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Figure.
Intraepithelial Tubal Metastasis Mimicking a Serous Tubal Intraepithelial Carcinoma (STIC) in a Patient With Uterine Endometrial Carcinoma (UEC)
Intraepithelial Tubal Metastasis Mimicking a Serous Tubal Intraepithelial Carcinoma (STIC) in a Patient With Uterine Endometrial Carcinoma (UEC)

A, Low-power photomicrograph of the fimbriated end of the distal fallopian tube of patient 1 (hematoxylin-eosin, original magnification ×40). Black arrowhead indicates the presence of STIC (noninvasive into the underlying stroma); white arrowhead, normal adjacent tubal epithelium. B, High-power photomicrograph of the fimbriated end of the distal fallopian tube of patient 4 (hematoxylin-eosin, original magnification ×400). Inset panel shows low-power (×40) micrograph of same specimen with asterisk marking area of magnification. Black arrowhead identifies area of significant cytologic atypia, morphologically consistent with a tubal intraepithelial carcinoma (TIC); white arrowhead, adjacent normal tubal epithelium. C, Immunohistochemical (IHC) staining for p53 in the TIC with clonal type overexpression (black arrowhead); white arrowhead, a wild-type (WT) p53 staining pattern (original magnification ×400). D, IHC for Ki-67 in the TIC (black arrowhead) shows a mitotic index of approximately 40%. White arrowhead shows low staining in the normal epithelium (original magnification ×400). E, IHC staining for PAX8 in the TIC (black arrowhead) shows strong nuclear reactivity. White arrowhead indicates normal epithelium (original magnification ×400). F, IHC staining for WT-1 in the TIC (black arrowhead) shows absent nuclear expression. White arrowhead indicates WT expression in normal epithelium (original magnification ×400). G, Low-power photomicrograph of patient 4’s primary UEC (hematoxylin-eosin, original magnification ×40). H, High-power photomicrograph of the area indicated by the rectangle in panel G (hematoxylin-eosin, original magnification ×400). I, IHC for p53 in the primary UEC shows a WT staining pattern (original magnification ×400). J, IHC staining for Ki-67 in the primary UEC shows a mitotic index of approximately 20% (original magnification ×400). K, IHC staining for PAX8 in the primary UEC shows patchy nuclear positivity (original magnification ×400). L, IHC staining for WT-1 in the primary UEC shows absent nuclear expression. Next-generation sequencing detected the presence of a TP53 mutation exclusively in the tubal lesion from patient 4, consistent with IHC results (compare panels C and I), supporting the tubal lesion from patient 4 as a mucosal micrometastasis/implant from the patient’s primary UEC rather than a STIC (original magnification ×400).

Table 1.  
Clinicopathologic and Coverage Data for Sequenced Samples
Clinicopathologic and Coverage Data for Sequenced Samples
Table 2.  
Prioritized Mutations From Next-Generation Sequencing of STICs and Synchronous Gynecologic Malignant Neoplasms
Prioritized Mutations From Next-Generation Sequencing of STICs and Synchronous Gynecologic Malignant Neoplasms
1.
Siegel  RL, Miller  KD, Jemal  A.  Cancer statistics, 2015.  CA Cancer J Clin. 2015;65(1):5-29.PubMedGoogle ScholarCrossref
2.
Kurman  RJ, Shih  IeM.  Pathogenesis of ovarian cancer: lessons from morphology and molecular biology and their clinical implications.  Int J Gynecol Pathol. 2008;27(2):151-160.PubMedGoogle Scholar
3.
Kurman  RJ, Shih  IeM.  The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.  Am J Surg Pathol. 2010;34(3):433-443.PubMedGoogle ScholarCrossref
4.
Kurman  RJ, Shih  IeM.  Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer: shifting the paradigm.  Hum Pathol. 2011;42(7):918-931.PubMedGoogle ScholarCrossref
5.
Ahmed  AA, Etemadmoghadam  D, Temple  J,  et al.  Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary.  J Pathol. 2010;221(1):49-56.PubMedGoogle ScholarCrossref
6.
Kindelberger  DW, Lee  Y, Miron  A,  et al.  Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma: evidence for a causal relationship.  Am J Surg Pathol. 2007;31(2):161-169.PubMedGoogle ScholarCrossref
7.
Piek  JM, van Diest  PJ, Zweemer  RP,  et al.  Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer.  J Pathol. 2001;195(4):451-456.PubMedGoogle ScholarCrossref
8.
Piek  JM, Verheijen  RH, Kenemans  P, Massuger  LF, Bulten  H, van Diest  PJ.  BRCA1/2-related ovarian cancers are of tubal origin: a hypothesis.  Gynecol Oncol. 2003;90(2):491.PubMedGoogle ScholarCrossref
9.
Lee  Y, Miron  A, Drapkin  R,  et al.  A candidate precursor to serous carcinoma that originates in the distal fallopian tube.  J Pathol. 2007;211(1):26-35.PubMedGoogle ScholarCrossref
10.
Cani  AK, Hovelson  DH, McDaniel  AS,  et al.  Next-gen sequencing exposes frequent MED12 mutations and actionable therapeutic targets in phyllodes tumors.  Mol Cancer Res. 2015;13(4):613-619.PubMedGoogle ScholarCrossref
11.
Hovelson  DH, McDaniel  AS, Cani  AK,  et al.  Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors.  Neoplasia. 2015;17(4):385-399.PubMedGoogle ScholarCrossref
12.
Conner  JR, Meserve  E, Pizer  E,  et al.  Outcome of unexpected adnexal neoplasia discovered during risk reduction salpingo-oophorectomy in women with germ-line BRCA1 or BRCA2 mutations.  Gynecol Oncol. 2014;132(2):280-286.PubMedGoogle ScholarCrossref
13.
O’Hara  AJ, Bell  DW.  The genomics and genetics of endometrial cancer.  Adv Genomics Genet. 2012;2012(2):33-47.PubMedGoogle Scholar
14.
Cancer Genome Atlas Research Network.  Integrated genomic analyses of ovarian carcinoma.  Nature. 2011;474(7353):609-615.PubMedGoogle ScholarCrossref
Brief Report
November 2015

