Virchows Arch (2014) 464:241–245 DOI 10.1007/s00428-013-1520-3

CASE REPORT

Increased ALK1 copy number and renal cell carcinoma—a case report Ciara Ryan & Nick Mayer & Joan Cunningham & Gordon Hislop & Norman Pratt & Stewart Fleming

Received: 31 October 2013 / Revised: 14 November 2013 / Accepted: 25 November 2013 / Published online: 7 December 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract There have been recent reports of a rare variant of renal cell carcinoma associated with upregulation of the anaplastic lymphoma kinase gene (ALK) arising as a consequence of chromosomal translocations. The tumours were described as having a characteristic morphology. Here, we describe a case with similar morphology characterised by eosinophilic cells, abundant intracytoplasmic lumina and scattered large ganglionlike tumour cells. There was focal staining for ALK demonstrated by immunohistochemistry. However, rather than exhibiting a chromosomal translocation involving ALK, the use of FISH and a break-apart probe demonstrated that there was increased copy number of intact 2p23, the chromosomal region containing the ALK gene. Furthermore, the use of comparative genomic hybridisation showed increase of the whole of chromosome 2 along with chromosomes 6 and 17. There was no evidence of loss of 3p nor of trisomy of 7 associated with clear cell and papillary carcinoma, respectively. We suggest that this demonstrates a novel mechanism of upregulation of ALK activity by increased copy number occurring during the development of a renal carcinoma with the characteristic ALK-associated morphology. Keywords ALK . Copy number . Renal cell carcinoma

Introduction The anaplastic lymphoma kinase gene (ALK) encodes a receptor tyrosine kinase of the insulin receptor family so named C. Ryan : N. Mayer Cork University Hospital, Cork, Ireland J. Cunningham : G. Hislop : N. Pratt : S. Fleming (*) Ninewells Hospital and Dundee University Medical School, Dundee, Scotland e-mail: [email protected]

because of the finding of its involvement in anaplastic lymphomas [1]. The gene is silenced in most adult tissues but overexpression is detected by immunocytochemistry in anaplastic or diffuse large B cell lymphoma and in 50 % of inflammatory myofibroblastic tumours [2, 3]. The most common chromosomal alteration which results in a fusion depends on the tumour type: in T cell anaplastic lymphoma, t(2;5)(p23;q35) NPM1-ALK; in ALK + ve DLBCL, t(2;17)(p23;q23) CLTC-ALK; in non-small cell lung carcinoma, inv[2](p23p21) EML4-ALK. Further ALK rearrangements have been described involving NPM-ALK, KIF5BALK and TPM4-ALK in non-small cell lung carcinoma, oesophageal carcinoma and a small proportion of both breast and colo-rectal carcinomas [4]. The overexpression of the ALK oncogene as a result of these chromosomal alterations frequently leads to constitutive activation of its tyrosine kinase activity with consequent cellular changes. ALK-associated renal cell carcinoma is a relatively recent addition to ALK pathology. Two types have been described, either ALK alterations as potentially causative genetic events or as progression factors associated with other renal tumour types. To date, reports have identified pathogenic ALK translocations resulting in oncogenic fusion products in six patients. Two children of African American or West African origin and both carriers of sickle cell trait had renal tumours of a morphologically distinctive ALK-associated RCC involving vinculin (VCL) as a new fusion partner. The fusion oncogene, the result of a t(2;10)(p23:q22) translocation, comprises a fusion of vinculin and ALK retaining the cellular localisation signal of vinculin and constitutively active ALK tyrosine kinase [5, 6]. Two Japanese cases of ALK-associated RCC in adults have been described involving alternative fusion partners—one TPM3-ALK and the other EML4-ALK[6, 7]. Neither of these patients had evidence of sickle cell trait. Most recently, two cases of papillary RCC have been described, also in adults, with an ALK rearrangement, currently unspecified,

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but not an ALK-VCL translocation [8]. ALK-associated renal cell carcinoma is now recognised as a distinct lesion in the recent International Society of Urological Pathology (ISUP) Vancouver classification of renal neoplasia [9]. Subsequent papers have suggested that ALK alterations may be found additional to 3p loss or trisomy 7 and 17 in clear cell or papillary renal cell carcinoma, respectively. A recent large study of 534 clear cell and papillary RCCs found that approximately 10 % of cases had increased ALK gene copy numbers but these cases did not have the morphology previously described in ALK-associated RCC [5]. Increased ALK gene copy number has been described in ovarian carcinoma [10] and neuroblastoma [11]. Here, we provide evidence of increased ALK gene copy number in a case of ALK-associated RCC which harboured no other genetic abnormality known to be associated with the development of RCC.

Case report Clinical history A 36-year-old African male, who did not carry sickle cell trait, presented with an incidental heterogenous 6-cm renal mass, detected by abdominal ultrasound performed during investigation for hypertension and hyperlipidaemia. A triphasic CT scan was subsequently performed confirming an enhancing mass consistent with renal cell carcinoma. Staging CT of thorax revealed multiple small nodules; these were not deemed suspicious for metastatic disease. An open right radical nephrectomy was performed. Post-operative complications included a right-sided varicocoele and one episode of frank haematuria, both of which resolved. One year later, he is alive and well.

