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Diagnostic application of KRAS mutation testing in uterine microglandular proliferations
Human Pathology (2015) 46, 1000–1005
www.elsevier.com/locate/humpath
Original contribution
Diagnostic application of KRAS mutation testing in uterine microglandular proliferations☆ Wei Hong MD, PhD, Rita Abi-Raad MD, Ahmed K. Alomari MD, Pei Hui MD, PhD, Natalia Buza MD ⁎ Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023 Received 27 January 2015; revised 14 March 2015; accepted 18 March 2015
Keywords: KRAS mutation; Microglandular hyperplasia; Endometrial adenocarcinoma; Endometrial complex mucinous lesions; Single-starnd conformation polymorphism
Summary Microglandular proliferations often pose a diagnostic challenge in small endocervical and endometrial biopsies. Microglandular hyperplasia (MGH) is one of the most common pseudoneoplastic glandular proliferations of uterine cervix, which can closely mimic endometrial adenocarcinomas (EAC) with a microglandular pattern (microglandular EAC). Although MGH is typically characterized by relatively uniform nuclei and rare to absent mitoses, atypical forms with architectural and/or cytologic deviation from the usual morphology have been previously described. Recently, a series of MGH with high mitotic activity has also been documented. Although careful morphological assessment and immunohistochemical workup can resolve the diagnostic dilemma in some cases, additional differential diagnostic tools are needed to separate both the common and atypical variants of MGH from EAC with microglandular pattern. Frequent KRAS mutation has been previously reported in endometrial complex mucinous lesions and endometrial mucinous carcinomas. However, the diagnostic utility of KRAS mutation analysis has not yet been explored in the context of cervical and endometrial microglandular lesions. Twelve mitotically active MGH cases and 15 cases of EAC with microglandular growth pattern were selected for the study. KRAS mutation analysis was performed in all cases by highly sensitive single-strand conformation polymorphism analysis. Clinical history and follow-up data were retrieved from electronic medical records. KRAS mutation was absent in all MGH cases, whereas 9 (60%) of 15 microglandular EAC cases tested positive for KRAS mutation. Our data indicate that KRAS mutation analysis may offer additional discriminatory power in separating benign MGH from EAC with microglandular pattern. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Microglandular hyperplasia (MGH) of the cervix – first described nearly 5 decades ago by Taylor et al [1] – is a ☆ Disclosures: The authors have no conflicts of interest to disclose. The research was funded by the authors’ intradepartmental research fund. ⁎ Corresponding author at: Department of Pathology, Yale University School of Medicine, 310 Cedar Street LH 108, PO Box 208023, New Haven, CT 06520-8023. E-mail address: [email protected] (N. Buza).
http://dx.doi.org/10.1016/j.humpath.2015.03.010 0046-8177/© 2015 Elsevier Inc. All rights reserved.
common, benign proliferation of tightly packed, irregular endocervical glands with scant intervening stroma, often resulting in a cribriform architectural pattern. The glands are typically lined by bland, flattened, or cuboidal cells, usually with very low mitotic activity. However, atypical variants of MGH have also been described and may show reticular, solid (sheetlike) or pseudoinfiltrative growth pattern, nuclear pleomorphism, hobnail and signet ring cells, or increased mitotic activity [2,3]. The morphological features of MGH may closely mimic endometrial adenocarcinomas (EACs) with mucinous
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differentiation or with microglandular pattern (microglandular adenocarcinoma), especially in limited biopsy specimens [4-9]. In addition to careful morphological examination, immunohistochemical studies – including p16, vimentin, estrogen receptor (ER), carcinoembryonic antigen (CEA) and Ki-67 – may be helpful in the differential diagnosis. Cervical MGH tends to be negative for both vimentin and CEA, positive for ER, and may show focal p16 expression [10-12]. The Ki-67 proliferation index is typically lower in MGH compared with carcinomas, although it may be up to 15% in mitotically active MGH cases [11]. On the other hand, there is significant overlap between the immunoprofile of MGH and microglandular EAC of the endometrium, often making it difficult to separate the 2 entities even when immunohistochemistry is used. KRAS mutation is a common pathogenetic event in various human cancers, for example, pancreatic, colon, lung, and ovarian adenocarcinomas. Recent studies also suggest that KRAS mutation analysis may be a helpful adjunct in the diagnosis and risk stratification of complex mucinous endometrial lesions and mucinous EACs, as more than half of complex endometrial mucinous lesions and more than 80% of mucinous endometrial carcinomas have been shown to harbor KRAS mutations [13-15]. However, KRAS mutation has not been previously evaluated in MGH or in microglandular EAC. In the current study, we aimed to investigate the role of KRAS mutational status in the differential diagnosis of endometrial microglandular adenocarcinoma and benign cervical MGH.
2. Materials and methods Twelve cases of mitotically active MGH and 15 cases of EACs showing at least focal microglandular pattern were retrieved from our departmental archives after approval from the institutional review board. All available hematoxylin and eosin slides were reviewed, and the morphological parameters – nuclear atypia, mitotic activity – were assessed.