Next-Generation Sequencing of Tubal Intraepithelial Carcinomas

Author Affiliations
  • 1Michigan Center for Translational Pathology, Department of Pathology, University of Michigan, Ann Arbor
  • 2Department of Pathology, Massachusetts General Hospital, Boston
  • 3Computational Medicine & Bioinformatics, University of Michigan, Ann Arbor
  • 4Department of Urology, University of Michigan, Ann Arbor
  • 5Comprehensive Cancer Center, University of Michigan, Ann Arbor
  • 6Department of Internal Medicine, University of Michigan, Ann Arbor
JAMA Oncol. 2015;1(8):1128-1132. doi:10.1001/jamaoncol.2015.1618
Abstract

Importance  High-grade serous carcinoma (HGSC) is the most prevalent and lethal form of ovarian cancer. HGSCs frequently arise in the distal fallopian tubes rather than the ovary, developing from small precursor lesions called serous tubal intraepithelial carcinomas (TICs, or more specifically, STICs). While STICs have been reported to harbor TP53 mutations, detailed molecular characterizations of these lesions are lacking.

Observations  We performed targeted next-generation sequencing (NGS) on formalin-fixed, paraffin-embedded tissue from 4 women, 2 with HGSC and 2 with uterine endometrioid carcinoma (UEC) who were diagnosed as having synchronous STICs. We detected concordant mutations in both HGSCs with synchronous STICs, including TP53 mutations as well as assumed germline BRCA1/2 alterations, confirming a clonal association between these lesions. Next-generation sequencing confirmed the presence of a STIC clonally unrelated to 1 case of UEC, and NGS of the other tubal lesion diagnosed as a STIC unexpectedly supported the lesion as a micrometastasis from the associated UEC.

Conclusions and Relevance  We demonstrate that targeted NGS can identify genetic alterations in minute lesions, such as TICs, and confirm TP53 mutations as early driving events for HGSC. Next-generation sequencing also demonstrated unexpected associations between presumed STICs and synchronous carcinomas, providing evidence that some TICs are actually metastases rather than HGSC precursors.

Introduction

Ovarian cancers will account for over 14 000 estimated deaths in the United States in 2015, with nearly two-thirds presenting at high stage with a poor 5-year overall survival rate (27%).1 Despite advances in surgery, medicine, and imaging, ovarian cancer mortality has changed little over several decades, and the need for early detection of cancers at a curable stage remains unmet. A dualistic model of ovarian cancer pathogenesis posits a heterogeneous group of low-grade, clinically indolent, genomically stable tumors (type I, which account for approximately 25% of all ovarian carcinomas) and a high-grade, clinically aggressive group with high risk for distant metastases (type II, comprising the remaining carcinomas, roughly 75%).2-4 These groups can also be broadly distinguished on morphologic grounds, with high-grade serous carcinoma (HGSC) comprising most type II cancers, the most common and lethal ovarian carcinoma group. In contrast to type I ovarian carcinomas,3,4 type II tumors are characterized by very frequent TP53 mutations (>95%)5 and associated genomic instability.