Material and methods Histopathology On macroscopic examination of the nephrectomy specimen, there was a 6.5-cm encapsulated tumour arising in the lower pole of the right kidney. It had a heterogenous cut surface composed predominantly of a soft cream solid component admixed with haemorrhagic areas with focal cystic change adjacent to a single gelatinous area. Histologically, the tumour was mainly composed of polygonal or cuboidal cells arranged in a solid architectural pattern and supported by a rich capillary vascular supply. The cells had eosinophilic granular cytoplasm with indistinct cell borders and some discohesion. The most distinctive feature was that many cells were expanded by a clear intracytoplasmic

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lumen occasionally with an intraluminal brush border (Fig. 1). Nuclei were centrally placed with a moderate degree of pleomorphism and with small solitary nucleoli. Mitoses were present but infrequent. Scattered foci of psammomatous calcification were noted along with occasional collections of foamy histiocytes. In places, the tumour cells adopted a more spindle cell morphology but remained of similar grade to the polygonal cells. There were also areas in which the sheets of polygonal cells were admixed with large multinucleated ganglion-like cells with pale eosinophilic granular cytoplasm but with nuclei of a similar morphology to the rest of the tumour. There were no areas of either clear cell of papillary RCC morphology. The tumour cells were diffusely positive for RCC, MNF and Vimentin. AE1/3, CAM5.2, CK7 and E-cadherin were focally positive. CD10 shows sparse areas of positivity while EMA, Melan-A, HMB45, C-kit, S100, CD34 and Chromogranin were all negative. Some of the intermediate filament stains showed accentuation around the cytoplasmic lumina. Of note, there was focal cytoplasmic staining for ALK1 (Fig. 1) but as is often the case with non-lymphoid lesions, the staining was weak. Cytogenetics Interphase FISH studies on paraffin sections were performed with an ALK break-apart probe (Abbott, Vysis) located on the short arm of chromosome 2 (2p23). This showed an intact pattern but with additional intact copies of ALK in the tumour cells. This intact pattern showed no evidence of disruption of ALK and excluded the following ALK rearrangements: t[2, 5] NPM1-ALK; t[1, 2] TPM3-ALK; inv [2], EML4–ALK; and t[2, 10], VCL–ALK. DNA from the tumour, extracted from the paraffin block, was used in an array-based comparative genomic hybridisation. These data showed low level gain of the whole of chromosome 2. There was no evidence of the 3p loss characteristic of clear cell RCC, the trisomy of chromosome 7 found in papillary RCC, nor of the multiple chromosomal losses encountered in chromophobe RCC (Fig. 2). These CGH data also showed gains of chromosomes 2, 6, 16 and 17 which were confirmed by FISH using centromeric probes for the relevant chromosomes (Abbott, Vysis).

Discussion Due to recent developments highlighting ALK tyrosine kinase receptor as a therapeutic target in lung adenocarcinomas, ALK has become the focus of many studies aiming to detect ALK rearrangements in other tumours thereby increasing therapeutic options. Our case is unique as it represents the first case with morphology of an ALK-associated RCC associated with gain of the intact ALK gene without evidence of break-apart

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Fig. 1 a Low-power examination shows the solid architecture composed of eosinophilic cells many of which have cytoplasmic lumina. (H&E×4) b The ALK-associated renal cell carcinoma consists of cuboidal eosinophilic tumour cells frequently showing the presence of intracytoplasmic lumina. c In other areas of the tumour, large ganglion-like cells were present. (Both H&E×20 lens magnification) d There was focal weak staining of tumour cells for ALK in the cytoplasm (IHC×20 lens magnification)

of ALK or of fusion products. We have also confirmed lack of genetic changes seen in other types of renal cell carcinoma including 3p loss, trisomy of chromosome 7 and chromosomal losses associated with chromophobe RCC. To date, six morphologically similar cases of renal tumours associated with chromosomal abnormalities involving the ALK gene and resulting in oncogenic ALK fusion products have been described. Two of these, in a paediatric population, were African males (like our patient) with sickle cell trait and a VCL-ALK translocation forming a novel fusion product [5, 6]. One was finally categorised as indeterminate subtype (with mixed features including those of medullary, chromophobe and transitional cell carcinomas) and the second as a renal medullary carcinoma. Both of these cases showed the striking intracytoplasmic lumen formation. Vinculin, the other component of the fusion protein, is a protein essential for the assembly of cell adhesion focal contacts. Two Japanese cases described by Sugawara et al. were in the adult population with different translocations—TPM3-ALK (found in anaplastic large cell lymphoma and inflammatory myofibroblastic tumour) and EML4-ALK (reported to be identified in lung, breast and colon adenocarcinomas)—and different morphology [7]. Most recently, two cases of papillary RCC in adults were identified in a 534-patient study showing unspecified but VCL-negative ALK rearrangements [8]. Sukov et al. [8] identified increased ALK copy number as a poor prognostic marker in 54 of 532 cases of both clear cell and papillary variants of RCC. Increased copy number of the ALK gene has recently been shown to be pathogenic in ovarian carcinoma

