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• W1483007989 abstract "Recently, Tiacci et al (2011) documented for the first time that 100% of hairy-cell leukaemia (HCL) carry the BRAF-V600E mutation (Tiacci et al, 2011). Given the extraordinarily high frequency along with potential treatment implications (Cantwell-Dorris et al, 2011), there is an implicit understanding that BRAF testing could be part of the laboratory diagnosis of HCL (Takahashi et al, 2011; Tiacci et al, 2011). The next steps in the cascade of clinical implementation are: confirmation by independent groups, determination of diagnostic performance in routine samples, and identification of the optimal method for testing. Confirmation by independent groups (Boyd et al, 2011; Pardanani & Tefferi, 2011) as well as detection by Sanger sequencing (Tiacci et al, 2011), allele-specific polymerase-chain-reaction (Tiacci et al, 2011a) and melting-curve analysis (Boyd et al, 2011) have begun; however, the assessment of diagnostic performance in routine clinical specimens has, to our knowledge, not been specifically addressed. As part of our implementation strategy for diagnostic tests, we performed validation experiments for the use of BRAF V600E pyrosequencing in a series of routine clinical samples from HCL patients. Here, we report the diagnostic performance measures in combination with published data as well as the unexpected finding of a variant HCL that was BRAF wild-type. From our files, we identified a consecutive series of 18 HCL-patients with formalin-fixed, decalcified and paraffin-embedded bone marrow biopsies. The median age of the cohort was 56 (range: 40–74) years and 16 patients were male. Immunophenotyping showed positivity for CD20, DBA-44 and CD11c whereas CD23 or cyclin-D1 was negative. Microdissection of regions with leukaemic infiltrates between 20% and 80% was followed by DNA extraction and pyrosequencing [PyroMark Q24 (Qiagen, Hilden, Germany), protocol according to manufacturer]. In one of the 18 cases, the quality of DNA was insufficient and repeat-examination was inconclusive. In 16 of the 17 remaining informative cases we found BRAF V600E (Fig 1A) whereas one case was BRAF wild-type (Fig 1B). The test performance was calculated as follows: analytical sensitivity 94% (n = 17/18), overall detection 88% (n = 16/18) and diagnostic sensitivity 94% (n = 16/17). In synopsis with literature findings, three aspects of these results caught our attention: Pyrosequencing of BRAF V600E in Hairy cell leukaemia. (A) Representative reverse pyrogram demonstrates both BRAF V600E mutant- and wild-type alleles (WT). Quantification of peak-heights in this case demonstrates ˜63% wild-type (WT; CAC=GTG) and ˜37% mutant (V600E; CTC=GAG) alleles. (B) Lack of BRAF V600E in one of 17 informative HCL-cases. (C, D) Case shown in B stained for CD11c (C) or CD5 (D) using alkaline-phosphatase immunohistochemistry at ×600 magnification. (E) Correlation of leukaemic infiltrate (as determined by H&E or CD20 staining) with peak-height quantification from pyrosequencing in 16 BRAF V600E mutant cases. Case shown in (A) is highlighted in magenta. First, the case without BRAF V600E (Fig 1B) was that of a 74-year-old Caucasian man who was the oldest patient in our cohort. Immunolabelling demonstrated strong CD20 and CD11c positivity (Fig 1C) whereas Cyclin-D1 was negative. Interestingly, CD5 demonstrated strong reactivity in the leukaemic infiltrate (Fig 1D) and, together with the clinical course, this case resembled a report of so-called variant HCL (Usha et al, 2000; Chen et al, 2006; Foucar et al, 2008). While overall rare, the other V600E mutated HCL cases reported here were CD5-negative and the one wild-type case suggests that CD5-positive variant-HCL may represent an exception with respect to BRAF V600E in HCL (Tiacci et al, 2011). Second, we were interested in the overall performance of detecting BRAF V600E as a diagnostic test for HCL. Currently available data of 2019 cases [COSMIC database; http://www.sanger.ac.uk/genetics/CGP/cosmic/; last accessioned: Sept, 19, 2011; (Boyd et al, 2011; Pardanani & Tefferi, 2011; Tiacci et al, 2011) and our cohort] show 112 true positive, 45 false positive, one false negative and 1861 true negative cases with respect to BRAF V600E. The Youden index for BRAF V600E in HCL is 0·97 and the specificity 97·6%. The deviation of specificity was caused by 35 cases of Langerhans cell histiocytosis (Pardanani & Tefferi, 2011), four cases of acute myeloid leukaemia, three cases of myeloma, one case of post-transplant lymphoproliferative disorder, one case of B-cell lymphoma (unclassified) and one case of diffuse-large B-cell lymphoma from various studies (COSMIC database) that were assigned ‘false-positive’. Altogether the positive predictive value was 71·3% (95% confidence interval: 63·8–77·8) and the negative predictive value was 99·9% (95% confidence interval: 99·7–99·99) for HCL when BRAF V600E is detected or not respectively. Even when the one variant case (reported here) is counted as ‘false-negative’, the sensitivity was 99·11% for BRAF V600E testing in lymphoid and myeloid neoplasms. Thus, even when including all reported entities in these lineages (Arcaini et al, 2011), the remarkable diagnostic performance supports the notion that inclusion of BRAF V600E status could be part of the laboratory diagnosis of HCL. Third, we were interested whether quantification of mutant peak heights (by pyrosequencing) can function as a surrogate for the estimation of leukaemic involvement in the bone marrow [generally determined by CD20 or haematoxylin & eosin (H&E) staining]. Comparisons of both methods demonstrated significant correlation (Spearman r = 0·66; P = 0·006; Fig. 1E) and suggest that molecular detection of BRAF V600E may simultaneously enable quantification of this tumour-specific marker, for example for disease monitoring during treatment or follow-up. However, given the excellent results of current therapeutic regimens (Else et al, 2005), it remains to be determined whether and when detection and/or quantification of BRAF V600E would add clinically useful information. In summary, the analytical and diagnostic performance of pyrosequencing for BRAF V600E assessment in routine HCL samples is sufficient to warrant quick, reliable and cost-effective implementation into clinical practice. Using this approach we identified one case of variant-HCL that lacks the V600E mutation. While the effect of such an outlier on the exceptional diagnostic performance of BRAF V600E testing is marginal, it revives the interest in variant-HCL (Arcaini et al, 2011; Tiacci et al, 2011, 2011a). Whether alternative methods can surpass the speed, simplicity, and certainty of pyrosequencing needs to be determined; however, ultimately clinical cost-benefit analyses will determine whether BRAF V600E testing should become part of the laboratory diagnosis of HCL. The authors would like to thank Sonja Jung and Elena Moser for help with DNA extraction and mutational analysis as well as Julia Melzner and Iwona Nerbas for technical assistance with immunohistochemistry. JKL and PM designed the study, JKL and BK analysed pathology, RM performed mutational testing and all authors interpreted the data and wrote the paper. JKL was supported by the Else-Kröner-Fresenius Foundation. Nothing to declare." @default.
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• W1483007989 title "Pyrosequencing of BRAF V600E in routine samples of Hairy Cell Leukaemia identifies CD5+ variant Hairy Cell Leukaemia that lacks V600E" @default.
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• W1483007989 doi "https://doi.org/10.1111/j.1365-2141.2011.08963.x" @default.
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