Human Pathology (2015) 46, 1226–1231
www.elsevier.com/locate/humpath
Case study
Broadening the spectrum of SMARCB1-associated malignant tumors: a case of uterine leiomyosarcoma in a patient with schwannomatosis☆ Irene Paganini MSc a , Roberta Sestini PhD a , Matilde Cacciatore MD b , Gabriele L. Capone MSc a , Luisa Candita MD a , Concetta Paolello MD c , Marta Sbaraglia MD b , Angelo P. Dei Tos MD b,⁎, Sabrina Rossi MD, PhD b,1 , Laura Papi MD, PhD a,1 a
Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, 50139, Florence, Italy Department of Pathology and Molecular Genetics, Treviso General Hospital, 31100,Treviso, Italy c Department of Oncology, Treviso General Hospital, 31100, Treviso, Italy b
Received 22 January 2015; revised 8 April 2015; accepted 14 April 2015
Keywords: SMARCB1; LZTR1; NF2; Schwannomatosis; Leiomyosarcoma
Summary Schwannomatosis is a tumor predisposition syndrome characterized by development of multiple intracranial, spinal, and peripheral schwannomas. Constitutional alterations in either SMARCB1 or LZTR1 on 22q are responsible of the phenotype. We describe a 34-year-old woman who developed multiple benign peripheral sheath tumors and a uterine leiomyosarcoma. The patient carried a de novo constitutional alteration in exon 8 of SMARCB1, c.1118G N A, which destroyed the splice donor site of intron 8. Two schwannomas and the leiomyosarcoma of the patient retained the SMARCB1 mutation; in addition, the tumors showed loss of the normal chromosome 22. In conclusion, our findings enlarged the spectrum of SMARCB1-predisposing tumors and demonstrated, for the first time, the association of a malignant smooth muscle tumor to schwannomatosis. Therefore, clinicians should definitely be aware that a constitutional SMARCB1 mutation, which mainly predisposes to benign nerve sheath tumors, may also predispose to aggressive neoplasms throughout life, within an unexpected spectrum. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Schwannomatosis is a tumor predisposition syndrome characterized by development of multiple intracranial, spinal, and peripheral schwannomas, without involvement of vestib☆ Funding/Support: This study was supported by grants from Istituto Toscano Tumori (to L.P.). ⁎ Corresponding author at: Department of Pathology and Molecular Genetics, Treviso General Hospital, Piazza Ospedale 1, 31100, Treviso, Italy. E-mail address: [email protected] (A. P. Dei Tos). 1 These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.humpath.2015.04.008 0046-8177/© 2015 Elsevier Inc. All rights reserved.
ular nerve, which is the pathognomonic sign of neurofibromatosis type 2 (NF2) [1]. For a long time, patients with NF2 and schwannomatosis have been confused with each other because of common clinical features. For this reason, prevalence of schwannomatosis is not yet certainly defined, but it is estimated to be about 1 in 40 000 births. Schwannomatosis is a genetic condition, but its transmission pattern is not completely clarified. Molecular analysis of NF2 gene in patients with schwannomatosis has demonstrated the presence of inactivating mutations in tumor tissues but no evidence of germline mutations as found in patients with NF2 [2]. Therefore, the hallmark of
SMARCB1-associated malignant tumors schwannomatosis is the evidence of different somatic mutations in multiple tumors from the same patient with schwannomatosis. In 2007, studying these patients, Hulsebos et al [3] identified germline mutations in SMARCB1, a gene located on 22q centromeric to NF2, encoding for a protein implicated in chromatin remodeling. Although SMARCB1 is considered the first predisposing gene for schwannomatosis, its constitutional mutations are found only in 40% to 50% of familial cases and 8% to 10% of sporadic cases [4]. Very recently, constitutional alterations in a novel gene, LZTR1, also located on 22q in centromeric position to SMARCB1, have been found in a proportion of patients with schwannomatosis lacking germline variants of SMARCB1 [5,6], pointing to LZTR1 and SMARCB1 mutations as alternative pathogenetic mechanisms. The tumorigenesis of schwannomatosis-associated schwannomas follows a “4-hits/3-steps” mechanism with multistep biallelic inactivation of SMARCB1 or LZTR1 and NF2 genes: SMARCB1 or LZTR1 germline mutation followed by an NF2 somatic mutation and loss of heterozygosity (LOH) for chromosome 22 represent the stages of the 4-hits/3-steps mechanism [7]. Remarkably, immunohistochemical studies have demonstrated a mosaic pattern of SMARCB1 protein expression, with mixed positive and negative nuclei, in most tumors from patients with familial schwannomatosis and in a large proportion (~55%) of tumors from sporadic schwannomatosis [8]. Differently, schwannomas occurring in nonsyndromic context generally show intact immunohistochemical expression of SMARCB1 [8]. Notably, most schwannomatosis-associated tumors are benign; very rarely, malignant peripheral nerve sheath tumors may occur, with only 4 bona fide cases published so far [9,10]. SMARCB1 is also involved in the development of atypical teratoid/rhabdoid tumors (AT/RT) in the setting of the rhabdoid tumor predisposition syndrome (RTPS1); a few RTPS1 families include members affected either by AT/RT or multiple schwannomas, but to the best of our knowledge, no cooccurence of AT/RT and schwannomatosis in the same patient has been reported so far (reviewed by Sredni and Tomita [11]). The clinical spectrum of schwannomatosis has been further expanded by the description of the occurrence of benign tumors showing other lines of differentiation, as meningiomas [12] and, very recently, a case of uterine leiomyoma [13]. In these tumors, the presence of a SMARCB1 mutation as well as LOH of 22q indicate their contribution to schwannomatosis clinical features. We report a case of a patient with schwannomatosis with a novel germline SMARCB1 mutation, who developed multiple benign peripheral nerve sheath tumors and a uterine leiomyosarcoma.