Representative blocks were selected for KRAS mutation analysis. Pertinent clinical history and follow-up information were obtained from the patients’ electronic medical records. Eight of the 12 MGH cases were included in a previous case series by our group with detailed morphological and immunohistochemical analysis [3]. KRAS mutation analysis was performed as previously described [16]. Five-micrometer-thick sections were cut from formalin-fixed, paraffin-embedded tissue blocks and were manually dissected into 1.5-mL centrifuge tubes. KRAS mutation analysis was performed by polymerase chain reaction (PCR) single-strand conformational polymorphism (SSCP). Briefly, DNA was extracted from unstained tissue sections using Qiagen tissue kit according to the manufacturer’s instruction (Qiagen tissue kit, Qiagen; Los Angeles, CA). Exon 2 of the KRAS gene was amplified by PCR using flanking primers: forward 5′-GACTGAATATAAACTTGTGG-3′ and reverse 5′-CTGTATCAAAGAATGGTCCT-3′ in a 50-μL PCR reaction solution containing 1× PCR buffer, 0.1 mmol/L deoxynucleotide, 1.5 mmol/L MgCl2, and 2.5 U of AmpliTaq Gold DNA polymerase. Polymerase chain reaction started with initial denaturation at 95°C for 8 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute and synthesis at 72°C for 2 minutes, and finished by a final extension at 72°C for 10 minutes (ABI Veriti Thermal Cycler, Applied Biosystems; Foster City, CA). The PCR product was analyzed by SSCP using 4 μL of the PCR product on MDE nondenaturing gel (Lonza Rockland, Inc., Rockland, ME). Electrophoresis was carried out on ice for 2 hours and 45 minutes at 325 volts. The SSCP gel was then stained with SYBR Gold (Molecular Probes; Life Technologies, Eugene, OR) 1:10000 in tris-EDTA added for 20 minutes and imaged by Biorad GelDoc UV System (BioRad; Hercules, CA). The presence of KRAS mutation was determined by comparing the SSCP banding patterns with known KRAS mutations as positive controls. Because of very similar SSCP banding patterns of codon 12 GAT mutation and codon 13 GAC mutation, in any case showing a GAT mutation pattern on SSCP, the abnormal bands were cut out of the SSCP gel and Sanger sequencing was performed.
Table 1
Clinicopathological parameters and KRAS mutation status of mitotically active cervical microglandular hyperplasia cases
Case no.
Age (y)
Menopausal status
Site/specimen
Nuclear atypia
Mitotic activity/10 HPF
KRAS result
Follow-up (mo)
1 2 3 4 5 6 7 8 9 10 11 12
46 43 43 37 56 52 42 33 42 50 33 41
PreM NA PreM PreM PreM PostM PreM PreM PreM PostM PreM PreM
Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical
Moderate Mild Mild Mild Mild Mild Moderate Mild Mild Mild Moderate Moderate
5 5 6 6 7 8 8 8 10 11 11 11
WT WT WT WT WT WT WT WT WT WT WT WT
62.9 21 51.8 NA 73.2 96.9 12.2 15.1 16.3 69.1 NA 8.5
bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC
Abbreviations: PreM, premenopausal; PostM, postmenopausal; NA, not available; bx, biopsy; ECC, endocervical curettage; WT, wild type; HPF, high power field.
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Fig. 1 Microscopic features of mitotically active MGH. A and B, Case 1: MGH in a 46-year-old patient, the total mitotic count was 5/10 HPF. Follow-up showed no evidence of malignancy at 62.9 months, and KRAS mutation was absent. C and D, Case 6: MGH in a 52-year-old postmenopausal patient with 8 mitoses/10 HPF. Follow-up showed no evidence of malignancy at 96.9 months, and KRAS mutation was absent. Arrows indicate mitotic figures. Hematoxylin and eosin stain; A and C, original magnification ×100; B and D, ×400.
3. Results All 12 mitotically active MGH cases were diagnosed in endocervical biopsy/curettage or cervical polypectomy specimens. The patients’ age ranged from 33 to 56 years with a median of 42.5 years (mean, 43.2 years). Menopausal status was available for 11 patients, only 2 of which were postmenopausal. Clinical follow-up information was available for 10 patients – follow-up period ranging from 9 to 97 months, with a median follow-up of 36.4 months – all of whom were alive and well, without evidence of endometrial or endocervical malignancy (Table 1). Microscopic examination on low magnification showed characteristic architectural features of cervical MGH. On high magnification, focal mild to moderate nuclear atypia was observed and the mitotic count ranged from 5 to 11/10 high-power field (HPF) (Fig. 1). KRAS mutation was absent in all 12 MGH cases (Fig. 2). In the EAC group, the patients’ age at diagnosis ranged between 52 and 86 years (mean, 65 years; median, 64 years). Fourteen patients underwent hysterectomy and staging surgery; however, in 1 case (case 10), no residual carcinoma was identified (Table 2). Microscopically, all 15 EAC cases were of endometrioid histology and showed microglandular pattern, at least focally. Ten tumors were well differentiated (overall FIGO grade 1), and 5 tumors were moderately differentiated (overall
FIGO grade 2) (Fig. 3). Grade 1 nuclear atypia was encountered in 4 cases, whereas the remaining cases showed grade 2 nuclear atypia. The mitotic activity ranged from 1 to 18/10 HPF, with an average of 7.1 mitoses per 10 HPF. KRAS mutation analysis was performed on the hysterectomy specimen in 4 cases and on the endometrial biopsy/curettage in 11 cases. Nine (60%) of the 15 EACs with microglandular pattern harbored KRAS muta-
Fig. 2 KRAS mutation analysis by PCR SSCP showed banding patterns identical to the negative control (wild type, GGT) in a mitotically active cervical MGH case (case 6). Known positive controls show distinct banding patterns unique to each mutation (KRAS mutations GTT, AGT, GAT, GCT, and TGT).
