Novel BRAF and KRAS Mutations in Papillary Thyroid Carcinoma Arising in Struma Ovarii A. Tan, C. J. R. Stewart, K. L. Garrett, M. Rye & P. A. Cohen
Endocrine Pathology ISSN 1046-3976 Volume 26 Number 4 Endocr Pathol (2015) 26:296-301 DOI 10.1007/s12022-015-9394-3
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Author's personal copy Endocr Pathol (2015) 26:296–301 DOI 10.1007/s12022-015-9394-3
Novel BRAF and KRAS Mutations in Papillary Thyroid Carcinoma Arising in Struma Ovarii A. Tan 1 & C. J. R. Stewart 1 & K. L. Garrett 1 & M. Rye 1 & P. A. Cohen 2
Published online: 11 September 2015 # Springer Science+Business Media New York 2015
Abstract Papillary carcinomas of thyroid type rarely arise within struma ovarii. There are limited data on the immunohistochemical and molecular features of these tumors. Three cases of papillary carcinoma arising in struma ovarii (PCSO) were identified. The clinicopathological features were reviewed and immunohistochemical staining for HBME-1, cytokeratin (CK) 19, and CD56 was performed. Tumor DNA was sequenced for somatic mutations using a panel of 26 oncogenes, with a particular focus on BRAF and KRAS mutations. The patients were aged 22, 48, and 55 years. All cases were FIGO stage IA. Two tumors were of classical histological type, and one was a follicular variant papillary carcinoma. All tumors expressed HBME-1 and two were positive for CK19. CD56 was negative in all three cases. One tumor demonstrated a BRAF G469A mutation in exon 11, and in a second case, a KRAS Q61K double base mutation in exon 3 was detected. These mutations have not been described previously in PCSO. No mutations were detected in the benign follicular components of the tumors adjacent to the malignant papillary tissue. None of the patients had tumor recurrence on clinical follow-up (range 11 months to 8½years). HBME-1, CK19, and CD56 are useful immunohistochemical markers of PCSO. Novel BRAF and KRAS mutations were identified in two of three tumors suggesting that mutations in PCSO may differ from those commonly identified in papillary carcinoma of the eutopic thyroid. The clinical significance of these
* A. Tan
[email protected] 1
St John of God Pathology, 12 Salvado Road, Subiaco 6008, WA, Australia
2
St John of God Hospital, 12 Salvado Road, Subiaco 6008, WA, Australia
mutations is uncertain but follow-up data in this small series support the generally good prognosis of PCSO. Keywords Papillary thyroid carcinoma . Malignant struma ovarii . Immunohistochemistry . Molecular . Ovary
Introduction Approximately 20 % of ovarian tumors are teratomas, and up to 20 % of teratomas contain thyroid tissue [1]. However, only 5 % of teratomas are struma ovarii which by definition comprise at least 50 % thyroid-type tissue [1–3]. Patients with struma ovarii typically present with a pelvic mass, lower abdominal pain, abnormal vaginal bleeding, menstrual irregularities, or ascites [4]. Clinical and biochemical hyperthyroidism is rare, occurring in less than 5 % of cases [4, 5]. Most struma ovarii are clinically benign but a small proportion of cases show malignant histological features resembling those seen in the eutopic thyroid. Such malignant struma ovarii (MSO) occur most commonly in the 5th and 6th decades, are usually unilateral (94 %) and more commonly affect the left ovary [3–6]. As in the thyroid gland, papillary carcinoma is the most common histological type of MSO accounting for 70 % of cases and of these one third are follicular variant papillary carcinomas. Papillary carcinoma arising in struma ovarii (PCSO) can be clinically malignant in the absence of conventional histological features of malignancy but most such lesions demonstrate follicular growth patterns and lack papillary cytological features [7]. Malignant struma ovarii generally has a favorable prognosis with overall survival of 92, 85, and 79 %, at 5, 10 and 25 years, respectively, in one series [3]. However, tumor recurrences have been documented up to