1227 past and family history was not relevant. Between 24 and 31 years of age, she underwent 5 additional surgeries for the occurrence of multiple benign peripheral nerve sheath tumors, which were located in the pelvis, right brachial plexus, retroperitoneum, and right thigh. Tumors size ranged between 2 and 15 cm, the largest being the pelvic mass. The tumors were diagnosed as schwannomas, with the exception of the ones located in the brachial plexus, which were interpreted as hybrid neurofibromas/schwannomas. Periodic cranial magnetic resonance imaging (MRI) scans were always negative for vestibular lesions. At 33 years of age, she underwent hysteroannessiectomy for a 10-cm tumor located in the posterior wall of the uterine body. The tumor, identified by MRI during follow-up, had rapidly enlarged, with a 4-fold increase in size over 11 months. Based on its histologic features, a diagnosis of uterine leiomyosarcoma was made. During the same surgery, an additional schwannoma of the right psoas was excised. The patient received 6 cycles of chemotherapy (gemcitabine, 1000 mg/m2 on days 1 and 8, and docetaxel, 75 mg/m2 on day 8). Thirteen months after surgery, a control MRI identified a 3-cm hepatic nodule and 2 bone lesions at the right ileum, which were confirmed by scintigraphy. A subsequent control 3 months later showed a clear-cut increase in size of these lesions as well as additional multiple bone lesions (vertebral and left femoral) and a 3.3-cm retroperitoneal nodule. The biopsy of the hepatic lesion showed a malignant tumor with morphological and immunophenotypic features consistent with metastatic leiomyosarcoma. The patient has been currently treated with chemotherapy for metastatic disease (doxorubicin hydrochloride, 40 mg/m2 every 4 weeks) and radiotherapy at the right pelvis for pain control (3000 cGy in 10 fractions). Written informed consent was obtained from the patient for publication of this case report.
2.2. DNA and RNA extraction DNA was extracted from blood leukocytes and from fresh frozen tumor tissue using standard procedures with phenol/ chloroform extraction and ammonium acetate/ethanol precipitation. DNA from formalin-fixed, paraffin-embedded tissues was isolated according to manufacturers’ manual of QIAamp DNA FFPE Tissue Kit (Qiagen; Helden, Germany). RNA was extracted from blood sample using PAXgene blood RNA Kit (PreAnalytix, Qiagen).
2.3. Sanger sequencing
2. Materials and methods 2.1. Case history The patient presented her first clinical manifestation at 18 years old with the development of a scalp tumor, which was removed 2 years later and interpreted as schwannoma. Her
The entire coding sequences of SMARCB1, LZTR1, and NF2 as well as MED12 exon 2 were sequenced with polymerase chain reaction and capillary sequencing. Primer sequences are available on request. Capillary sequencing was performed on 310 Capillary DNA Analyzer (Life Technologies; Carlsbad, CA). Raw and analyzed sequence results were visualized on Sequence Scanner v1.0 (Life Technologies).
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2.4. Microsatellite analysis Loss of heterozygosity on 22q was investigated using microsatellites D22S420, D22S1174, D22S1154, D22S1163, and D22S277 from the ABI PRISM Linkage Mapping set version 2.5 (Life Technologies).
2.5. Multiplex ligation-dependent probe amplification Copy number changes (deletions or duplications) of SMARCB1 and NF2 loci and flanking genes were analyzed by multiplex ligation-dependent probe amplification (MLPA). SMARCB1, NF2 and 22q11 MLPA test kits (P258_C1, P044_B1, and P324_A2, MRC-Holland; Amsterdam, The Netherlands) were used, and electrophoresis data were analyzed using GeneMapper software (Life Technologies).
2.6. Immunohistochemistry To test SMARCB1 expression, 4-μm paraffin-embedded tissue sections from the benign nerve sheath tumors and the leiomyosarcoma were immunostained (clone 25/baf47,1:25, pH9, BD Transduction Laboratories Biosciences San Diego, CA, USA) by an automated immunostainer (Dako Autostainer, DakoCytomation; Glostrup, Denmark). Cells lacking nuclear staining were considered as negative. Lack of nuclear staining in a fraction of neoplastic cells was considered as reduced (mosaic) expression.
