André Fehr

Analysis of MYB expression and MYB-NFIB gene fusions in adenoid cystic carcinoma and other salivary neoplasms.

Recent studies have shown that the recurrent t(6;9)(q22–23;p23–24) translocation in adenoid cystic carcinoma results in a novel fusion of the MYB proto-oncogene with the transcription factor gene NFIB. To determine the frequency of this finding, we used RT-PCR assays of the MYB and MYB-NFIB fusion transcripts, and immunohistochemistry for the MYB protein, to study adenoid cystic carcinomas and other epithelial tumors of the salivary glands, and head and neck region. MYB-NFIB fusion transcript was detected in 25 of 29 (86%) frozen adenoid cystic carcinoma tumor samples, and in 14 of 32 (44%) formalin-fixed paraffin-embedded adenoid cystic carcinoma tumor specimens. In contrast, the MYB-NFIB fusion was not expressed in non-adenoid cystic carcinoma neoplasms of the head and neck, confirming the high specificity of the MYB-NFIB fusion. Adenoid cystic carcinomas from various anatomic sites, including salivary gland, sinonasal cavity, tracheobronchial tree, larynx, breast, and vulva were repeatedly fusion-positive, indicating that adenoid cystic carcinomas located in different anatomic sites not only have important morphologic features in common, but also probably evolve through activation of the same molecular pathways. Studies of the expression of MYB revealed that 89% of the tumors, including both fusion-positive and fusion-negative cases, overexpressed MYB RNA. Similarly, 82% of adenoid cystic carcinomas stained positive for MYB protein, compared with 14% of non-adenoid cystic carcinoma neoplasms, indicating that MYB immunostaining may be useful for the diagnosis of adenoid cystic carcinoma, but that neoplasms sometimes in the differential diagnosis are also labeled. The latter are, however, fusion-negative. In summary, our studies show that MYB activation through gene fusion or other mechanisms is a major oncogenic event in adenoid cystic carcinoma occurring at various anatomic sites. In addition to being a diagnostically useful biomarker for adenoid cystic carcinoma, MYB and its downstream effectors are also novel potential therapeutic targets.

Mammary analogue secretory carcinoma of the salivary glands with ETV6-NTRK3 gene fusion.

To the Editor:We read with great interest the recent publication in Am J Surg Pathol by Skalova et al7 regarding a new tumor entity in salivary glands. In this excellent paper the authors have described a previously unrecognized tumor type in salivary glands with histologic and immunohisto

The MYB-NFIB gene fusion-a novel genetic link between adenoid cystic carcinoma and dermal cylindroma.

We have recently shown that the recurrent t(6;9)(q22 ∼ 23;p23 ∼ 24) translocation in adenoid cystic carcinoma (ACC) of the breast and head and neck results in a fusion of the two transcription factor genes MYB and NFIB. Here we demonstrate, for the first time, that benign sporadic, dermal cylindromas also express the MYB–NFIB gene fusion. RT–PCR and immunohistochemical analyses revealed that eight of 12 analysed tumours (67%) expressed MYB–NFIB fusion transcripts and/or stained positive for MYB protein. Nucleotide sequence analyses confirmed that the composition of the chimeric transcript variants identified was identical to that in ACC, suggesting a similar molecular mechanism of activation of MYB in cylindroma as in ACC. In contrast, no evidence for the presence of the MYB–NFIB fusion was found in other types of basaloid skin and salivary gland tumours, indicating that the fusion indeed has a restricted expression pattern. Our findings broaden the spectrum of neoplasms associated with MYB oncogene activation and reveal a novel genetic link between ACC and dermal cylindroma. These results, together with our previous observations, further strengthen the evidence for common molecular pathways of importance for the development of both benign and malignant breast, salivary and adnexal tumours. Copyright

Adenoid cystic carcinoma of the lacrimal gland: MYB gene activation, genomic imbalances, and clinical characteristics.