Many, if not most, ovarian HGSCs are derived from precursor lesions arising from epithelium in the fimbriated end of the fallopian tube.6-8 These precursor lesions, termed serous tubal intraepithelial carcinomas (TICs and, more specifically, STICs), demonstrate atypical histologic changes that are reminiscent of HGSC. Furthermore, STICs harbor clonal TP53 mutations,6,9 indicating that this alteration is an early event in the oncogenesis of HGSC. However, comprehensive sequencing-based assessment of molecular alterations in TICs has not been reported in large part owing to their minute size. Likewise, it is unclear if some presumed STICs may in fact represent metastatic tubal deposits.

To more comprehensively assess somatic alterations in TICs and assess associations between TICs and synchronous carcinomas, we performed a pilot study of targeted next-generation sequencing (NGS) on a series of 4 TICs (2 discovered incidentally) in patients undergoing total abdominal hysterectomy/bilateral salpingo-oophorectomy (TAH/BSO) for gynecologic malignant neoplasms.

Box Section Ref ID

At a Glance

  • Serous tubal intraepithelial carcinomas (STICs) are precursors of the most common type of ovarian cancer, but detailed genomic characterization of these microscopic lesions has been lacking.

  • Targeted next-generation sequencing on 4 tubal intraepithelial carcinomas (TICs) from patients with concurrent ovarian or uterine cancers was performed.

  • TP53 mutations were present in all TICs

  • In patients with ovarian cancer, concordant mutations in TP53 and BRCA1/2 in the STIC and paired cancer confirm clonal associations between these lesions.

  • Some TICs are actually mucosal metastases from carcinomas arising outside of the fallopian tube, as shown in a patient with uterine cancer.

Methods
Tissue Samples

Four cases of gynecologic malignant neoplasms diagnosed in 2011 to 2012 with coexisting TIC were selected from the case files of the Department of Pathology at the University of Michigan following institutional review board approval. Review of hematoxylin-eosin–stained slides by experienced gynecologic pathologists (J.N.S. and K.R.C.) confirmed the diagnosis in each case. Available demographic and clinicopathologic data were obtained from the medical record.

Immunohistochemical Analysis

Immunohistochemical (IHC) analysis was performed using the Ventana Benchmark System (Ventana Medical Systems) on formalin-fixed, paraffin-embedded (FFPE) tissue sections cut to a thickness of 4 µm. Antibody clones and staining evaluation are described in the eMethods in the Supplement.

Targeted NGS

Ten-micron FFPE sections (10 per sample) were cut from representative blocks from the primary tumor and the tubal lesion from each case. Tumor tissue containing high estimated tumor content (ranging from 60% to 80% tumor nuclei) was macrodissected. DNA isolation, NGS using the DNA component of the Oncomine Comprehensive Panel (OCP), and data analysis to identify prioritized nonsynonymous mutations were performed essentially as described.10,11 We have extensively validated this OCP workflow performance using molecular standards and routine tissue samples,11 and detailed information is provided in the eMethods in the Supplement.

Results
Clinicopathologic Characteristics

We identified 4 cases from 2011 to 2012 in which TICs (all presumed to be serous) were identified in TAH/BSO specimens resected for gynecologic cancer (Table 1 and the Figure, A and B). Patients 1 and 2 were diagnosed as having HGSC (stage IIC and IIIC, respectively), and patients 3 and 4 demonstrated uterine endometrioid carcinoma (UEC), Féderation Internationale de Gynécologie et d'Obstétrique (FIGO) grade 1 (stage IIIA and IB, respectively). Additional clinical information is provided in the eResults in the Supplement. Immunohistochemical analysis for p53 was performed for each presumed STIC, showing strong diffuse nuclear expression in patients 2 to 4, and a total lack of expression in patient 1 (TP53 IHC analysis for patient 4 is shown in the Figure, C).

NGS Results

Manual macrodissection was used to isolate TICs and matched invasive carcinomas from each case (eFigure 1 in the Supplement). Targeted NGS was performed on 5 to 20 ng of genomic DNA, using a custom, multiplexed polymerase chain reaction–based Ion Torrent Ampliseq panel comprising 2462 amplicons covering 135 cancer-related genes (the DNA component of the OCP; the genes are in the eTable in the Supplement) with sequencing performed on the IonTorrent PGM Sequencer. Detailed information on DNA yield and sequencing statistics, including germline single nucleotide polymorphism concordance in paired cases (≥92% per paired sample), is provided in Table 1 and eFigure 2, the eResults, and the eReferences in the Supplement.