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Table 2 Clinicopathological parameters and KRAS mutation status of microglandular EAC Case Age Specimen no. (y)
FIGO Nuclear Mitotic KRAS grade grade activity/10 result HPF
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
2 2 1 1 1 1 2 1 1 2 1 2 1 1 1
86 77 53 67 58 52 77 52 58 60 61 64 72 71 67
EMB EMC EMC EMC EMC Hysterectomy Hysterectomy EMB EMC EMC EMB Hysterectomy Hysterectomy EMC EMB
2 2 1 2 1 2 2 1 2 2 1 2 2 2 2
9 18 5 5 7 10 4 5 13 2 1 3 12 3 10
GAT GAT GAT WT WT WT WT WT GAC a GAT GTT GAT GAT TGT WT
Abbreviations: EMB, endometrial biopsy; EMC, endometrial curretting; WT, wild type; HPF, high power field. a Codon 13 mutation; all other mutations are codon 12 mutations of exon 2.
tions, including 6 GAT, 1 GTT, and 1 TGT mutations in codon 12 and 1 GAC mutation in codon 13, all of which are known activating mutations (Fig. 4).
Comparing the clinicopathological parameters between the 2 groups, patients with MGH presented at a younger age (median age, 42.5 years versus 64 years with microglandular EAC) and displayed moderate nuclear atypia less frequently than EAC. The average mitotic activity on the other hand was found to be higher in mitotically active MGH than in microglandular EAC (Table 3).
4. Discussion Microglandular proliferations are commonly encountered in the routine gynecologic pathology practice. Most cases represent benign endocervical MGH and can be readily diagnosed as such if it presents in the right clinical context and shows characteristic microscopic features of the lesion. That is, most patients with MGH are young – within their reproductive years, and up to half of them report oral contraceptive use or concurrent pregnancy [1,17-23]. Typical morphological features of MGH include tightly packed, small glands with scant intervening stroma; bland, cuboidal, or low columnar mucinous glandular epithelium; and rare to absent mitotic figures [2]. Abundant acute and chronic inflammatory cells are usually seen both within the stroma and intraluminal mucin. However, MGH may also present in postmenopausal patients [20,22,23] and/or may show atypical histology,
Fig. 3 Endometrial adenocarcinoma with microglandular growth pattern, mimicking benign MGH. A and B, Case 10: 60-year-old patient with FIGO grade 2 EAC in the endometrial curettage; KRAS mutation (GAT) in codon 12 was identified. C and D, Case 9: 58-year-old patient with FIGO grade 1 EAC in endometrial curettage; KRAS mutation (GAC) in codon 13 was identified. Hematoxylin and eosin stain; A and C, ×100; B and D, ×200.
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Fig. 4 Microglandular EAC with KRAS mutation (GAT) of codon 12 (case 10), identical with the banding pattern of a known positive control (GAT). Negative control (wild type, GGT) with a distinct banding pattern on the left and known positive controls (KRAS mutations GTT, AGT, GAT, GCT, and TGT) on the right.
causing a significant diagnostic dilemma. Atypical morphological features may include sheetlike proliferation, pseudoinfiltrative pattern, abundant stromal hyalinization, signet ring and hobnail cells, moderate nuclear atypia, and increased mitotic activity [2,3]. Small fragments of cervical MGH within an endometrial biopsy – especially if showing some nuclear atypia and mitotic activity – may be misdiagnosed as microglandular or mucinous EAC [8,9,11]. This potential diagnostic pitfall was first described by Young and Scully [8] in 1992 in a series of 6 uterine (5 endometrial and 1 cervical) carcinomas, simulating MGH. All 6 cases showed closely packed glands, most of them “microglandular”, and mucinous intraluminal secretions containing acute inflammatory cells. Mild to moderate nuclear atypia and low mitotic activity (b1/10 HPF) were also common features among their cases. Additional endometrial carcinoma cases with similar morphological features have been subsequently reported by other authors emphasizing their architectural similarity to MGH, deceptively bland cytologic appearance and low mitotic activity [9,24-28]. Zaloudek et al [9] coined the term microglandular adenocarcinoma for EACs with prominent microglandular changes mimicking MGH. Although many of the morphological features – microglandular architecture, prominent neutrophilic infiltrate, relatively bland cytology – show significant overlap between the 2 entities, presence of subnuclear vacuoles, immature squamous metaplasia, and Table 3 Diagnosis