PURPOSE To investigate genetic alterations in lacrimal gland adenoid cystic carcinomas (ACCs) with emphasis on the MYB-NFIB fusion oncogene and its downstream targets, MYB rearrangements, and copy number alterations in relation to clinical data and survival. DESIGN Experimental study. PARTICIPANTS AND CONTROLS Fourteen patients with primary lacrimal gland ACC were included. As a control, we also studied the expression of MYB-NFIB in 19 non-ACC lacrimal gland tumors. METHODS The expression and identity of MYB-NFIB fusion transcripts were studied using reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequence analyses. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to evaluate the expression of MYB/MYB-NFIB target genes. High-resolution array-based comparative genomic hybridization (arrayCGH) and fluorescence in situ hybridization were used to study copy number alterations and MYB rearrangements. MAIN OUTCOME MEASURES mRNA or protein expression of MYB-NFIB, MYB, and its down stream targets; copy number alterations; and genomic rearrangements. RESULTS The median age of the patients was 43 years (equal gender distribution), and the median time of survival was 8.6 years. The MYB-NFIB fusion was expressed in 7 of 14 ACCs. In contrast, all non-ACC tumors were fusion-negative. All 13 ACCs tested stained positive for the MYB protein, and for the MYB targets KIT and BCL2, 12 were positive for MYC and CCNE1, and 9 were positive for CCNB1. Rearrangements of MYB were detected in 8 of 13 cases, including 2 cases with gain of an apparently intact MYB gene. The arrayCGH analysis revealed recurrent copy number alterations with losses involving 6q23-q27, 12q12-q14.1, and 17p13.3-p12, and gains involving 19q12, 19q13.31-qter, 8q24.13-q24.21, 11q12.3-q14.1, and 6q23.3. Neither MYB-NFIB fusion nor any copy number alteration correlated with survival. CONCLUSIONS Lacrimal gland ACCs are frequently positive for the MYB-NFIB fusion, overexpress MYB and its downstream targets, and have genomic profiles characterized by losses involving 6q, 12q, and 17p, and gains involving 19q, 8q, and 11q. Our findings show that lacrimal gland ACCs are genetically and clinically similar to their salivary gland counterparts and that MYB-NFIB is a clinically useful diagnostic biomarker for ACC. Our data also suggest that MYB and its downstream targets are potential therapeutic targets for these tumors. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Lacrimal gland pleomorphic adenoma and carcinoma ex pleomorphic adenoma: genomic profiles, gene fusions, and clinical characteristics.

PURPOSE To study genetic alterations in lacrimal gland pleomorphic adenoma (PA) and carcinoma ex pleomorphic adenoma (Ca-ex-PA) with focus on copy number changes and expression patterns of the translocation target genes PLAG1, HMGA2, and CRTC1-MAML2 in relation to clinical data. DESIGN Experimental study. PARTICIPANTS A total of 36 tumors from 32 patients with lacrimal gland PA or Ca-ex-PA were included in the study. METHODS Genome wide, high-resolution array-based comparative genomic hybridization (arrayCGH) and immunohistochemistry were used to study the genomic profiles and expression patterns of the translocation targets PLAG1, HMGA2, and CRTC1-MAML2. MAIN OUTCOME MEASURES Copy number alterations (gains/losses) and protein expression of PLAG1, HMGA2, and CRTC1-MAML2. RESULTS Genome-wide arrayCGH analysis revealed normal genomic profiles in 10 of 17 PA samples. The average number of genomic imbalances per tumor was 3.25 (range, 1-7) in primary and recurrent PAs with alterations compared with 7.7 (range, 4-12) in Ca-ex-PAs. Five recurrent copy number alterations were identified in PAs, including losses of 1pter-p31.3, 6q22.1-q24.3, 8q24.22-q24.3, and 13q21.31-q21.33, and gain of 9p23-p22.3. Gain of 9p23-p22.3 also was seen in a Ca-ex-PA. In Ca-ex-PA, gain of 22q12.3-qter was the only recurrent alteration. Detailed analysis of the array data identified NFIB and PDGFB as the 2 major candidate target oncogenes that may be activated as a result of copy number gains involving 9p and 22q. Both genes have been implicated in the pathogenesis of PA and other types of salivary gland tumors. Immunohistochemical analysis revealed frequent overexpression of the translocation target gene PLAG1 in PAs and in 1 Ca-ex-PA. In contrast, overexpression of HMGA2 was observed in only a small subset of PAs. The CRTC1-MAML2 fusion oncoprotein was overexpressed in 2 mucoepidermoid Ca-ex-PAs. CONCLUSIONS Lacrimal and salivary gland PAs and Ca-ex-PAs have similar genomic profiles and frequently overexpress the PLAG1 oncoprotein. Copy number gains involving 9p23-p22.3 (NFIB) and 22q12-qter (PDGFB) may be of importance for disease progression in a subset of lacrimal gland PAs.

CRTC1-MAML2 gene fusion in mucoepidermoid carcinoma of the lacrimal gland

Epithelial tumors of the lacrimal gland are histologically similar to salivary gland tumors. Here we report on a rare case of mucoepidermoid carcinoma (MEC) in a 73‑year-old man with a swelling of the left lacrimal gland. The tumor had a microscopic appearance consistent with a classical low-grade MEC of the lacrimal gland. There were no signs of recurrence or metastases during a five-year follow-up. Using RT-PCR and FISH we demonstrated that the tumor was positive for the CRTC1-MAML2 gene fusion previously shown to be associated with in particular low-grade salivary MECs with favorable prognosis. By immunohistochemistry we showed that the majority of tumor cells, including epidermoid, intermediate and mucous producing cells, expressed the CRTC1-MAML2 fusion protein. In contrast, 15 non-MEC lacrimal neoplasm were fusion-negative. Our findings show that lacrimal MEC is not only clinically and morphologically but also genetically identical to MECs originating from other exocrine glands, including those of the lung, thyroid, cervix and salivary glands. Taken together, the present and previous studies further emphasize the fundamental biologic and genetic similarities among MECs developing from different anatomical sites and organs. Moreover, our findings indicate that the CRTC1-MAML2 fusion may be a useful diagnostic and prognostic biomarker for lacrimal MEC.