The NGS variant calls were filtered using predefined criteria (see the eMethods in the Supplement) to nominate potential somatic driving alterations. Somatic TP53 mutations were present in all 4 tubal lesions (Table 2), with the 2 patients with HGSC showing evidence of a clonal association between the TICs and the primary serous ovarian carcinomas (details are provided in the eMethods in the Supplement). For the 2 patients who presented with incidental TICs in the context of UEC, NGS demonstrated that the 2 lesions in patient 3 were genetically heterogeneous, with the TIC harboring a single TP53 mutation and the UEC showing somatic MTOR, PTEN, KRAS, PIK3CA, and ATM mutations (Table 2), consistent with a genetically distinct STIC and UEC. In patient 4, who harbored a TIC and a UEC (histologic images are shown in the Figure, B, G, and H), the tubal lesion surprisingly demonstrated somatic PTEN and CTNNB1 mutations in addition to a TP53 mutation; the matched UEC also demonstrated concordant PTEN and CTNNB1 mutations, but no TP53 mutation was noted (despite >200 covering reads at that position). Correspondingly, IHC for p53 showed a clonal staining pattern in the TIC, with wild-type (WT) staining observed in the primary tumor (Figure C vs I). Additional IHC stains (Figure, D-F and J-L) demonstrate both the primary tumor and TIC to be positive for PAX8 and negative for WT-1. The TIC showed a proliferative index around 40% (as assessed by Ki-67 IHC), while the primary tumor demonstrated 20% to 30% Ki-67 staining. As described in the next section, this genomic profile and immunophenotype supports the tubal lesion as a micrometastasis (tube mucosal implant) from the UEC rather than a STIC.

Discussion

Herein, we report using targeted NGS on macrodissected routine FFPE archival tissue for a comprehensive investigation of somatic driving mutations associated with fallopian tube TICs and their association with synchronous gynecologic malignant neoplasms. In all tubal lesions, somatic TP53 mutations were identified, consistent with findings of previous reports that TP53 mutation occurs early in the pathogenesis of HGSC.9 and the common use of TP53 immunostaining in STIC diagnosis. The genetic concordance between the STICs and HGSCs in patients 1 and 2 supports a clonal association between the 2 lesions. In contrast, the mutational discordance between the STIC and UEC in patient 3 implies separate and independent neoplastic processes in the fallopian tube and uterus. Patients 1 and 2 harbored germline BRCA1/2 mutations, and STICs are frequently found in the fallopian tubes of patients with germline BRCA mutation with concomitant HGSC and in approximately 5% of those undergoing prophylactic TAH/BSO for HGSC risk-reduction.12 The STIC in patient 3 is potentially sporadic because no germline cancer predisposing mutations in BRCA1, BRCA2,or MSH2 were identified; however, additional cancer predisposing loci were not assessed because the OCP was designed to interrogate somatic driving alterations.

Patient 4 demonstrated a small tubal lesion that resembled a STIC by morphologic characteristics and immunophenotype (clonal TP53); however, NGS sequencing demonstrated PTEN and CTNNB1 mutations in addition to TP53. PTEN and CTNNB1 mutations are characteristic of UEC13 but are uncommon in HGSC.4,14 Given that the TIC and primary UEC present in patient 4 harbored concomitant PTEN and CTNNB1 mutations, we propose that the fallopian tube lesion represents a mucosal UEC micrometastasis mimicking a STIC rather than an independent STIC. The TP53 mutation present in the tubal lesion as supported by both NGS and IHC, but not in the endometrial primary, provides further support that the tube lesion represents a micrometastasis. These findings highlight that although STICs may be precursors to HGSC, not all high-grade TICs are of tubal origin. Likewise, the fallopian tube can harbor metastases from other sites, even in an apparent intraepithelial fashion, and the possibility that a TIC could represent a metastasis should be considered even in the context of serous histologic findings (eg, some “STICs” may represent metastases from primary peritoneal HGSCs). Reliance on clonal p53 expression by IHC analysis for the diagnosis of STIC, as with this patient, can be a potential pitfall complicating the identification of fallopian tube metastases.

Conclusions

This pilot study demonstrates the feasibility of targeted NGS on very small epithelial lesions, such as TICs, with analysis performed on as little as 5 ng of input genomic DNA isolated by macrodissection from FFPE tissue. Although macrodissection in this context is challenging, the clonal TP53 IHC expression in each TIC supports the use of the TP53 variant allele frequency (detected by NGS) to estimate tumor content and supports our approach. Further studies including larger cohorts of synchronous and asynchronous TIC/HGSC cases using this methodology or laser capture microdissection and more comprehensive sequencing may be useful in identifying possible lesions driving progression and potential biomarkers to assist with early detection or minimal residual disease monitoring. Finally, as shown in patient 4, characterization of additional cases diagnosed as STIC may help identify micrometastases to the tubal mucosa that can morphologically mimic true STICs.