reserve cell hyperplasia are helpful features in favor of MGH. Accurate identification of the specimen source – endocervical versus endometrial – based on contiguous tissue fragments is another helpful morphological clue. Immunohistochemical stains may provide assistance in the differential diagnosis; however, the immunoprofiles of MGH and microglandular EAC show significant overlap. Vimentin is typically negative in MGH, whereas most EAC is vimentin positive. Both lesions are usually negative for CEA immunostain, whereas both of them stain positive for ER and at least focally for p16 [10-12]. The proliferation index by Ki-67 immunostain tends to be lower in MGH, although it may be as high as 15% in mitotically active MGH cases [3,11,29]. KRAS – a member of the Ras protein family – plays an important role in human carcinogenesis, with up to 20% to 30% of all human cancers – including pancreatobiliary, colorectal, lung, and ovarian cancer – harboring KRAS activating mutations [30]. More than 95% of pathogenic mutations are localized to hotspots in exon 2 (codons 12 and 13) of the KRAS gene [31]; mutations in codon 61 are much less frequent. KRAS mutation analysis is a routinely used ancillary test in the diagnosis of pancreatic adenocarcinoma and for predicting therapeutic response in other cancer types [30]. Recent studies identified KRAS mutations in nearly 30% of endometrioid EAC [32], in 86% of mucinous EAC, and in up to 89% of complex atypical mucinous lesions of the endometrium [13,15]. It has also been suggested that KRAS mutational status may be helpful to refine the risk stratification of patients with endometrial mucinous lesions [13]. However, KRAS mutation analysis has not yet been reported in cervical MGH and microglandular EAC. Our study explored the diagnostic utility of KRAS mutation analysis in a series of mitotically active cervical MGH and microglandular EAC by PCR SSCP. KRAS mutation was absent in all 12 mitotically active MGH cases, whereas 60% of microglandular EAC tested positive for KRAS mutation. Because endometrial and endocervical biopsy/curettage specimens often contain only small amounts of tissue, a highly sensitive and specific method for KRAS mutation testing – such as SSCP in the current study – is essential. In conclusion, our data suggest that KRAS mutation analysis could be a valuable adjunct test in diagnostically challenging small endometrial or endocervical biopsy specimens with microglandular growth pattern. Based on the current and other recent studies, a positive KRAS mutation test result is a strong indicator of a neoplastic
Comparison of mitotically active cervical MGH and EAC with microglandular features Age (y, range) Nuclear atypia Mild (n, %)
MA-MGH 43.2 (33-56) MG-EAC 65 (52-86)
Mitotic activity/10 HPF; mean (range) KRAS mutation positive; n (%) Moderate (n, %)
8/12 (66.7%) 4/12 (33.3%) 4/15 (27%) 11/15 (73%)
8 (5-11) 7.1 (1-18)
0/12 (0%) 9/15 (60%)
Abbreviations: MA-MGH, mitotically active microgandular hyperplasia; MG-EAC, microglandular endometrial adenocarcinoma; HPF, high power field.
KRAS in uterine microglandular proliferations (microglandular EAC) or preneoplastic (complex atypical endometrial mucinous proliferation) process, although absence of KRAS mutation does not rule out the possibility of malignancy. Additional studies of larger cohorts are important to confirm our molecular findings.
References [1] Taylor HB, Irey NS, Norris HJ. Atypical endocervical hyperplasia in women taking oral contraceptives. JAMA 1967;202:637-9. [2] Young RH, Scully RE. Atypical forms of microglandular hyperplasia of the cervix simulating carcinoma. A report of five cases and review of the literature. Am J Surg Pathol 1989;13:50-6. [3] Abi-Raad R, Alomari A, Hui P, Buza N. Mitotically active microglandular hyperplasia of the cervix: a case series with implications for the differential diagnosis. Int J Gynecol Pathol 2014;33:524-30. [4] Copeland LJ, Sneige N, Ordonez NG, et al. Endodermal sinus tumor of the vagina and cervix. Cancer 1985;55:2558-65. [5] McCluggage WG. Endocervical glandular lesions: controversial aspects and ancillary techniques. J Clin Pathol 2003;56:164-73. [6] Nucci MR. Pseudoneoplastic glandular lesions of the uterine cervix: a selective review. Int J Gynecol Pathol 2014;33:330-8. [7] Fukunaga M. Mucinous endometrial adenocarcinoma simulating microglandular hyperplasia of the cervix. Pathol Int 2000;50:541-5. [8] Young RH, Scully RE. Uterine carcinomas simulating microglandular hyperplasia. A report of six cases. Am J Surg Pathol 1992;16:1092-7. [9] Zaloudek C, Hayashi GM, Ryan IP, Powell CB, Miller TR. Microglandular adenocarcinoma of the endometrium: a form of mucinous adenocarcinoma that may be confused with microglandular hyperplasia of the cervix. Int J Gynecol Pathol 1997;16:52-9. [10] Zamecnik M. Hormone receptors in microglandular hyperplasia of the uterine cervix. Int J Gynecol Pathol 2002;21:424-5. [11] Qiu W, Mittal K. Comparison of morphologic and immunohistochemical features of cervical microglandular hyperplasia with lowgrade mucinous adenocarcinoma of the endometrium. Int J Gynecol Pathol 2003;22:261-5. [12] Roh MH, Agoston E, Birch C, Crum CP. P16 immunostaining patterns in microglandular hyperplasia of the cervix and their significance. Int J Gynecol Pathol 2009;28:107-13. [13] Alomari A, Abi-Raad R, Buza N, Hui P. Frequent KRAS mutation in complex mucinous epithelial lesions of the endometrium. Mod Pathol 2014;27:675-80. [14] Xiong J, He M, Jackson C, et al. Endometrial carcinomas with significant mucinous differentiation associated with higher frequency of k-ras mutations: a morphologic and molecular correlation study. Int J Gynecol Cancer 2013;23:1231-6.