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29 years after initial diagnosis necessitating long-term follow up in these patients [8]. Immunohistochemical staining with HBME-1 (Hector Battifora mesothelial cell 1) and cytokeratin (CK) 19, which is characteristically positive in papillary thyroid carcinoma, may help to confirm the diagnosis of PCSO. More recently, absent CD56 expression has also been found to be highly sensitive and specific for papillary thyroid carcinoma [9, 10]. However, the application of these three antibodies in combination is not known in PCSO. Primary thyroid carcinomas have also been shown to harbor a variety of genetic alterations in the BRAF, RAS (KRAS, NRAS, and HRAS), PIK3CA, and RET genes, including point mutations and rearrangements [11–13]. Of these, BRAF mutations are seen in 40–45 % of papillary thyroid carcinoma and the great majority (up to 98 %) are V600E mutations. The V600E mutation and other closely located mutations (K601E and TV599_600M deletion) have also been identified in cases of PCSO, and there are isolated reports of NRAS and HRAS mutations in these tumors [11]. However, data on the molecular changes in MSO including PCSO remain extremely limited. In the present study, we have performed a histological review of three cases of PCSO with immunohistochemical analysis of HBME-1, CK19, CD56, and tumor DNA sequencing for somatic mutations in a panel of 26 oncogenes, with particular focus on BRAF and KRAS mutations.
Methods Following ethical approval from the St. John of God Subiaco Hospital Human Research Ethics Committee, cases of struma ovarii and MSO were identified from the database of the Western Australian Gynaecologic Oncology Multidisciplinary Meetings between January 1st 2005 and 31st December 2014. Clinical data were extracted from the patient medical records. All specimens had been fixed in neutral buffered formalin and processed routinely to paraffin wax. The haematoxylin and eosin (H&E)-stained slides were reviewed, and the tumors were assessed for papillary carcinoma morphology, the presence of ovarian surface involvement or vascular invasion. Sections 2–3 μm were stained for immunohistochemistry with antibodies against HBME-1, CK19, and CD56 using an autoimmunostainer (DAKO) according to the manufacturer’s instructions (Table 1). DNA was extracted from the formalin-fixed paraffin-embedded (FFPE) tumor blocks and assessed for somatic mutation status using the TruSight Tumor panel (Illumina) on a Miseq next generation sequencer. The panel covers hotspot mutations in 26 genes including AKT1, ALK, APC, BRAF, CDH1, CTNNB1, EGFR, ERBB2, FBXW7, FGFR2, FOXL2,
297 Table 1
Details of primary antibodies
Antibody
Source
Clone
Dilution
CK 19
Dako (Botany, NSW, Australia)
RCK 108
Ready to use
HBME-1
Dako (Botany, NSW, Australia)
HBME-1
1:50
CD56
Dako (Botany, NSW, Australia)
123C3
Ready to use
GNAQ, GNAS, KIT, KRAS, MAP2K1, MET, MSH6, NRAS, PIK3CA, PDGFRA, PTEN, SMAD4, SRC, STK11, and TP53. HRAS is not included in this panel. Analysis of variants was performed using Variant studio software (Illumina) and reviewed using Varseq software (Golden Helix).