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Article Information

Corresponding Author: Scott A. Tomlins, MD, PhD, University of Michigan Medical School, 1524 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (tomlinss@umich.edu).

Accepted for Publication: April 24, 2015.

Published Online: June 18, 2015. doi:10.1001/jamaoncol.2015.1618.

Author Contributions: Drs McDaniel and Tomlins had access to all data and were responsible for primary data analysis. Drs McDaniel and Stall contributed equally to this manuscript.

Study concept and design: McDaniel, Stall, Tomlins, Cho.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: McDaniel, Tomlins, Cho.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: McDaniel, Hovelson.

Obtained funding: Tomlins, Cho.

Administrative, technical, or material support: Stall, Tomlins.

Study supervision: Tomlins.

Conflict of Interest Disclosures: Dr Tomlins has a sponsored research agreement with ThermoFisher Scientific that provided access to the next-generation sequencing panel used herein. ThermoFisher Scientific had no other role in the study design, collection of data, analysis, drafting/review of the manuscript, or decision to submit for publication.

Funding/Support: This work was supported in part by the A. Alfred Taubman Medical Research Institute (SAT).

Role of the Funder/Sponsor: The A. Alfred Taubman Medical Research Institute had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

References
1.
Siegel  RL, Miller  KD, Jemal  A.  Cancer statistics, 2015.  CA Cancer J Clin. 2015;65(1):5-29.PubMedGoogle ScholarCrossref
2.
Kurman  RJ, Shih  IeM.  Pathogenesis of ovarian cancer: lessons from morphology and molecular biology and their clinical implications.  Int J Gynecol Pathol. 2008;27(2):151-160.PubMedGoogle Scholar
3.
Kurman  RJ, Shih  IeM.  The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.  Am J Surg Pathol. 2010;34(3):433-443.PubMedGoogle ScholarCrossref
4.
Kurman  RJ, Shih  IeM.  Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer: shifting the paradigm.  Hum Pathol. 2011;42(7):918-931.PubMedGoogle ScholarCrossref
5.
Ahmed  AA, Etemadmoghadam  D, Temple  J,  et al.  Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary.  J Pathol. 2010;221(1):49-56.PubMedGoogle ScholarCrossref
6.
Kindelberger  DW, Lee  Y, Miron  A,  et al.  Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma: evidence for a causal relationship.  Am J Surg Pathol. 2007;31(2):161-169.PubMedGoogle ScholarCrossref
7.
Piek  JM, van Diest  PJ, Zweemer  RP,  et al.  Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer.  J Pathol. 2001;195(4):451-456.PubMedGoogle ScholarCrossref
8.
Piek  JM, Verheijen  RH, Kenemans  P, Massuger  LF, Bulten  H, van Diest  PJ.  BRCA1/2-related ovarian cancers are of tubal origin: a hypothesis.  Gynecol Oncol. 2003;90(2):491.PubMedGoogle ScholarCrossref
9.
Lee  Y, Miron  A, Drapkin  R,  et al.  A candidate precursor to serous carcinoma that originates in the distal fallopian tube.  J Pathol. 2007;211(1):26-35.PubMedGoogle ScholarCrossref
10.
Cani  AK, Hovelson  DH, McDaniel  AS,  et al.  Next-gen sequencing exposes frequent MED12 mutations and actionable therapeutic targets in phyllodes tumors.  Mol Cancer Res. 2015;13(4):613-619.PubMedGoogle ScholarCrossref
11.
Hovelson  DH, McDaniel  AS, Cani  AK,  et al.  Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors.  Neoplasia. 2015;17(4):385-399.PubMedGoogle ScholarCrossref
12.
Conner  JR, Meserve  E, Pizer  E,  et al.  Outcome of unexpected adnexal neoplasia discovered during risk reduction salpingo-oophorectomy in women with germ-line BRCA1 or BRCA2 mutations.  Gynecol Oncol. 2014;132(2):280-286.PubMedGoogle ScholarCrossref
13.
O’Hara  AJ, Bell  DW.  The genomics and genetics of endometrial cancer.  Adv Genomics Genet. 2012;2012(2):33-47.PubMedGoogle Scholar
14.
Cancer Genome Atlas Research Network.  Integrated genomic analyses of ovarian carcinoma.  Nature. 2011;474(7353):609-615.PubMedGoogle ScholarCrossref
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