was identified as a possible target which inhibition may lead to reduced tumor growth. Meanwhile there exist two types of EGFR-inhibitors which are approved for clinical application, i.e. the therapeutical anti- EGFR antibodies cetuximab (Erbitux ®) ) and Panitumumab (Vectibix ® ) as well as the tyrosine kinase inhibitors (TKI) erlotinib (Tarceva ® )) and gefitinib (Iressa ® ). In several clinical studies it became obvious that not all tumors respond equally but that certain genetic characteristics are the prerequisite to clinical benefit. This was the background to link the application of the drugs to pre-therapeutic eligibility tests and that in Europe KRAS mutation testing as well as EGFR mutation testing became a prerequisite for anti-epidermal growth factor receptor therapy of metastatic colorectal cancer since the end of 2007 2 3 and metastatic non-small cell lung cancer (NSCLC) in 2009, respectively. Since the analyses have to be performed using carefully selected tumor tissue the tests should be done only in Institutes of Pathology where pathologists are able to check istology, select the tumor tissue adequate for molecular testing and sign out a combined morphological/molecular report. In addition each institute should participate in interdisciplinary ring trials (round robin tests) which should be lead by an independent organisation. For that purpose the German Society of Pathology (DGP) and the German Association of Pathologist have (BDP) created the QuiP (Quality in Pathology) – initiative which organizes interlaboratory tests and confers the respected certification. Only if a reliable morphological diagnosis is combined with a solid genetic analysis a robust basis for targeted therapy is given. Methods. A multitude of procedures and methods are available for detecting KRAS/EGFR mutations in tumor samples (Weichert 4-11). In the Berlin Institute the following selection of techniques has been found useful, reliable and applicable for routine molecular pathology. Tissue selection. As shown in several studies 12 13 a precise selection of the tissue to be analysed is mandatory. This should be done by an experienced pathologist indicating the area on H&E-stained slides estimating the percentage of tumor in relation to the whole tissue section. Subsequently a manual microdissection has to be done by the technician. Tissue samples can be stored for years prior to molecular analyses. DNA preparation. For DNA preparation the three unstained slides were used. The putative tumor areas corresponding to 157 the marks on the H&E slide were microdissected. DNA preparation was done as described previously 14 . In brief, microdisseceted tissue was transferred to 180 µl ATL-buffer (Qiagen, Hilden, Germany) and kept for 10 min at 95°C. After cooling down to room temperature, 20 µl of proteinase K solution were added. After gentle mixing, the sample was incubated at 55°C until complete lysis (after about 2 h). The further steps of isolation of DNA follow the protocol “Tissue Protocol- QIAamp DNA Mini Kit” (Qiagen). The nucleic acids were eluted at a volume of 60-100 µl and DNA content was estimated with a Nanodrop 1000 (PeqLab, Erlangen Germany). For sequence analyses the techniques of Sanger sequencing, pyro-sequencing, chip analyses and melting curve analyses were applied (technical details see Weichert et al. 15 ). Results. Institute of Pathology, Berlin. For the analysis of the somatic KRAS genotype of 263 patients we used Sangersequencing, pyrosequencing, melting curve analysis and chip hybridization in parallel. We used Sanger sequencing as the reference method. For all cases DNA of sufficient quality was prepared. Overall mean (± SD) DNA yield was 196.8 ng/µl (± 142.5 ng/µl). Array analysis, melting curve analysis and pyrosequencing, using DNA from the same preparation, was performed in 233, 188 and 136 cases, respectively. Using Sanger sequencing 108 out of 260 cases (41.5%) were found to have a mutation in either codon 12 (31.9%) or codon 13 (9.6%) of the KRAS gene. The most frequent mutations were p.G12D (12.7%), p.G12V (10.8%) and p.G13D (9.2%). Similar distributions were seen with the other three methods. The array analysis identified 104 out of 223 cases (44.6%) to harbor somatic mutations, melting curve analysis found 77 out of 188 cases (41%) to be mutated, and by pyrosequencing 51 out of 136 cases (37.5%) were reported to carry mutations. A crossover comparison of the four methods yielded k values exceeding 0.9 (for more details see 15 ). Analogue test comparisons were done for EGFR mutation analyses using tissue from NSCLC after surgical tumor resection revealing similar results. QuiP-Initiative. Detailed descriptions of the results regarding round robin tests for KRAS- and EGFR-testing are published elsewhere. In summary, around Germany currently there exist over 60 Institutes of Pathology certified for KRAS testing and 53 certified for EGFR testing (for details visit the home page of the DGP 17 ). Discussion. Somatic gain-of-function mutations in the KRAS gene of CRCs predict the lack of response to anti-EGFR therapy with cetuximab and panitumumab, and KRAS mutational screening prior to treatment with either drug has become mandatory in Europe since the end of 2007. A similar situation become relevant in 2009 when clinical approval of gefitinib was limited to “tumours that have tested positive for EGFR with an activating mutation” (EMEA 2009;2,3). This resulted in a fast growing completely new branch of routine predictive diagnostic molecular pathology. The pre-therapeutical analyses which guide patients’ therapy are chance and challenge for pathologists as a new personal responsibility is given. To fulfil this following points have to be considered: 1. The test has to be done only in certified Institutes of Pathology, e.g. with a QuiP-certicate, for details see ref. 17 . 2. The responsible pathologists should be experienced in morphology and molecular testing. 3. The responsible pathologists should: • re-confirm the histological diagnosis on an H&E slide and