Studies of genomic imbalances and the MYB-NFIB gene fusion in polymorphous low-grade adenocarcinoma of the head and neck.

Polymorphous low-grade adenocarcinoma (PLGA) is a malignancy predominantly originating from the minor salivary glands. The molecular events underlying the pathogenesis of PLGA is poorly understood and no recurrent genetic aberrations have so far been identified. We used genome-wide, high-resolution aCGH analysis to explore genomic imbalances in 9 cases of PLGA. Because of the well-known morphologic similarities between PLGA and adenoid cystic carcinoma (ACC) we also analyzed all tumors for expression of the recently identified ACC-associated MYB-NFIB gene fusion. aCGH analysis revealed that the PLGA genome contains comparatively few copy number alterations (CNAs). Gains/losses of whole chromosomes or chromosome arms were more than twice as common as partial CNAs. Two cases showed gain of chromosome 8 and one case each gain of chromosome 9, loss of chromosome 22 and loss of the Y chromosome. One case showed loss of the entire 6q arm and one case an interstitial deletion of a 33-Mb segment within 6q22.1-q24.3. This region contains the MYB oncogene and the candidate tumor suppressor gene PLAGL1. RT-PCR analysis revealed that one of the 9 PLGAs expressed the ACC-associated MYB-NFIB gene fusion, illustrating the diagnostic difficulties associated with the diagnosis of these morphologically partly overlapping entities. Taken together, our findings indicate that the PLGA genome is genetically stable and contains comparatively few CNAs which is in line with the clinical observation that PLGA is a slow-growing, low-grade carcinoma with low metastatic potential.

YWHAE-FAM22 gene fusion in clear cell sarcoma of the kidney.