1005 [15] Yoo SH, Park BH, Choi J, et al. Papillary mucinous metaplasia of the endometrium as a possible precursor of endometrial mucinous adenocarcinoma. Mod Pathol 2012;25:1496-507. [16] Dillon DA, Johnson CC, Topazian MD, Tallini G, Rimm DL, Costa JC. The utility of Ki-ras mutation analysis in the cytologic diagnosis of pancreatobiliary neoplasma. Cancer J 2000;6:294-301. [17] Kyriakos M, Kempson RL, Konikov NF. A clinical and pathologic study of endocervical lesions associated with oral contraceptives. Cancer 1968;22:99-110. [18] Govan AD, Black WP, Sharp JL. Aberrant glandular polypi of the uterine cervix associated with contraceptive pills: pathology and pathogenesis. J Clin Pathol 1969;22:84-9. [19] Graham J, Graham R, Hirabayashi K. Reversible “cancer" and the contraceptive pill. Report of a case. Obstet Gynecol 1968;31:190-2. [20] Medeiros F, Bell DA. Pseudoneoplastic lesions of the female genital tract. Arch Pathol Lab Med 2010;134:393-403. [21] Wilkinson E, Dufour DR. Pathogenesis of microglandular hyperplasia of the cervix uteri. Obstet Gynecol 1976;47:189-95. [22] Nucci MR. Symposium part III: tumor-like glandular lesions of the uterine cervix. Int J Gynecol Pathol 2002;21:347-59. [23] Young RH, Clement PB. Pseudoneoplastic glandular lesions of the uterine cervix. Semin Diagn Pathol 1991;8:234-49. [24] Zamecnik M, Skalova A, Opatrny V. Microglandular adenocarcinoma of the uterus mimicking microglandular cervical hyperplasia. Ann Diagn Pathol 2003;7:180-6. [25] Karateke A, Haliloglu B, Atay V, Gurbuz A, Kir G. A case of microglandular adenocarcinoma of the endometrium. Gynecol Oncol 2005;99:778-81. [26] McCluggage WG, Perenyei M. Microglandular adenocarcinoma of the endometrium. Histopathology 2000;37:285-7. [27] Da Forno PD, McGregor AH, Brown LJ. Microglandular hyperplasia: a pitfall in the diagnosis of microglandular type endometrioid adenocarcinoma. Histopathology 2005;46:346-8. [28] Giordano G, D'Adda T, Gnetti L, Merisio C, Melpignano M. Endometrial mucinous microglandular adenocarcinoma: morphologic, immunohistochemical features, and emphasis in the human papillomavirus status. Int J Gynecol Pathol 2006;25:77-82. [29] Cina SJ, Richardson MS, Austin RM, Kurman RJ. Immunohistochemical staining for Ki-67 antigen, carcinoembryonic antigen, and p53 in the differential diagnosis of glandular lesions of the cervix. Mod Pathol 1997;10:176-80. [30] Perincheri S, Hui P. KRAS mutation testing in clinical practice. Expert Rev Mol Diagn 2014:1-10. [31] Anderson SM. Laboratory methods for KRAS mutation analysis. Expert Rev Mol Diagn 2011;11:635-42. [32] Lax SF, Kendall B, Tashiro H, Slebos RJ, Hedrick L. The frequency of p53, K-ras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma: evidence of distinct molecular genetic pathways. Cancer 2000;88:814-24.
www.elsevier.com/locate/humpath
Original contribution
Diagnostic application of KRAS mutation testing in uterine microglandular proliferations☆ Wei Hong MD, PhD, Rita Abi-Raad MD, Ahmed K. Alomari MD, Pei Hui MD, PhD, Natalia Buza MD ⁎ Department of Pathology, Yale University School of Medicine, New Haven, CT 06520-8023 Received 27 January 2015; revised 14 March 2015; accepted 18 March 2015
Keywords: KRAS mutation; Microglandular hyperplasia; Endometrial adenocarcinoma; Endometrial complex mucinous lesions; Single-starnd conformation polymorphism
Summary Microglandular proliferations often pose a diagnostic challenge in small endocervical and endometrial biopsies. Microglandular hyperplasia (MGH) is one of the most common pseudoneoplastic glandular proliferations of uterine cervix, which can closely mimic endometrial adenocarcinomas (EAC) with a microglandular pattern (microglandular EAC). Although MGH is typically characterized by relatively uniform nuclei and rare to absent mitoses, atypical forms with architectural and/or cytologic deviation from the usual morphology have been previously described. Recently, a series of MGH with high mitotic activity has also been documented. Although careful morphological assessment and immunohistochemical workup can resolve the diagnostic dilemma in some cases, additional differential diagnostic tools are needed to separate both the common and atypical variants of MGH from EAC with microglandular pattern. Frequent KRAS mutation has been previously reported in endometrial complex mucinous lesions and endometrial mucinous carcinomas. However, the diagnostic utility of KRAS mutation analysis has not yet been explored in the context of cervical and endometrial microglandular lesions. Twelve mitotically active MGH cases and 15 cases of EAC with microglandular growth pattern were selected for the study. KRAS mutation analysis was performed in all cases by highly sensitive single-strand conformation polymorphism analysis. Clinical history and follow-up data were retrieved from electronic medical records. KRAS mutation was absent in all MGH cases, whereas 9 (60%) of 15 microglandular EAC cases tested positive for KRAS mutation. Our data indicate that KRAS mutation analysis may offer additional discriminatory power in separating benign MGH from EAC with microglandular pattern. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Microglandular hyperplasia (MGH) of the cervix – first described nearly 5 decades ago by Taylor et al [1] – is a ☆ Disclosures: The authors have no conflicts of interest to disclose. The research was funded by the authors’ intradepartmental research fund. ⁎ Corresponding author at: Department of Pathology, Yale University School of Medicine, 310 Cedar Street LH 108, PO Box 208023, New Haven, CT 06520-8023. E-mail address: [email protected] (N. Buza).