Results One hundred six cases of struma ovarii and three cases of MSO, all of which were papillary carcinomas, were identified during the study period. The clinicopathological findings and the immunohistochemical analyses are summarised in Table 2. All tumors arose within otherwise benign struma ovarii and additional mature germ cell components were present in all cases. Two tumors arose in the left ovary, and all were contained within the ovary (no ovarian surface involvement). None of the cases had been considered suspicious for malignancy preoperatively or intraoperatively. All tumors showed histological features of papillary carcinoma of thyroid-type. Two tumors exhibited classical morphology while another showed features of follicular variant of papillary carcinoma. Lymphovascular invasion was not present. The light microscopic features of the tumors on H&E-stained sections are shown in Fig. 1. Postoperative thyroid function tests were normal in all cases. Two patients subsequently had thyroid ultrasound, one of whom had a nodule which showed benign appearances on fine needle aspiration cytology. All patients are free of disease on follow-up (range 11 months to 8½years). On immunohistochemistry, all tumors were positive for HBME-1 and two cases expressed CK 19 (Fig. 2). All three cases lacked CD56 immunostaining. A BRAF mutation G469A (NM_004333.4 c.1406G > C, NP_004324.2 p.Gly469Ala) was identified in exon 11 in patient #1 and a KRAS mutation Q61K (NM_033360.2: c.180_181delTCinsAA, NP_203524.1:p.Gln61Lys) was identified in exon 3 in patient #2 (Table 3). These mutations have not been reported previously in PCSO or in MSO generally. No KRAS or BRAF mutations were identified in the final tumor (Patient #3, Table 3). In addition to the malignant structures, the benign components of the tumors were also assessed for mutational status to verify if the observed somatic mutations were also evident in the non-malignant cells (Fig. 1). Patient #1 was initially
Author's personal copy 298 Table 2 Summary of clinicopathological and immunohistochemical findings in three cases of papillary carcinoma in struma ovarii
Endocr Pathol (2015) 26:296–301
Age at diagnosis Disease free interval
Patient #1
Patient #2
Patient #3
55 years 11 months
22 years 16 months
48 years 8½years
Site of tumor
Left ovary
Right ovary
Left ovary
Thyroid function test Thyroid ultrasound
Normal Normal
Normal Normal
Diagnosis
Normal 14 mm nodule in right lobe and 7 mm nodule on isthmus (benign on FNA) Multifocal FV PC
Classical PC
Classical PC
HBME-1
Positive
Positive
Positive
CK 19 CD 56
Positive Negative
Negative Negative
Positive Negative
PC papillary carcinoma, FV follicular variant, FNA fine needle aspiration
assessed from a mixed population of cells containing papillary and benign follicular components. An isolated ‘follicular-only’ portion of tissue was also evaluated for mutations. Patient
Patient #1
Patient #1
Patient #2
Patient #2
Fig. 1 Light microscopy of H&E stained sections. Patient #1—Low power light microscopy of an H&E stained section of the left ovary which demonstrates a struma ovarii (marked). The thyroid tissue showed a range of appearances including variably sized follicles, some with conspicuous central colloid. The tumor was surrounded by normal ovarian tissue and did not involve the ovarian capsule (FIGO Stage 1A). Patient #1—High power light microscopy of an H&E stained section which showed a follicular variant of papillary thyroid carcinoma. The MSO retained a follicular architecture with papillary nuclear features, including pale overlapping nuclei, nuclear grooves, and intranuclear inclusions. Patient #2—Low power light microscopy of an H&E stained section of the right ovary demonstrating a multi-cystic teratoma containing mature thyroid tissue and a central focus of classical papillary
#2 contained a purely papillary lesion surrounded by struma ovarii tissue, and an area of thyroid follicles was also assessed for mutations. Patient #3 contained a mixed population of
Patient #3
Patient #3
thyroid carcinoma. The circles represent the tissue sampled for mutation testing (larger circle; papillary thyroid carcinoma, smaller circle; adjacent non-malignant tissue). Patient #2—High power light microscopy of an H&E stained section showed well developed papillary architecture and malignant nuclear features of papillary thyroid carcinoma. Patient #3— Low power light microscopy of an H&E stained section of the left ovary, much of which had been replaced by mature thyroid tissue. Numerous colloid-filled follicles of varying size and shape were seen. Papillary nuclear features were present throughout the thyroid tissue. The area marked was sampled for mutation testing. Patient #3—High power light microscopy of an H&E stained section which demonstrated well developed papillary projections lined by columnar epithelium with nuclear features of papillary thyroid carcinoma
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Fig. 2 Examples of positive HBME-1 and CK 19 expression in papillary thyroid carcinoma of struma ovarii
HBME-1 (mag x100)
cellular components, but no areas devoid of malignant structures could be isolated from this archival sample for comparative analysis. No mutations were detected in the adjacent non-malignant components from patients #1 and #2.