158 • he should identify and mark the tumor area for enrichment of tumor cells. 4. This is followed by manual microdissection to assure that at least 40% of the material for the molecular analysis is indeed tumor tissue. 5. The selected tumor tissue then should be analyzed following the procedure and recommendations described above and by others. 6. Finally the responsible pathologists should prepare a combined report giving details on the histology and the molecular result. If these criteria are met molecular pathology is facing an excellent future coming closer to clinical decisions and thus to the patients. references 1 Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58:71-96. 2 http://www.emea.europa.eu/humandocs/Humans/EPAR/erbitux/erbitux.htm 3 http://www.emea.europa.eu/humandocs/Humans/EPAR/vectibix/ vectibix.htm 4 Simi L, Pratesi N, Vignoli M, et al. High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer. Am J Clin Pathol 2008;130(2):247-53. 5 Ogino S, Kawasaki T, Brahmandam M, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn. 2005;7:413-21. 6 Clayton SJ, Scott FM, Walker J, et al. K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification. Clin Chem 2000;46:1929-38. The role of the pathologist in the assessment of kidney adequacy G. Monga, G. Mazzucco * Dipartimento di Scienze Mediche. Facoltà di Medicina e Chirurgia. Università del Piemonte Orientale, Amedeo Avogadro. Novara; * Dipartimento di Scienze Biomediche e Oncologia Umana. Facoltà di Medicina e Chirurgia. Università di Torino Every year, no more than 1/3-1/4 of patients awaiting kidney transplant can receive the graft. This shortage of grafts has led to an ever increasing use of kidneys from marginal deceased donors (subjects aged ≥ 55 years or < 55 years with a history of hypertension and/or diabetes, or deceased after a cerebrovascular incident). At present, pretransplant renal biopsy (PTRB) is the most reliable method available to assess the kidney state. However, there are several problems connected to this diagnostic procedure: 1. Morphologic evaluation of fixed and paraffin embedded samples vs frozen tissue. The former procedure is greatly favoured. Indeed, the frozen sections technique allows for a faster evaluation, offering briefer diagnostic times. However, the price is paid by the quality of the material, which is less satisfactory than that available after fixation and paraffin embedding. 2. Semiquantitative vs morphometric evaluation of the morphologic changes. The latter procedure is more accurate, but 5 th triennial congress of the italian society of anatomic Pathology and diagnostic cytoPathology Transplantation pathology Moderators: F.W. Grigioni (Bologna), M. Rugge (Padova) 7 Lilleberg SL, Durocher J, Sanders C, et al. High sensitivity scanning of colorectal tumors and matched plasma DNA for mutations in APC, TP53, K-RAS, and BRAF genes with a novel DHPLC fluorescence detection platform. Ann NY Acad Sci 2004;1022:250-6. 8 Rothschild CB, Brewer CS, Loggie B, et al. Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay. J Immunol Methods 1997;206:11-9. 9 Emanuel JR, Damico C, Ahn S, et al. Highly sensitive nonradioactive single-strand conformational polymorphism: detection of Ki-ras mutations. Diagn Mol Pathol 1996;5:260-4. 10 Keohavong P, Zhu D, Whiteside TL, et al. Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues. Anal Biochem 1997;247:394-403. 11 Ward R, Hawkins N, O’Grady R, et al. Restriction endonucleasemediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples. Am J Pathol 1998, 153:373-9. 12 Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008;26(10):1626-34. 13 Lièvre A, Bachet JB, Boige V, et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008;26(3):374-9. 14 Petersen I, Schewe C, Schlüns K, et al. Inter-laboratory validation of PCR-based HPV detection in pathology specimens. Virchows Arch 2007;451:701-16. 15 Weichert W, Schewe C, Lehmann A, et al. KRAS Genotyping of Paraffin-Embedded Colorectal Cancer Tissue in Routine: Diagnostics Comparison of Methods and Impact of Histology. J Mol Diagn <strong>2010</strong>;12:35-42. 16 Neumann J, Zeindl-Eberhart E, Kirchner T, et al. Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathol Res Pract. 2009;205(12):858-62. 17 http://www.dgp-berlin.de time consuming and, therefore, unsuitable in an emergency diagnostic setting. It follows that semiquantitative evaluation must be used. 3. Bioptical procedure: needle (NB) vs wedge (WB) biopsy. Opinions as to the primacy are conflicting among both surgeons and pathologists. WB offers larger amounts of tissue, but, according to several authors, increases the risk of an overestimation of glomerular sclerosis (GS) and interstitial fibrosis and allows for only limited sampling of the deeper renal tissue where larger arteries are present. 4. The choice of the morphological parameters for the grading of the kidney damage. Global GS alone has been used, but opinions on this procedure are conflicting. At present, besides GS, three other parameters (interstitial fibrosis, tubular atrophy and vascular arterio-arteriolosclerotic damage) are usually included in different scoring systems. PTRB was considered useful in this setting in the prediction of short- and long-term graft outcome, in supplying a reference frame in the interpretation of subsequent graft biopsies and mandatory in the assessment of kidney adequacy for single and/or double transplant or its being discarded 1 . Since PTRB is justified on the assumption that it represents the real state of the whole kidney, it follows that the critical issue is its reliability, i.e. how accurately it represents the true histology of the whole kidney. This question prompted a study which has already been published 2 dealing with a comparative evaluation, according to the Karpinski et al. scoring system 3
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lectures Azienda sanitaria dell’A
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lectures The Convention on Human Ri
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lectures Case n. 1 Aggressive psamm
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lectures Discussion. In most cases,
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lectures Hiroshima K, Shibuya K, Sh
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lectures Deficit of DNA mismatch re
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238 ultrastructural features. Lung
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240 Dideoxysequencing. The DNA to b
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242 of error. Nevertheless, the num