and in occasional sporadic neuroblastomas; therefore, its potential in tumourigenesis has been established [10, 11]. Our case adds to the literature documenting the possibility of a unique mechanism of ALK upregulation, by chromosomal numerical alteration, in the development of a tumour with the morphology previously seen in cases with chromosomal translocations involving ALK . Morphology may be a clue to identifying ALK-associated RCCs, and our case had some distinct features similar to those previously reported with ALK rearrangements that may be unique to this specific tumour subtype. Of particular note are the presence of large cytoplasmic lumina, ganglion-like cells and focally prominent vacuolated cells. The localisation of ALK kinase activity in the focal contacts with phosphorylation of focal contact components would be predicted to disrupt cell polarity leading to the formation of intracytoplasmic lumina as has been demonstrated previously in in vitro experiments [12]. The intracytoplasmic lumina were less conspicuous in the two adult Japanese cases—described as having occasional deeply eosinophilic intracytoplasmic inclusions resulting in rhabdoid features in the TPM3-ALK case and rare pseudonuclear inclusions in the EML4-ALK case [7]. An awareness of the association between intracytoplasmic lumina and ALK amplification in renal tumours may guide the pathologist towards molecular testing if this feature is noted on histologic examination. The paediatric renal medullary carcinoma and both recently described ALK-associated papillary RCCs did not contain this feature. Psammomatous calcifications were found in our case and in three of the previously

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Fig. 2 a FISH on tumour cells with an ALK Break Apart probe (located at 2p23) showing additional intact signals is consistent with either gain of copy number of 2p23 or chromosome 2. b Comparative genomic

hybridization shows that the gains involve the whole of chromosome 2 with additional gains of chromosome 6 and 17

reported adult cases [7]. ALK immunohistochemistry showed focal weak cytoplasmic positivity, staining patterns were not specified in the two paediatric cases [5], the Japanese cases had diffuse strong ALK positivity and the cases of Sukov et al, without specific translocations, showed variable staining— weak cytoplasmic positivity in one and moderate to strong in the other [7, 8]. Our patient is alive with no evidence of disease 1 year postsurgery. Sukov et al. found a correlation between number of

copies of ALK and prognosis—the presence of more than five copies was associated with a poorer cancer-specific survival in clear cell RCC but not in papillary RCC [8].

Conclusion We suggest that some cases of this unusual renal malignancy can arise by increased copy number of the ALK gene rather

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than rearrangement, as seen in ovarian carcinoma and neuroblastoma. The recognition of this rare tumour is important because of the availability of ALK-based chemotherapy.

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245 6. Marino-Enriquez A, Ou WB, Weldon CB, Fletcher JA, Perez-Atayde AR (2011) ALK rearrangement in sickle cell trait-associated renal medullary carcinoma. Genes Chromosomes Cancer 50:146–153 7. Sugawara E, Togashi Y, Kuroda N, Sakata S, Hatano S, Asaka R, Yuasa T, Yonese J, Kitagawa M, Mano H, Ishikawa Y, Takeuchi K (2012) Identification of anaplastic lymphoma kinase fusions in renal cancer: large-scale immunohistochemical screening by the intercalated antibody-enhanced polymer method. Cancer 118:4427–4436 8. Sukov WR, Hodge JC, Lohse CM, Akre MK, Leibovich BC, Thompson RH, Cheville JC (2012) ALK alterations in adult renal cell carcinoma: frequency, clinicopathologic features and outcome in a large series of consecutively treated patients. Mod Pathol 25:1516–1525 9. Srigley JR, Delahunt B, Eble JN, Egevad L, Epstein JI, Grignon D, Hes O, Moch H, Montironi R, Tickoo SK, Zhou M, Argani P, Panel IRT (2013) The International Society of Urological Pathology (ISUP) Vancouver Classification of Renal Neoplasia. Am J Surg Pathol 37: 1469–1489 10. Ren H, Tan ZP, Zhu X, Crosby K, Haack H, Ren JM, Beausoleil S, Moritz A, Innocenti G, Rush J, Zhang Y, Zhou XM, Gu TL, Yang YF, Comb MJ (2012) Identification of anaplastic lymphoma kinase as a potential therapeutic target in ovarian cancer. Cancer Res 72:3312–3323 11. Bagci O, Tumer S, Olgun N, Altungoz O (2012) Copy number status and mutation analyses of anaplastic lymphoma kinase (ALK) gene in 90 sporadic neuroblastoma tumors. Cancer Lett 317:72–77 12. Rahilly MA, Fleming S (1992) A tumour promoter induces alterations in vinculin and actin distribution in human renal epithelium. J Pathol 166:283–288

Increased ALK1 copy number and renal cell carcinoma-a case report.

There have been recent reports of a rare variant of renal cell carcinoma associated with upregulation of the anaplastic lymphoma kinase gene (ALK) ari...
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