Discussion We encountered only three cases of MSO out of a total of 106 cases of struma ovarii over a 10-year period illustrating the rarity of such tumors in routine diagnostic practice. All tumors were papillary carcinomas histologically, showing similar histological features to those more commonly encountered in the eutopic thyroid gland including one case of follicular variant papillary carcinoma. Consistent with most previous reports, the diagnosis of PCSO was not suspected prior to histological examination of the resected ovarian masses. None of the patients had evidence of tumor recurrence during follow-up ranging from 11 months to 8½years but it should be noted that MSO generally follows an indolent clinical course with prolonged intervals prior to the development of metastasis [8].
Table 3
Cell type
#2
#3
Important population-level analysis of a large retrospective series of 68 patients by Goffredo et al. [14] showed overall survival rates at 5, 10, and 20 years of 96.7, 94.3, and 84.9 %, respectively. In this patient cohort, there was only one death attributed to MSO. Six patients (8.8 %) had a concomitant or subsequent diagnosis of thyroid cancer and all were alive at the end of follow-up. At present, there are no reliable prognostic factors in these tumors and hence all patients require indefinite follow up. Although the diagnosis of papillary carcinoma in the thyroid gland is usually straightforward, some cases present diagnostic difficulty, particularly in situations where assessment of the characteristic cytological features may be compromised, for example, as a result of suboptimal fixation or degenerative changes. Furthermore, many variants of papillary carcinoma are now recognised some of which may mimic benign lesions or other types of malignancy. Thus, immunohistochemistry is frequently used to support a diagnosis of malignancy generally, or papillary carcinoma more specifically, using HBME-1, CK19, and CD56. It should be noted that none of these antibodies are completely sensitive or specific, but they have
Summary of somatic mutations found in the MSO specimens
Patient Malignant tissue tested
#1
CK 19 (mag x100)
Mutation
Adjacent non-malignant tissue tested Methods used to confirm
Cell type
Mutation
Methods used to confirm
Follicular BRAF G469A (exon 11) NGS* Trusight panel Pure adjacent No mutation NGS Trusight variant of PCSO (depth 54335× 36.3 % thyroid follicles (depth covered 2321×) panel, Sanger allele freq), Sanger (BRAF exon 11) (BRAF exon 11) Classical PCSO KRAS Q61K (exon 3) NGS Trusight panel Pure adjacent No mutation NGS Trusight (depth 2427× 54.6 % thyroid follicles (depth covered 2896×) panel, Sanger allele freq. Repeat (KRAS exon 3) DNA extraction depth 2944× 46.73 %), Sanger (KRAS exon 3) Classical PCSO No mutation detected NGS Trusight panel, Sanger NA- unable to find NA NA and CAST PCR specifically discrete thyroid looking for V600 mutations follicles in sample
NGS* =next generation sequencing
Author's personal copy 300 Table 4
Endocr Pathol (2015) 26:296–301 Summary of somatic mutation analyses from published PCSO case reports and case series
Publication
No. of cases
Histo-type
BRAF mutations
RAS mutations
RET/PTC rearrangements
Flavin et al. 2007
1
Classical PC
T1779A
None
None
Boutross-Tadross et al. 2007
10
FV PC
None
None
Schmidt et al. 2007
6
Classical PC
None
Wolff et al. 2010 Coyne and Nikiforov 2010
1 1
FV PC FV PC
2 cases: V600E 1 case: K601E 1 case: TV599-600 M deletion K601E None
Present in 7 out of 10 cases None
None HRAS Codon 61
None None
Stanojevic et al. 2012
1
FV PC
None
KRAS G12V
None