3. Results 3.1. Molecular findings LZTR1 and SMARCB1 were directly sequenced in DNA extracted from blood lymphocytes of the patient. No mutation was identified in LZTR1; on the contrary, SMARCB1 sequencing analysis showed the constitutional mutation c.1118G N A, involving the last base of the exon 8. This alteration was predicted to disrupt the donor splice site and was absent in the healthy parents of the patient, confirming that it occurred de novo. The alteration of the splicing process was confirmed by RNA analysis that showed the retaining of intron 8 in messenger RNA (Fig. 1A); the derivative RNA is predicted to code for an elongated protein with a new translation stop site after 48 amino acids (Fig. 1A). Sanger sequencing of SMARCB1 exon 8 in 2 schwannomas and in the leiomyosarcoma of the patient showed the loss of the wild-type G allele (Fig. 1B), confirmed by LOH analysis in the 2 FFPE schwannomas and by MLPA analysis for the fresh frozen tissue of leiomyosarcoma (Fig. 1C). Furthermore, we analyzed the entire coding sequence and exon-intron junctions of NF2 gene in the 2 schwannomas and leiomyosarcoma. One schwannoma (T692) showed a
I. Paganini et al. somatic variant of NF2 gene: c.418delC, p.(Leu140Serfs*34); whereas no NF2 alterations were detected in the other schwannoma and in the leiomyosarcoma. Finally, based on the increasingly recognized role of MED12 gene in uterine smooth muscle tumors [14], we sought to sequence its exon 2 hotspot region in DNA extracted from the leiomyosarcoma, but no variants were identified.
3.2. Pathologic findings Most of the benign nerve sheath tumors showed the classic features of schwannoma. Differently, the tumors located at the brachial plexus were histologically characterized by the presence of schwannoma-like nodules composed of Antoni A regions, surrounded by a neurofibroma-like component with abundant collagen and myxoid changes, fulfilling the criteria for the diagnosis of hybrid neurofibromas/schwannomas [15] (Fig. 2A). The uterine tumor was composed by spindle cells with eosinophilic cytoplasm showing a clear-cut smooth muscle differentiation. No rhabdoid features were seen. The tumor was hypercellular throughout and exhibited a diffuse moderate to severe nuclear atypia. The mitotic index was high, with up to 26 mitoses per 10 high-power fields (Fig. 2B). Foci of neoplastic necrosis were present (Fig. 2C). At immunohistochemical analysis, the neoplasm showed diffuse expression of smooth muscle actin and caldesmon as well as focal expression of desmin, confirming the smooth muscle differentiation of the tumor. All the other markers tested, as S-100, GFAP, synaptophysin, myogenin, CKAE1/AE3, CKMNF116, CD117, and DOG1, were negative. Interestingly, in the schwannomas, the hybrid schwannoma/ neurofibromas of the radial plexus and the uterine leiomyosarcoma SMARCB1 was not homogeneously expressed, and areas with SMARCB1-negative cells were present (Fig. 2D and E), in line with previous studies [8].
4. Discussion Schwannomatosis is a rare genetic syndrome characterized by the development of multiple peripheral schwannomas, without the involvement of vestibular nerves. The clinical findings in patients with schwannomatosis have been widened in the last few years, and currently, the spectrum of associated tumors includes meningiomas [12] and leiomyomas [13]. However, to date, malignant tumors have been reported only in few cases, and they are invariably represented by malignant peripheral nerve sheath tumors [9,10]. We describe a patient affected by SMARCB1-related sporadic schwannomatosis, who developed a uterine leiomyosarcoma in addition to the typical benign peripheral nerve sheath tumors. We demonstrated the presence of SMARCB1 germline mutation and, more importantly, documented loss of the SMARCB1 wildtype allele in the leiomyosarcoma, proving the role of this genetic alteration in the tumor pathogenesis.
SMARCB1-associated malignant tumors
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Fig. 1 A, Partial sequence of SMARCB1 complementary DNA from leukocytes of the patient. B, The mutation causes retaining of intron 8, and it is predicted to create a new translation stop site after 48 amino acids. Partial sequences of SMARCB1 in blood and tumors of patient 299. The SMARCB1 mutation occurs in the last base of exon 8. This alteration is retained in the 2 schwannomas (T691, T692) and in the leiomyosarcoma (T791) with loss of the wild-type allele. C, Allelic losses on 22q in the 2 formalin-fixed, paraffin-embedded schwannomas. Microsatellite analysis shows LOH of 22q. The arrows indicate the lost alleles in tumor DNA. The 2 schwannomas lost the same allele in all the analyzed microsatellites. DNA extracted from fresh tumor tissue of leiomyosarcoma was analyzed by MLPA (kit P324_A2). Normalized data from Coffalyzer.net software show a deletion of 22q. The deletion is highlighted by the red dots that indicate the deleted probes (ratio of 0.5).
Immunohistochemical analysis showed loss of SMARCB1 expression in a minority of neoplastic nuclei. Notably, among schwannomatosis-related schwannomas, there is a remarkable intertumoral variability with cases showing less than 10% negative nuclei [8]. We believe that the pattern of SMARCB1 expression in our case may belong to the low end of the mosaic spectrum. SMARCB1 somatic changes can be found in other malignant neoplasms including epithelioid sarcoma, epithelioid malignant peripheral nerve sheath tumor, myoepithelial carcinoma, extraskeletal myxoid chondrosarcoma, and renal
medullary carcinoma indicating that it is involved in their pathogenesis [16]. Uterine leiomyosarcoma is the most common uterine sarcoma, accounting for approximately 2% of all uterine malignancies [17]. Leiomyosarcoma shares several characteristics with leiomyoma, including smooth muscle morphology and immunophenotype as well as the frequent arising within the uterine corpus in middle-aged women. However, they are considered biologically unrelated tumors because of their disparate clinical, cytogenetic, and molecular features [14].