http://dx.doi.org/10.1016/j.humpath.2015.03.010 0046-8177/© 2015 Elsevier Inc. All rights reserved.
common, benign proliferation of tightly packed, irregular endocervical glands with scant intervening stroma, often resulting in a cribriform architectural pattern. The glands are typically lined by bland, flattened, or cuboidal cells, usually with very low mitotic activity. However, atypical variants of MGH have also been described and may show reticular, solid (sheetlike) or pseudoinfiltrative growth pattern, nuclear pleomorphism, hobnail and signet ring cells, or increased mitotic activity [2,3]. The morphological features of MGH may closely mimic endometrial adenocarcinomas (EACs) with mucinous
KRAS in uterine microglandular proliferations
1001
differentiation or with microglandular pattern (microglandular adenocarcinoma), especially in limited biopsy specimens [4-9]. In addition to careful morphological examination, immunohistochemical studies – including p16, vimentin, estrogen receptor (ER), carcinoembryonic antigen (CEA) and Ki-67 – may be helpful in the differential diagnosis. Cervical MGH tends to be negative for both vimentin and CEA, positive for ER, and may show focal p16 expression [10-12]. The Ki-67 proliferation index is typically lower in MGH compared with carcinomas, although it may be up to 15% in mitotically active MGH cases [11]. On the other hand, there is significant overlap between the immunoprofile of MGH and microglandular EAC of the endometrium, often making it difficult to separate the 2 entities even when immunohistochemistry is used. KRAS mutation is a common pathogenetic event in various human cancers, for example, pancreatic, colon, lung, and ovarian adenocarcinomas. Recent studies also suggest that KRAS mutation analysis may be a helpful adjunct in the diagnosis and risk stratification of complex mucinous endometrial lesions and mucinous EACs, as more than half of complex endometrial mucinous lesions and more than 80% of mucinous endometrial carcinomas have been shown to harbor KRAS mutations [13-15]. However, KRAS mutation has not been previously evaluated in MGH or in microglandular EAC. In the current study, we aimed to investigate the role of KRAS mutational status in the differential diagnosis of endometrial microglandular adenocarcinoma and benign cervical MGH.
2. Materials and methods Twelve cases of mitotically active MGH and 15 cases of EACs showing at least focal microglandular pattern were retrieved from our departmental archives after approval from the institutional review board. All available hematoxylin and eosin slides were reviewed, and the morphological parameters – nuclear atypia, mitotic activity – were assessed.
Representative blocks were selected for KRAS mutation analysis. Pertinent clinical history and follow-up information were obtained from the patients’ electronic medical records. Eight of the 12 MGH cases were included in a previous case series by our group with detailed morphological and immunohistochemical analysis [3]. KRAS mutation analysis was performed as previously described [16]. Five-micrometer-thick sections were cut from formalin-fixed, paraffin-embedded tissue blocks and were manually dissected into 1.5-mL centrifuge tubes. KRAS mutation analysis was performed by polymerase chain reaction (PCR) single-strand conformational polymorphism (SSCP). Briefly, DNA was extracted from unstained tissue sections using Qiagen tissue kit according to the manufacturer’s instruction (Qiagen tissue kit, Qiagen; Los Angeles, CA). Exon 2 of the KRAS gene was amplified by PCR using flanking primers: forward 5′-GACTGAATATAAACTTGTGG-3′ and reverse 5′-CTGTATCAAAGAATGGTCCT-3′ in a 50-μL PCR reaction solution containing 1× PCR buffer, 0.1 mmol/L deoxynucleotide, 1.5 mmol/L MgCl2, and 2.5 U of AmpliTaq Gold DNA polymerase. Polymerase chain reaction started with initial denaturation at 95°C for 8 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute and synthesis at 72°C for 2 minutes, and finished by a final extension at 72°C for 10 minutes (ABI Veriti Thermal Cycler, Applied Biosystems; Foster City, CA). The PCR product was analyzed by SSCP using 4 μL of the PCR product on MDE nondenaturing gel (Lonza Rockland, Inc., Rockland, ME). Electrophoresis was carried out on ice for 2 hours and 45 minutes at 325 volts. The SSCP gel was then stained with SYBR Gold (Molecular Probes; Life Technologies, Eugene, OR) 1:10000 in tris-EDTA added for 20 minutes and imaged by Biorad GelDoc UV System (BioRad; Hercules, CA). The presence of KRAS mutation was determined by comparing the SSCP banding patterns with known KRAS mutations as positive controls. Because of very similar SSCP banding patterns of codon 12 GAT mutation and codon 13 GAC mutation, in any case showing a GAT mutation pattern on SSCP, the abnormal bands were cut out of the SSCP gel and Sanger sequencing was performed.
Table 1
Clinicopathological parameters and KRAS mutation status of mitotically active cervical microglandular hyperplasia cases
Case no.