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244 Background. In cases of breast
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246 Background. TNM stage I CRC is
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248 Background. We evaluated the ex
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250 Armstrong C (eds). Myology. Thi
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252 to report the occurrence of the
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254 ovarian SCC HC-type showed nucl
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256 predisposition for Eastern and
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258 and prominent nucleoli. Areas o
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260 sarcoma in a young lady which w
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262 Methods. p-AKT (Ser473) and p-m
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264 cell cycle during G1-S. Additio
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266 up: the patient was given cours
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268 The mosaic pattern of INI1/SMAr
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270 Incidence of O6-methylguanine D
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272 leiomyomas are usually found bo
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274 drial disorder which involves t
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276 CLDN5 is claimed to be specific
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278 Results 1 st year (179 nodules)
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280 6)Anatomia Patologica Ed Oncolo
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282 Udine, Italy 6)Department of On
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284 Aberrant S-100 protein expressi
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286 phy white respect to lesion int
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288 a bulk of wt tumor could impact
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290 to the primary OSCC and develop
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292 peared to be encapsulated. On c
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294 ate intensity. Lack of EGFR and
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296 Analysis of microsatellite inst
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298 IDH-1/IDH-2, TP53, and C-MYC/N-
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300 Thus, it is possible that the h
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302 feasibility of fluorescence in
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304 sina, Italia 4)Dipartimento Di
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306 The histological analysis highl
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308 demonstrated no significative r
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310 rhage, acute tubular necrosis,
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312 performed in order to exclude t
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314 use of IGK gene rearrangement a
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316 evaluation of DNA ploidy in car
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320 Conclusioni. Dall’analisi del
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322 Pelvic epidermal cyst of the ro
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324 sive recent hemorrhage was seen
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326 expression of HMlH1 and HMSH2 i
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328 Kaushik D, Gulamjat SM, Thangja
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330 Results. The gastric lesion (7
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332 Methods. Macroscopically, left
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334 Spontaneous cutaneous cholester
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336 leptogenesis 3 but this possibi
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338 Complete cytoreduction and hype
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340 alternative NFKB pathways. On t
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342 Primary synovial sarcoma of kid
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344 Solitary extramedullary plasmac
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346 type (p < 0.05), with a prevale
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348 Squamous metaplasia was weakly
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350 in 3.3%). P63 gene gain was fou
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352 BCl10 expression in peripheral
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354 Breast lump in a male, first ma
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356 blastema compared to the neopla
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358 lack fat and are associated wit
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360 neuroendocrine tumors arising i
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362 latory cytokine/chemokine gene
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364 references 1 Capella C, Solcia
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366 Methods. Gastric biopsy routine
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368 sential prerequisite to make an
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370 nodule (DN)”, is commonly bel
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372 Summary of results. By studying
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374 (C5) was 60%, because C4 cases,
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376 composed of ammonium acid urate
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378 Background. Spindle cell lipoma
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380 TCl1A and MNDA expression in sp
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382 Methods. Immunohistochemistry w
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author indeX Faquin W., 310 Faralli
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author indeX Pandolfi P.P., 370 Pan
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