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I. Paganini et al.
Fig. 2 A, A schwannoma-like nodule surrounded by a neurofibroma-like component with remarkable myxoid changes in 1 of the tumor interpreted as hybrid neurofibroma/schwannoma. The uterine leiomyosarcoma was highly cellular and showed moderate to severe nuclear atypia with brisk mitotic activity (B) and tumor necrosis (C). Some of the neoplastic cells lacked SMARCB1 nuclear staining in the schwannoma (D) and the leiomyosarcoma (E).
Recently, challenging this perspective, mutations in exon 2 of MED12 gene have been reported in both leiomyoma (50%-70%) and leiomyosarcoma (b30%) [14]. Leiomyosarcoma of our schwannomatosis patient did not show any variant in exon 2 of MED12, ruling out its possible pathogenetic role and, indirectly, strengthening the causative role of the SMARCB1 biallelic inactivation, in our case. To our knowledge, only 1 case of retroperitoneal leiomyosarcoma lacking SMARCB1 expression has been reported in the literature, but data on SMARCB1 mutational status have not been provided [18]. Our observation may suggest to investigate thoroughly sporadic leiomyosarcomas for SMARCB1 deficiency. In conclusion, our findings enlarged the spectrum of SMARCB1-predisposing tumors and demonstrated, for the first time, the association of a malignant tumor with smooth muscle differentiation to schwannomatosis. Therefore, clini-
cians should definitely be aware that a constitutional SMARCB1 mutation, which mainly predisposes to benign nerve sheath tumors, may also predispose to aggressive neoplasms throughout life, within an unexpected spectrum.
References [1] Jacoby LB, Jones D, Davis K, et al. Molecular analysis of the NF2 tumor-suppressor gene in schwannomatosis. Am J Hum Genet 1997; 61:1293-302. [2] MacCollin M, Willett C, Heinrich B, et al. Familial schwannomatosis: exclusion of the NF2 locus as the germline event. Neurology 2003;60: 1968-74. [3] Hulsebos TJ, Plomp AS, Wolterman RA, Robanus-Maandag EC, Baas F, Wesseling P. Germline mutation of INI1/SMARCB1 in familial schwannomatosis. Am J Hum Genet 2007;80:805-10. [4] Hadfield KD, Newman WG, Bowers NL, et al. Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis. J Med Genet 2008;45:332-9.
SMARCB1-associated malignant tumors [5] Piotrowski A, Xie J, Liu YF, et al. Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas. Nat Genet 2014;46:182-7. [6] Paganini I, Chang VY, Capone GL, et al. Expanding the mutational spectrum of LZTR1 in schwannomatosis. Eur J Hum Genet 2014 [Epub ahead of print]. [7] Sestini R, Bacci C, Provenzano A, Genuardi M, Papi L. Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas. Hum Mutat 2008;29:227-31. [8] Patil S, Perry A, Maccollin M, et al. Immunohistochemical analysis supports a role for INI1/SMARCB1 in hereditary forms of schwannomas, but not in solitary, sporadic schwannomas. Brain Pathol 2008;18:517-9. [9] Gonzalvo A, Fowler A, Cook RJ, et al. Schwannomatosis, sporadic schwannomatosis, and familial schwannomatosis: a surgical series with long-term follow-up. Clinical article. J Neurosurg 2011;114:756-62. [10] Carter JM, O'Hara C, Dundas G, et al. Epithelioid malignant peripheral nerve sheath tumor arising in a schwannoma, in a patient with "neuroblastoma-like" schwannomatosis and a novel germline SMARCB1 mutation. Am J Surg Pathol 2012;36:154-60. [11] Sredni ST, Tomita T. Rhabdoid tumor predisposition syndrome. Pediatr Dev Pathol 2015;18:49-58.
1231 [12] Bacci C, Sestini R, Provenzano A, et al. Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation. Neurogenetics 2010;11:73-80. [13] Hulsebos TJ, Kenter S, Siebers-Renelt U, Hans V, Wesseling P, Flucke U. SMARCB1 involvement in the development of leiomyoma in a patient with schwannomatosis. Am J Surg Pathol 2014; 38:421-5. [14] Ravegnini G, Mariño-Enriquez A, Slater J, et al. MED12 mutations in leiomyosarcoma and extrauterine leiomyoma. Mod Pathol 2013;26: 743-9. [15] Harder A, Wesemann M, Hagel C, et al. Hybrid neurofibroma/ schwannoma is overrepresented among schwannomatosis and neurofibromatosis patients. Am J Surg Pathol 2012;36:702-9. [16] Hollmann TJ, Hornick JL. INI1-deficient tumors: diagnostic features and molecular genetics. Am J Surg Pathol 2011;35:47-63. [17] Kurman RJ, Carcangiu ML, Herrington CS, Young RH. WHO Classification of Tumors of Female Reproductive Organs. 4th ed. Lyon: IARC; 2014. [18] Perry A, Fuller CE, Judkins AR, Dehner LP, Biegel JA. INI1 expression is retained in composite rhabdoid tumors, including rhabdoid meningiomas. Mod Pathol 2005;18:951-8.