Age (y)
Menopausal status
Site/specimen
Nuclear atypia
Mitotic activity/10 HPF
KRAS result
Follow-up (mo)
1 2 3 4 5 6 7 8 9 10 11 12
46 43 43 37 56 52 42 33 42 50 33 41
PreM NA PreM PreM PreM PostM PreM PreM PreM PostM PreM PreM
Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical Cervical
Moderate Mild Mild Mild Mild Mild Moderate Mild Mild Mild Moderate Moderate
5 5 6 6 7 8 8 8 10 11 11 11
WT WT WT WT WT WT WT WT WT WT WT WT
62.9 21 51.8 NA 73.2 96.9 12.2 15.1 16.3 69.1 NA 8.5
bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC bx/ECC
Abbreviations: PreM, premenopausal; PostM, postmenopausal; NA, not available; bx, biopsy; ECC, endocervical curettage; WT, wild type; HPF, high power field.
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Fig. 1 Microscopic features of mitotically active MGH. A and B, Case 1: MGH in a 46-year-old patient, the total mitotic count was 5/10 HPF. Follow-up showed no evidence of malignancy at 62.9 months, and KRAS mutation was absent. C and D, Case 6: MGH in a 52-year-old postmenopausal patient with 8 mitoses/10 HPF. Follow-up showed no evidence of malignancy at 96.9 months, and KRAS mutation was absent. Arrows indicate mitotic figures. Hematoxylin and eosin stain; A and C, original magnification ×100; B and D, ×400.
3. Results All 12 mitotically active MGH cases were diagnosed in endocervical biopsy/curettage or cervical polypectomy specimens. The patients’ age ranged from 33 to 56 years with a median of 42.5 years (mean, 43.2 years). Menopausal status was available for 11 patients, only 2 of which were postmenopausal. Clinical follow-up information was available for 10 patients – follow-up period ranging from 9 to 97 months, with a median follow-up of 36.4 months – all of whom were alive and well, without evidence of endometrial or endocervical malignancy (Table 1). Microscopic examination on low magnification showed characteristic architectural features of cervical MGH. On high magnification, focal mild to moderate nuclear atypia was observed and the mitotic count ranged from 5 to 11/10 high-power field (HPF) (Fig. 1). KRAS mutation was absent in all 12 MGH cases (Fig. 2). In the EAC group, the patients’ age at diagnosis ranged between 52 and 86 years (mean, 65 years; median, 64 years). Fourteen patients underwent hysterectomy and staging surgery; however, in 1 case (case 10), no residual carcinoma was identified (Table 2). Microscopically, all 15 EAC cases were of endometrioid histology and showed microglandular pattern, at least focally. Ten tumors were well differentiated (overall FIGO grade 1), and 5 tumors were moderately differentiated (overall
FIGO grade 2) (Fig. 3). Grade 1 nuclear atypia was encountered in 4 cases, whereas the remaining cases showed grade 2 nuclear atypia. The mitotic activity ranged from 1 to 18/10 HPF, with an average of 7.1 mitoses per 10 HPF. KRAS mutation analysis was performed on the hysterectomy specimen in 4 cases and on the endometrial biopsy/curettage in 11 cases. Nine (60%) of the 15 EACs with microglandular pattern harbored KRAS muta-
Fig. 2 KRAS mutation analysis by PCR SSCP showed banding patterns identical to the negative control (wild type, GGT) in a mitotically active cervical MGH case (case 6). Known positive controls show distinct banding patterns unique to each mutation (KRAS mutations GTT, AGT, GAT, GCT, and TGT).
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Table 2 Clinicopathological parameters and KRAS mutation status of microglandular EAC Case Age Specimen no. (y)
FIGO Nuclear Mitotic KRAS grade grade activity/10 result HPF
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
2 2 1 1 1 1 2 1 1 2 1 2 1 1 1
86 77 53 67 58 52 77 52 58 60 61 64 72 71 67
EMB EMC EMC EMC EMC Hysterectomy Hysterectomy EMB EMC EMC EMB Hysterectomy Hysterectomy EMC EMB
2 2 1 2 1 2 2 1 2 2 1 2 2 2 2
9 18 5 5 7 10 4 5 13 2 1 3 12 3 10
GAT GAT GAT WT WT WT WT WT GAC a GAT GTT GAT GAT TGT WT
Abbreviations: EMB, endometrial biopsy; EMC, endometrial curretting; WT, wild type; HPF, high power field. a Codon 13 mutation; all other mutations are codon 12 mutations of exon 2.
tions, including 6 GAT, 1 GTT, and 1 TGT mutations in codon 12 and 1 GAC mutation in codon 13, all of which are known activating mutations (Fig. 4).
Comparing the clinicopathological parameters between the 2 groups, patients with MGH presented at a younger age (median age, 42.5 years versus 64 years with microglandular EAC) and displayed moderate nuclear atypia less frequently than EAC. The average mitotic activity on the other hand was found to be higher in mitotically active MGH than in microglandular EAC (Table 3).
4. Discussion Microglandular proliferations are commonly encountered in the routine gynecologic pathology practice. Most cases represent benign endocervical MGH and can be readily diagnosed as such if it presents in the right clinical context and shows characteristic microscopic features of the lesion. That is, most patients with MGH are young – within their reproductive years, and up to half of them report oral contraceptive use or concurrent pregnancy [1,17-23]. Typical morphological features of MGH include tightly packed, small glands with scant intervening stroma; bland, cuboidal, or low columnar mucinous glandular epithelium; and rare to absent mitotic figures [2]. Abundant acute and chronic inflammatory cells are usually seen both within the stroma and intraluminal mucin. However, MGH may also present in postmenopausal patients [20,22,23] and/or may show atypical histology,
Fig. 3 Endometrial adenocarcinoma with microglandular growth pattern, mimicking benign MGH. A and B, Case 10: 60-year-old patient with FIGO grade 2 EAC in the endometrial curettage; KRAS mutation (GAT) in codon 12 was identified. C and D, Case 9: 58-year-old patient with FIGO grade 1 EAC in endometrial curettage; KRAS mutation (GAC) in codon 13 was identified. Hematoxylin and eosin stain; A and C, ×100; B and D, ×200.