proven valuable in diagnostic practice. All three ovarian tumors in the present series expressed HBME-1 while two cases were CK19 positive. Lack of CD56 expression in all three tumors was also observed. These findings support the diagnostic value of these markers in PCSO similar to their use in papillary carcinoma of the thyroid gland. It has been established that the most common mutations in thyroid papillary carcinoma involve the BRAF, RAS, and Ret oncogenes [11–13, 15, 16]. One tumor in this series demonstrated a BRAF G468A mutation. The less common BRAF G468A mutation variant has been reported previously in non-Hodgkin lymphoma, non-small cell lung cancer and in a small number of colorectal cancers, but to our knowledge has not previously been identified in PCSO or in MSO generally [http://www.mycancergenome.org]. It has been suggested that BRAF mutation-positive papillary thyroid cancers, most commonly V600E, may show adverse prognostic features including invasive growth and lymph node metastasis and have worse clinical outcomes compared to papillary thyroid carcinomas which are negative for these mutations. Therefore, adjuvant radioactive iodine treatment may be considered in such tumors [17]. However, recent data suggest that while the presence of BRAF mutations may correlate with more extensive or aggressive disease, there is no correlation with recurrence free, or disease specific, survival [18]. The clinical significance of BRAF mutations in PCSO, including the unusual variant described herein, is uncertain. In general, there is lack of consensus on the management of MSO or PCSO, although some authors have suggested thyroidectomy and I131 therapy, particularly for patients with gross extraovarian extension or distant metastases [19, 20]. The second tumor in this series demonstrated a KRAS Q61K mutation in exon 3. This mutation is more commonly seen as a single base change but was detected in this sample as a more complex 2 base pair change resulting in the same overall amino acid variation. Mutations in the KRAS gene are seen in only approximately 3 % of thyroid carcinomas
and the Q61K variant accounts for only 2 % of all KRAS mutations [http://www.mycancergenome.org]. To our knowledge, this is a novel finding in the context of PCSO. In addition it is also a more complex double base mutation. As is the case with the BRAF mutation described above, the clinical significance of KRAS mutations in PCSO requires further study. It is of interest that no somatic mutations were observed in the 26 genes analysed in patient #3. The reasons for this are unclear. Analysis of somatic mutation status in ten follicular variant PCSO did not show any mutations in BRAF or KRAS [15]. The results of mutation analyses from four published case reports and two case series are summarized in Table 4 [11, 15, 16, 21–23]. The failure to detect the same mutations identified in the malignant papillary structures of patients #1 and #2 in the isolated benign follicular tissue within the struma ovarii lesions suggests that the mutations are particularly associated with the papillary malignant transition of these cells. Generally, classical PCSO more commonly show BRAF mutations and follicular variant PCSO, particularly the encapsulated forms, more frequently demonstrate KRAS mutations [11, 22]. Our findings are not consistent with this as the mutation in patient #1, which was a follicular variant PCSO, was in the BRAF gene and the mutation in patient #2, and which was a classical PCSO, was in the KRAS gene. It is possible that in non-thyroid locations other associations occur, including rare mutation types. Summary We describe three cases of PCSO, all with histological features of papillary carcinoma, encountered in Western Australia over a 10-year period. Immunohistochemical staining for HBME-1 and CK19 was positive in three and two cases, respectively, and CD56 was absent in all three tumors, illustrating the value of these markers in the setting of ovarian thyroid-type malignancy. Novel mutations in BRAF and KRAS were identified in
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two cases and while additional larger studies are required, these findings suggest that there may be greater mutational diversity in PCSO than in eutopic thyroid papillary carcinoma. Acknowledgments We thank Drs. Stuart Salfinger, Jason Tan, Ganendra Raj Kader Ali Mohan, and Professor Yee Leung for the clinical data. Funding The work was supported by funding from the St John of God Pathology Research Group.
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