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Case study
Broadening the spectrum of SMARCB1-associated malignant tumors: a case of uterine leiomyosarcoma in a patient with schwannomatosis☆ Irene Paganini MSc a , Roberta Sestini PhD a , Matilde Cacciatore MD b , Gabriele L. Capone MSc a , Luisa Candita MD a , Concetta Paolello MD c , Marta Sbaraglia MD b , Angelo P. Dei Tos MD b,⁎, Sabrina Rossi MD, PhD b,1 , Laura Papi MD, PhD a,1 a
Department of Biomedical Experimental and Clinical Sciences, Medical Genetics, University of Florence, 50139, Florence, Italy Department of Pathology and Molecular Genetics, Treviso General Hospital, 31100,Treviso, Italy c Department of Oncology, Treviso General Hospital, 31100, Treviso, Italy b
Received 22 January 2015; revised 8 April 2015; accepted 14 April 2015
Keywords: SMARCB1; LZTR1; NF2; Schwannomatosis; Leiomyosarcoma
Summary Schwannomatosis is a tumor predisposition syndrome characterized by development of multiple intracranial, spinal, and peripheral schwannomas. Constitutional alterations in either SMARCB1 or LZTR1 on 22q are responsible of the phenotype. We describe a 34-year-old woman who developed multiple benign peripheral sheath tumors and a uterine leiomyosarcoma. The patient carried a de novo constitutional alteration in exon 8 of SMARCB1, c.1118G N A, which destroyed the splice donor site of intron 8. Two schwannomas and the leiomyosarcoma of the patient retained the SMARCB1 mutation; in addition, the tumors showed loss of the normal chromosome 22. In conclusion, our findings enlarged the spectrum of SMARCB1-predisposing tumors and demonstrated, for the first time, the association of a malignant smooth muscle tumor to schwannomatosis. Therefore, clinicians should definitely be aware that a constitutional SMARCB1 mutation, which mainly predisposes to benign nerve sheath tumors, may also predispose to aggressive neoplasms throughout life, within an unexpected spectrum. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Schwannomatosis is a tumor predisposition syndrome characterized by development of multiple intracranial, spinal, and peripheral schwannomas, without involvement of vestib☆ Funding/Support: This study was supported by grants from Istituto Toscano Tumori (to L.P.). ⁎ Corresponding author at: Department of Pathology and Molecular Genetics, Treviso General Hospital, Piazza Ospedale 1, 31100, Treviso, Italy. E-mail address: [email protected] (A. P. Dei Tos). 1 These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.humpath.2015.04.008 0046-8177/© 2015 Elsevier Inc. All rights reserved.
ular nerve, which is the pathognomonic sign of neurofibromatosis type 2 (NF2) [1]. For a long time, patients with NF2 and schwannomatosis have been confused with each other because of common clinical features. For this reason, prevalence of schwannomatosis is not yet certainly defined, but it is estimated to be about 1 in 40 000 births. Schwannomatosis is a genetic condition, but its transmission pattern is not completely clarified. Molecular analysis of NF2 gene in patients with schwannomatosis has demonstrated the presence of inactivating mutations in tumor tissues but no evidence of germline mutations as found in patients with NF2 [2]. Therefore, the hallmark of
SMARCB1-associated malignant tumors schwannomatosis is the evidence of different somatic mutations in multiple tumors from the same patient with schwannomatosis. In 2007, studying these patients, Hulsebos et al [3] identified germline mutations in SMARCB1, a gene located on 22q centromeric to NF2, encoding for a protein implicated in chromatin remodeling. Although SMARCB1 is considered the first predisposing gene for schwannomatosis, its constitutional mutations are found only in 40% to 50% of familial cases and 8% to 10% of sporadic cases [4]. Very recently, constitutional alterations in a novel gene, LZTR1, also located on 22q in centromeric position to SMARCB1, have been found in a proportion of patients with schwannomatosis lacking germline variants of SMARCB1 [5,6], pointing to LZTR1 and SMARCB1 mutations as alternative pathogenetic mechanisms. The tumorigenesis of schwannomatosis-associated schwannomas follows a “4-hits/3-steps” mechanism with multistep biallelic inactivation of SMARCB1 or LZTR1 and NF2 genes: SMARCB1 or LZTR1 germline mutation followed by an NF2 somatic mutation and loss of heterozygosity (LOH) for chromosome 22 represent the stages of the 4-hits/3-steps mechanism [7]. Remarkably, immunohistochemical studies have demonstrated a mosaic pattern of SMARCB1 protein expression, with mixed positive and negative nuclei, in most tumors from patients with familial schwannomatosis and in a large proportion (~55%) of tumors from sporadic schwannomatosis [8]. Differently, schwannomas occurring in nonsyndromic context generally show intact immunohistochemical expression of SMARCB1 [8]. Notably, most schwannomatosis-associated tumors are benign; very rarely, malignant peripheral nerve sheath tumors may occur, with only 4 bona fide cases published so far [9,10]. SMARCB1 is also involved in the development of atypical teratoid/rhabdoid tumors (AT/RT) in the setting of the rhabdoid tumor predisposition syndrome (RTPS1); a few RTPS1 families include members affected either by AT/RT or multiple schwannomas, but to the best of our knowledge, no cooccurence of AT/RT and schwannomatosis in the same patient has been reported so far (reviewed by Sredni and Tomita [11]). The clinical spectrum of schwannomatosis has been further expanded by the description of the occurrence of benign tumors showing other lines of differentiation, as meningiomas [12] and, very recently, a case of uterine leiomyoma [13]. In these tumors, the presence of a SMARCB1 mutation as well as LOH of 22q indicate their contribution to schwannomatosis clinical features. We report a case of a patient with schwannomatosis with a novel germline SMARCB1 mutation, who developed multiple benign peripheral nerve sheath tumors and a uterine leiomyosarcoma.