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Fig. 4 Microglandular EAC with KRAS mutation (GAT) of codon 12 (case 10), identical with the banding pattern of a known positive control (GAT). Negative control (wild type, GGT) with a distinct banding pattern on the left and known positive controls (KRAS mutations GTT, AGT, GAT, GCT, and TGT) on the right.
causing a significant diagnostic dilemma. Atypical morphological features may include sheetlike proliferation, pseudoinfiltrative pattern, abundant stromal hyalinization, signet ring and hobnail cells, moderate nuclear atypia, and increased mitotic activity [2,3]. Small fragments of cervical MGH within an endometrial biopsy – especially if showing some nuclear atypia and mitotic activity – may be misdiagnosed as microglandular or mucinous EAC [8,9,11]. This potential diagnostic pitfall was first described by Young and Scully [8] in 1992 in a series of 6 uterine (5 endometrial and 1 cervical) carcinomas, simulating MGH. All 6 cases showed closely packed glands, most of them “microglandular”, and mucinous intraluminal secretions containing acute inflammatory cells. Mild to moderate nuclear atypia and low mitotic activity (b1/10 HPF) were also common features among their cases. Additional endometrial carcinoma cases with similar morphological features have been subsequently reported by other authors emphasizing their architectural similarity to MGH, deceptively bland cytologic appearance and low mitotic activity [9,24-28]. Zaloudek et al [9] coined the term microglandular adenocarcinoma for EACs with prominent microglandular changes mimicking MGH. Although many of the morphological features – microglandular architecture, prominent neutrophilic infiltrate, relatively bland cytology – show significant overlap between the 2 entities, presence of subnuclear vacuoles, immature squamous metaplasia, and Table 3 Diagnosis
reserve cell hyperplasia are helpful features in favor of MGH. Accurate identification of the specimen source – endocervical versus endometrial – based on contiguous tissue fragments is another helpful morphological clue. Immunohistochemical stains may provide assistance in the differential diagnosis; however, the immunoprofiles of MGH and microglandular EAC show significant overlap. Vimentin is typically negative in MGH, whereas most EAC is vimentin positive. Both lesions are usually negative for CEA immunostain, whereas both of them stain positive for ER and at least focally for p16 [10-12]. The proliferation index by Ki-67 immunostain tends to be lower in MGH, although it may be as high as 15% in mitotically active MGH cases [3,11,29]. KRAS – a member of the Ras protein family – plays an important role in human carcinogenesis, with up to 20% to 30% of all human cancers – including pancreatobiliary, colorectal, lung, and ovarian cancer – harboring KRAS activating mutations [30]. More than 95% of pathogenic mutations are localized to hotspots in exon 2 (codons 12 and 13) of the KRAS gene [31]; mutations in codon 61 are much less frequent. KRAS mutation analysis is a routinely used ancillary test in the diagnosis of pancreatic adenocarcinoma and for predicting therapeutic response in other cancer types [30]. Recent studies identified KRAS mutations in nearly 30% of endometrioid EAC [32], in 86% of mucinous EAC, and in up to 89% of complex atypical mucinous lesions of the endometrium [13,15]. It has also been suggested that KRAS mutational status may be helpful to refine the risk stratification of patients with endometrial mucinous lesions [13]. However, KRAS mutation analysis has not yet been reported in cervical MGH and microglandular EAC. Our study explored the diagnostic utility of KRAS mutation analysis in a series of mitotically active cervical MGH and microglandular EAC by PCR SSCP. KRAS mutation was absent in all 12 mitotically active MGH cases, whereas 60% of microglandular EAC tested positive for KRAS mutation. Because endometrial and endocervical biopsy/curettage specimens often contain only small amounts of tissue, a highly sensitive and specific method for KRAS mutation testing – such as SSCP in the current study – is essential. In conclusion, our data suggest that KRAS mutation analysis could be a valuable adjunct test in diagnostically challenging small endometrial or endocervical biopsy specimens with microglandular growth pattern. Based on the current and other recent studies, a positive KRAS mutation test result is a strong indicator of a neoplastic
Comparison of mitotically active cervical MGH and EAC with microglandular features Age (y, range) Nuclear atypia Mild (n, %)
MA-MGH 43.2 (33-56) MG-EAC 65 (52-86)
Mitotic activity/10 HPF; mean (range) KRAS mutation positive; n (%) Moderate (n, %)
8/12 (66.7%) 4/12 (33.3%) 4/15 (27%) 11/15 (73%)
8 (5-11) 7.1 (1-18)
0/12 (0%) 9/15 (60%)
Abbreviations: MA-MGH, mitotically active microgandular hyperplasia; MG-EAC, microglandular endometrial adenocarcinoma; HPF, high power field.
KRAS in uterine microglandular proliferations (microglandular EAC) or preneoplastic (complex atypical endometrial mucinous proliferation) process, although absence of KRAS mutation does not rule out the possibility of malignancy. Additional studies of larger cohorts are important to confirm our molecular findings.
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