We read with great interest the recent publication in J Pathol by O’Meara et al, describing the identification of the target genes of the t(10;17)(q22;p13) translocation in clear cell sarcoma of the kidney (CCSK) [1]. In this excellent paper the authors show that the t(10;17) translocation in CCSK results in a YWHAE–FAM22 gene fusion in which exons 1–5 of YWHAE are linked to exons 2–7 of FAM22. The fusion was detected by FISH and/or RT–PCR in a previously published CCSK with a t(10;17) translocation, as well as in six of 50 further CCSKs tested (overall frequency 12%). Fusionpositive CCSKs were shown to have a significantly higher tumour cellularity compared to fusion-negative tumours (p < 0.001). CCSK is an aggressive paediatric renal cancer preferentially affecting young males [2]. The tumour is defined based on its typical light microscopic appearance, which deviates from that of mesoblastic nephroma and Wilms’ tumour. CCSK also shows a striking propensity to metastasize to bone, as opposed to Wilms’ tumour, which classically spreads to lymph nodes, lung and liver [2]. Of particular difficulty in the diagnosis of CCSK is the pronounced morphological diversity that these tumours may show, ranging from epithelioid to spindle-cell growth patterns, mimicking certain subtypes of Wilms’ tumour. Therefore, there is a need for diagnostically useful biomarkers which may assist in accurately identifying CCSK. As part of a comprehensive screen for tumourtype specific chromosome translocations in sarcomas at our institute, we previously identified a rare type of sarcoma in a 3 week-old boy with a large congenital tumour involving the soft tissues of the right neck (previously unpublished case). The clinicopathological characteristics of this case (designated case 1) are summarized in Table 1. Imaging studies revealed that the lesion extended from the foramen ovale near the cerebellum to the thoracic inlet. A fine needle aspirate was performed, followed by an incisional biopsy. Histopathologically, the tumour showed dense cellularity with uniform, small, primitive cells with poorly defined, lightly staining or clear cytoplasm (Figure 1A). The nuclei were round vesicular with a few small nucleoli. There were solid sheets of tumour cells in focal trabecular and multinodular arrangements. The tumour cells were intimately associated with a prominent vasculature resembling capillaries and venule-like channels. There were numerous mitoses and small foci of necrosis and vesicular invasion. Immunophenotypically, the tumour cells stained positive for vimentin and were negative for cytokeratins, LCA, HBA71 (CD99), NSE, muscle-specific actin (HHF35), S-100 protein, HMB45, desmin, α-smooth muscle actin, α-fetoprotein, chromogranin A, L26 (CD20), and UCHL1 (data not shown). The histopathology and immunoprofile were consistent with the diagnosis of an extrarenal CCSK [3,4]. The tumour did not respond to chemotherapy and the patient developed brain metastases 6 months later and died a few days thereafter. Cytogenetic analysis of the incisional biopsy from case 1 revealed a pseudodiploid male karyotype with two balanced translocations as the sole anomalies, t(10;17)(q22;p13) and t(8;11)(q22;p13) (Figure 1B). FISH analysis using painting probes confirmed the t(8;11) and t(10;17) translocations (data not shown). This is, to our knowledge, the first report of an extrarenal CCSK with a t(10;17) translocation and the fourth case of CCSK reported to date with this rearrangement. Inspired by the interesting findings of O’Meara and co-workers [1], we analysed our t(10;17)-positive CCSK for expression of the YWHAE–FAM22 gene fusion. Total RNA was isolated from frozen tumour cells and converted to cDNA as previously described [5]. RT–PCR analysis using previously described primer sequences [1] revealed expression of a 236 bp fragment (Figure 1C) which, after nucleotide sequence analysis, was shown to correspond to a YWHAE– FAM22 fusion transcript (Figure 1D) identical to that reported by O’Meara et al. [1]. Of interest, in agreement with O’Meara et al, this fusion-positive case was also a highly cellular CCSK at a late stage of the disease. We also searched the files of the Gothenburg Musculo-skeletal Tumour Centre for CCSKs and identifed three additional cases during the last 4 years. The samples were obtained with Institutional Review Board approval. The clinicopathological characteristics of these three cases are summarized in Table 1. Total RNA was extracted from five 10 μm sections obtained from paraffin blocks of each of these three cases and converted to cDNA as previously described [5]. Screening of these cDNAs for expression of YWHAE–FAM22 fusion transcripts, using primer sequences optimized for amplification of shorter transcript fragments in formalin-fixed paraffin-embedded material [1], revealed that all three tumours were negative for the YWHAE–FAM22 fusion (Figure 1C). Thus, one out of four (25%) of our CCSKs were fusion-positive, confirming that the YWHAE–FAM22 fusion is indeed a biomarker that identifies a subset of CCSK. In an effort to identify additional genetic alterations that may cooperate with the YWHAE–FAM22 fusion or characterize fusion-negative CCSKs, we also isolated DNA from three of our four cases of CCSK (there was no material available from case 2) and performed highresolution array CGH analyses using 244K arrays, as previously described [6]. Detailed analysis of the arrays

Overexpression of MYB drives proliferation of CYLD‐defective cylindroma cells

Cutaneous cylindroma is an adnexal tumour with apocrine differentiation. A predisposition to multiple cylindromas is seen in patients with Brooke–Spiegler syndrome, who carry germline mutations in the tumour suppressor gene CYLD. Previous studies of inherited cylindromas have highlighted the frequent presence of bi‐allelic truncating CYLD mutations as a recurrent driver mutation. We have previously shown that sporadic cylindromas express either MYB–NFIB fusion transcripts or show evidence of MYB activation in the absence of such fusions. Here, we investigated inherited cylindromas from several families with germline CYLD mutations for the presence of MYB activation. Strikingly, none of the inherited CYLD‐defective (n = 23) tumours expressed MYB–NFIB fusion transcripts. However, MYB expression was increased in the majority of tumours (69%) and global gene expression analysis revealed that well‐established MYB target genes were up‐regulated in CYLD‐defective tumours. Moreover, knock‐down of MYB expression caused a significant reduction in cylindroma cell proliferation, suggesting that MYB is also a key player and oncogenic driver in inherited cylindromas. Taken together, our findings suggest molecular heterogeneity in the pathogenesis of sporadic and inherited cutaneous cylindromas, with convergence on MYB activation.

Primary sinonasal adenoid cystic carcinoma presenting with skin metastases – genomic profile and expression of the MYB–NFIB fusion biomarker

Sir: Adenoid cystic carcinoma (ACC) of the head and neck is an aggressive neoplasm with a protracted clinical course and poor long-term survival, mainly owing to metastatic disease. ACC tends to metastasize to the lungs, bone, liver, and brain. Cutaneous metastases are very rare, and their exact incidence is underestimated, because they can be confused with other primary tumours of the skin. There are no specific immunohistochemical biomarkers for ACC, and if the primary malignancy is unknown, the index of suspicion may be low and the diagnosis may be missed. Recent studies have demonstrated that most cases of ACC, irrespective of anatomical location, harbour a t(6;9)(q22–23;p23–24) chromosomal translocation,