1227 past and family history was not relevant. Between 24 and 31 years of age, she underwent 5 additional surgeries for the occurrence of multiple benign peripheral nerve sheath tumors, which were located in the pelvis, right brachial plexus, retroperitoneum, and right thigh. Tumors size ranged between 2 and 15 cm, the largest being the pelvic mass. The tumors were diagnosed as schwannomas, with the exception of the ones located in the brachial plexus, which were interpreted as hybrid neurofibromas/schwannomas. Periodic cranial magnetic resonance imaging (MRI) scans were always negative for vestibular lesions. At 33 years of age, she underwent hysteroannessiectomy for a 10-cm tumor located in the posterior wall of the uterine body. The tumor, identified by MRI during follow-up, had rapidly enlarged, with a 4-fold increase in size over 11 months. Based on its histologic features, a diagnosis of uterine leiomyosarcoma was made. During the same surgery, an additional schwannoma of the right psoas was excised. The patient received 6 cycles of chemotherapy (gemcitabine, 1000 mg/m2 on days 1 and 8, and docetaxel, 75 mg/m2 on day 8). Thirteen months after surgery, a control MRI identified a 3-cm hepatic nodule and 2 bone lesions at the right ileum, which were confirmed by scintigraphy. A subsequent control 3 months later showed a clear-cut increase in size of these lesions as well as additional multiple bone lesions (vertebral and left femoral) and a 3.3-cm retroperitoneal nodule. The biopsy of the hepatic lesion showed a malignant tumor with morphological and immunophenotypic features consistent with metastatic leiomyosarcoma. The patient has been currently treated with chemotherapy for metastatic disease (doxorubicin hydrochloride, 40 mg/m2 every 4 weeks) and radiotherapy at the right pelvis for pain control (3000 cGy in 10 fractions). Written informed consent was obtained from the patient for publication of this case report.
2.2. DNA and RNA extraction DNA was extracted from blood leukocytes and from fresh frozen tumor tissue using standard procedures with phenol/ chloroform extraction and ammonium acetate/ethanol precipitation. DNA from formalin-fixed, paraffin-embedded tissues was isolated according to manufacturers’ manual of QIAamp DNA FFPE Tissue Kit (Qiagen; Helden, Germany). RNA was extracted from blood sample using PAXgene blood RNA Kit (PreAnalytix, Qiagen).
2.3. Sanger sequencing
2. Materials and methods 2.1. Case history The patient presented her first clinical manifestation at 18 years old with the development of a scalp tumor, which was removed 2 years later and interpreted as schwannoma. Her
The entire coding sequences of SMARCB1, LZTR1, and NF2 as well as MED12 exon 2 were sequenced with polymerase chain reaction and capillary sequencing. Primer sequences are available on request. Capillary sequencing was performed on 310 Capillary DNA Analyzer (Life Technologies; Carlsbad, CA). Raw and analyzed sequence results were visualized on Sequence Scanner v1.0 (Life Technologies).
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2.4. Microsatellite analysis Loss of heterozygosity on 22q was investigated using microsatellites D22S420, D22S1174, D22S1154, D22S1163, and D22S277 from the ABI PRISM Linkage Mapping set version 2.5 (Life Technologies).
2.5. Multiplex ligation-dependent probe amplification Copy number changes (deletions or duplications) of SMARCB1 and NF2 loci and flanking genes were analyzed by multiplex ligation-dependent probe amplification (MLPA). SMARCB1, NF2 and 22q11 MLPA test kits (P258_C1, P044_B1, and P324_A2, MRC-Holland; Amsterdam, The Netherlands) were used, and electrophoresis data were analyzed using GeneMapper software (Life Technologies).
2.6. Immunohistochemistry To test SMARCB1 expression, 4-μm paraffin-embedded tissue sections from the benign nerve sheath tumors and the leiomyosarcoma were immunostained (clone 25/baf47,1:25, pH9, BD Transduction Laboratories Biosciences San Diego, CA, USA) by an automated immunostainer (Dako Autostainer, DakoCytomation; Glostrup, Denmark). Cells lacking nuclear staining were considered as negative. Lack of nuclear staining in a fraction of neoplastic cells was considered as reduced (mosaic) expression.
3. Results 3.1. Molecular findings LZTR1 and SMARCB1 were directly sequenced in DNA extracted from blood lymphocytes of the patient. No mutation was identified in LZTR1; on the contrary, SMARCB1 sequencing analysis showed the constitutional mutation c.1118G N A, involving the last base of the exon 8. This alteration was predicted to disrupt the donor splice site and was absent in the healthy parents of the patient, confirming that it occurred de novo. The alteration of the splicing process was confirmed by RNA analysis that showed the retaining of intron 8 in messenger RNA (Fig. 1A); the derivative RNA is predicted to code for an elongated protein with a new translation stop site after 48 amino acids (Fig. 1A). Sanger sequencing of SMARCB1 exon 8 in 2 schwannomas and in the leiomyosarcoma of the patient showed the loss of the wild-type G allele (Fig. 1B), confirmed by LOH analysis in the 2 FFPE schwannomas and by MLPA analysis for the fresh frozen tissue of leiomyosarcoma (Fig. 1C). Furthermore, we analyzed the entire coding sequence and exon-intron junctions of NF2 gene in the 2 schwannomas and leiomyosarcoma. One schwannoma (T692) showed a
I. Paganini et al. somatic variant of NF2 gene: c.418delC, p.(Leu140Serfs*34); whereas no NF2 alterations were detected in the other schwannoma and in the leiomyosarcoma. Finally, based on the increasingly recognized role of MED12 gene in uterine smooth muscle tumors [14], we sought to sequence its exon 2 hotspot region in DNA extracted from the leiomyosarcoma, but no variants were identified.
3.2. Pathologic findings Most of the benign nerve sheath tumors showed the classic features of schwannoma. Differently, the tumors located at the brachial plexus were histologically characterized by the presence of schwannoma-like nodules composed of Antoni A regions, surrounded by a neurofibroma-like component with abundant collagen and myxoid changes, fulfilling the criteria for the diagnosis of hybrid neurofibromas/schwannomas [15] (Fig. 2A). The uterine tumor was composed by spindle cells with eosinophilic cytoplasm showing a clear-cut smooth muscle differentiation. No rhabdoid features were seen. The tumor was hypercellular throughout and exhibited a diffuse moderate to severe nuclear atypia. The mitotic index was high, with up to 26 mitoses per 10 high-power fields (Fig. 2B). Foci of neoplastic necrosis were present (Fig. 2C). At immunohistochemical analysis, the neoplasm showed diffuse expression of smooth muscle actin and caldesmon as well as focal expression of desmin, confirming the smooth muscle differentiation of the tumor. All the other markers tested, as S-100, GFAP, synaptophysin, myogenin, CKAE1/AE3, CKMNF116, CD117, and DOG1, were negative. Interestingly, in the schwannomas, the hybrid schwannoma/ neurofibromas of the radial plexus and the uterine leiomyosarcoma SMARCB1 was not homogeneously expressed, and areas with SMARCB1-negative cells were present (Fig. 2D and E), in line with previous studies [8].
4. Discussion Schwannomatosis is a rare genetic syndrome characterized by the development of multiple peripheral schwannomas, without the involvement of vestibular nerves. The clinical findings in patients with schwannomatosis have been widened in the last few years, and currently, the spectrum of associated tumors includes meningiomas [12] and leiomyomas [13]. However, to date, malignant tumors have been reported only in few cases, and they are invariably represented by malignant peripheral nerve sheath tumors [9,10]. We describe a patient affected by SMARCB1-related sporadic schwannomatosis, who developed a uterine leiomyosarcoma in addition to the typical benign peripheral nerve sheath tumors. We demonstrated the presence of SMARCB1 germline mutation and, more importantly, documented loss of the SMARCB1 wildtype allele in the leiomyosarcoma, proving the role of this genetic alteration in the tumor pathogenesis.
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Fig. 1 A, Partial sequence of SMARCB1 complementary DNA from leukocytes of the patient. B, The mutation causes retaining of intron 8, and it is predicted to create a new translation stop site after 48 amino acids. Partial sequences of SMARCB1 in blood and tumors of patient 299. The SMARCB1 mutation occurs in the last base of exon 8. This alteration is retained in the 2 schwannomas (T691, T692) and in the leiomyosarcoma (T791) with loss of the wild-type allele. C, Allelic losses on 22q in the 2 formalin-fixed, paraffin-embedded schwannomas. Microsatellite analysis shows LOH of 22q. The arrows indicate the lost alleles in tumor DNA. The 2 schwannomas lost the same allele in all the analyzed microsatellites. DNA extracted from fresh tumor tissue of leiomyosarcoma was analyzed by MLPA (kit P324_A2). Normalized data from Coffalyzer.net software show a deletion of 22q. The deletion is highlighted by the red dots that indicate the deleted probes (ratio of 0.5).
Immunohistochemical analysis showed loss of SMARCB1 expression in a minority of neoplastic nuclei. Notably, among schwannomatosis-related schwannomas, there is a remarkable intertumoral variability with cases showing less than 10% negative nuclei [8]. We believe that the pattern of SMARCB1 expression in our case may belong to the low end of the mosaic spectrum. SMARCB1 somatic changes can be found in other malignant neoplasms including epithelioid sarcoma, epithelioid malignant peripheral nerve sheath tumor, myoepithelial carcinoma, extraskeletal myxoid chondrosarcoma, and renal
medullary carcinoma indicating that it is involved in their pathogenesis [16]. Uterine leiomyosarcoma is the most common uterine sarcoma, accounting for approximately 2% of all uterine malignancies [17]. Leiomyosarcoma shares several characteristics with leiomyoma, including smooth muscle morphology and immunophenotype as well as the frequent arising within the uterine corpus in middle-aged women. However, they are considered biologically unrelated tumors because of their disparate clinical, cytogenetic, and molecular features [14].
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Fig. 2 A, A schwannoma-like nodule surrounded by a neurofibroma-like component with remarkable myxoid changes in 1 of the tumor interpreted as hybrid neurofibroma/schwannoma. The uterine leiomyosarcoma was highly cellular and showed moderate to severe nuclear atypia with brisk mitotic activity (B) and tumor necrosis (C). Some of the neoplastic cells lacked SMARCB1 nuclear staining in the schwannoma (D) and the leiomyosarcoma (E).
Recently, challenging this perspective, mutations in exon 2 of MED12 gene have been reported in both leiomyoma (50%-70%) and leiomyosarcoma (b30%) [14]. Leiomyosarcoma of our schwannomatosis patient did not show any variant in exon 2 of MED12, ruling out its possible pathogenetic role and, indirectly, strengthening the causative role of the SMARCB1 biallelic inactivation, in our case. To our knowledge, only 1 case of retroperitoneal leiomyosarcoma lacking SMARCB1 expression has been reported in the literature, but data on SMARCB1 mutational status have not been provided [18]. Our observation may suggest to investigate thoroughly sporadic leiomyosarcomas for SMARCB1 deficiency. In conclusion, our findings enlarged the spectrum of SMARCB1-predisposing tumors and demonstrated, for the first time, the association of a malignant tumor with smooth muscle differentiation to schwannomatosis. Therefore, clini-
cians should definitely be aware that a constitutional SMARCB1 mutation, which mainly predisposes to benign nerve sheath tumors, may also predispose to aggressive neoplasms throughout life, within an unexpected spectrum.
References [1] Jacoby LB, Jones D, Davis K, et al. Molecular analysis of the NF2 tumor-suppressor gene in schwannomatosis. Am J Hum Genet 1997; 61:1293-302. [2] MacCollin M, Willett C, Heinrich B, et al. Familial schwannomatosis: exclusion of the NF2 locus as the germline event. Neurology 2003;60: 1968-74. [3] Hulsebos TJ, Plomp AS, Wolterman RA, Robanus-Maandag EC, Baas F, Wesseling P. Germline mutation of INI1/SMARCB1 in familial schwannomatosis. Am J Hum Genet 2007;80:805-10. [4] Hadfield KD, Newman WG, Bowers NL, et al. Molecular characterisation of SMARCB1 and NF2 in familial and sporadic schwannomatosis. J Med Genet 2008;45:332-9.
SMARCB1-associated malignant tumors [5] Piotrowski A, Xie J, Liu YF, et al. Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas. Nat Genet 2014;46:182-7. [6] Paganini I, Chang VY, Capone GL, et al. Expanding the mutational spectrum of LZTR1 in schwannomatosis. Eur J Hum Genet 2014 [Epub ahead of print]. [7] Sestini R, Bacci C, Provenzano A, Genuardi M, Papi L. Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas. Hum Mutat 2008;29:227-31. [8] Patil S, Perry A, Maccollin M, et al. Immunohistochemical analysis supports a role for INI1/SMARCB1 in hereditary forms of schwannomas, but not in solitary, sporadic schwannomas. Brain Pathol 2008;18:517-9. [9] Gonzalvo A, Fowler A, Cook RJ, et al. Schwannomatosis, sporadic schwannomatosis, and familial schwannomatosis: a surgical series with long-term follow-up. Clinical article. J Neurosurg 2011;114:756-62. [10] Carter JM, O'Hara C, Dundas G, et al. Epithelioid malignant peripheral nerve sheath tumor arising in a schwannoma, in a patient with "neuroblastoma-like" schwannomatosis and a novel germline SMARCB1 mutation. Am J Surg Pathol 2012;36:154-60. [11] Sredni ST, Tomita T. Rhabdoid tumor predisposition syndrome. Pediatr Dev Pathol 2015;18:49-58.
1231 [12] Bacci C, Sestini R, Provenzano A, et al. Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation. Neurogenetics 2010;11:73-80. [13] Hulsebos TJ, Kenter S, Siebers-Renelt U, Hans V, Wesseling P, Flucke U. SMARCB1 involvement in the development of leiomyoma in a patient with schwannomatosis. Am J Surg Pathol 2014; 38:421-5. [14] Ravegnini G, Mariño-Enriquez A, Slater J, et al. MED12 mutations in leiomyosarcoma and extrauterine leiomyoma. Mod Pathol 2013;26: 743-9. [15] Harder A, Wesemann M, Hagel C, et al. Hybrid neurofibroma/ schwannoma is overrepresented among schwannomatosis and neurofibromatosis patients. Am J Surg Pathol 2012;36:702-9. [16] Hollmann TJ, Hornick JL. INI1-deficient tumors: diagnostic features and molecular genetics. Am J Surg Pathol 2011;35:47-63. [17] Kurman RJ, Carcangiu ML, Herrington CS, Young RH. WHO Classification of Tumors of Female Reproductive Organs. 4th ed. Lyon: IARC; 2014. [18] Perry A, Fuller CE, Judkins AR, Dehner LP, Biegel JA. INI1 expression is retained in composite rhabdoid tumors, including rhabdoid meningiomas. Mod Pathol 2005;18:951-8.
