Uterine smooth muscle tumors (SMT) are the most frequent neoplasms of the female genital tract, occurring in 77% of women 1,2. The main diagnostic entities are leiomyoma (LM) [including leiomyoma with bizarre nuclei (LBN), cellular LM, mitotically active LM among others], smooth muscle tumors of uncertain malignant potential (STUMP) and leiomyosarcoma (LMS) 3. A combination of histologic features, mitoses, cytologic atypia and tumor cell necrosis, are used to define the uncommon malignant SMT, LMS 4. However, there is an overlap in the defining histologic features of LMS with STUMP and LBN that frequently makes the definitive distinction challenging.
The exact pathogenesis of uterine and soft tissue LMS remains unclear despite multiple molecular studies. Available cytogenetic data suggests diverse alterations with complex numerical and structural chromosomal abnormalities 5–7. While no distinct cellular pathway is discovered to be directly involved in the SMT tumorigenesis, cell cycle/proliferation markers remain as the alternative best potential immunohistochemical markers valuable in the SMT diagnostic impasse. Lack of a reliable diagnostic tool beside histology is especially perceptible in evaluation of lesions showing cytologic atypia or borderline mitotic activity.
Discovering a reliable and easily accessible method to distinguish between the SMTs has been the subject of many studies in the past 2 decade. The diagnostic utility of various panels of immunohistochemical markers have been investigated including cell cycle regulatory proteins (p53, p16, p21, p27, bcl-2, bax), proliferation markers (Ki67, PHH3), ER, PR, and WT1 8–11 as well as other markers [IMP3 12, fascin 13, Stathmin 1 14]. In addition, molecular and cytogenetic aspects of smooth muscle malignancy are also well studied. Molecular alterations at the genomic and transcription level are compared in different categories of SMTs 6,15,16. Nevertheless, immunohistochemical and molecular measures have limited diagnostic utility in current practice and morphologic examination continues to be the foundation of the diagnosis in these tumors 17.
The minichromosome maintenance (MCM) family consists of 6 highly conserved proteins (MCM2–7) that play an essential role in the cell cycle progression, regulating the initiation and progression of DNA replication 18. MCMs form a protein complex as a key component of the prereplication complex during G1 phase and accordingly, their protein levels are reported to increase at G1 and peak at S1 phase 18–20. Therefore, these proteins are not only expressed in proliferating cells, but also in cells with proliferation intent 21. As the overexpression of MCM2 is demonstrated in multiple malignant neoplasms, it has recently gained a growing attention as a promising proliferation marker 18,21–24.
In 2004, Quade et al. 25, reported that MCM2 expression level increases significantly in uterine leiomyosarcoma, 28.2-fold normal myometrium (compared with 18.2-fold increase in LM). Previously, it was shown that nuclear positivity for MCM2 is significantly higher in high-grade compared with low-grade soft tissue leiomyosarcomas 26. Also, studying multiple malignant soft tissue neoplasms including LMS, Cunha et al. 27 showed that MCM2 has a higher mRNA expression compared with benign soft tissue tumors.
Cyclin D1 belongs to the highly conserved cyclin protein family that coordinate the sequential steps of cell division. Cyclin D1 activates cyclin dependant kinase (CDK4/CDK6) activity that leads to phosphorylation of RB and consequent progression of cell through the G1-S checkpoint. The overexpression of the corresponding gene, CCND1, is established in multiple malignancies 28,29. Cancer-associated mutations also occur and both nuclear and cytoplasmic localizations are reported in different malignancies following mutation 28.
Cyclin D1 expression has been studied in benign and malignant smooth muscle tumors. In 1999, Rao et al. 30 described all their uterine leiomyomata to be diffusely positive for cyclin D1. A similar study showed that cyclin D1 expression in leiomyomata was more than adjacent myometrium in premenopausal women, and similar to myometrium in menopausal women 31. More recently, Lee et al. 32, reported that among 80 primary uterine leiomyosarcomas and 39 metastatic leiomyosarcomas, only 1 case was positive for cyclin D1, with positivity criteria of >70% moderate to strong nuclear staining. In leiomyosarcomas of soft tissue, Dei Tos et al. 33 described cyclin D1 staining in 4 of 23 cases, ranging from 30% to 50% cell positivity.
The objective of this study was to explore the diagnostic utility of MCM2 and Cyclin D1 in differentiating the spectrum of SMTs. The focus of this study is on entities with pronounced cytologic atypia causing the most common diagnostic difficulties including LBN, STUMP, and LMS. The results are correlated with the well-established cell cycle markers p53, p16, and Ki67. Finally, we compare the values of MCM2, p16, and Ki67 parallel testing for reliable identification of LMS from benign SMTs.
MATERIALS AND METHODS
All cases of uterine spindle LMS (n=13), LBN (n=10), and STUMP (n=12) resected in The Ottawa Hospital after 2009 were included in the study. LMS inclusion was limited to spindle cell subtype. Ten recent cases of LM with no history of hormonal treatment were added as control.
The following primary antibodies were used for immunohistochemical staining, anti MCM2 antibody (Cell signaling # 12079, dilution 1/400); anti Cyclin D1 (Davis, #CRM307B, dilution 1:10); anti Ki67 antibody (Dako, #M7240, dilution 1:75), anti p53 antibody (Leica, #PA0057, ready to use); and anti p16 (Ventana, #9517, ready to use). All the reactions were performed using Leica BOND MAX automated slide staining instrument. Overall percentage of nuclear positivity was reported for each marker. For Ki67, a hotspot percentage count was performed. P53 nuclear positivity above 10% was recorded.
For specificity and sensitivity of combination models, parallel testing is performed and measures are reported for combination of markers using AND and OR functions.
Patient Demographics and Clinical Information
Patients’ age and lesions sizes are listed in Table 1. Frequently patients had >1 lesion resected. Therefore, exact size of the lesion was not available for some of the LM and LBNs listed as data incomplete. In LBN group, 2 patients were treated with selective progesterone receptor modulator (ulipristal acetate, also known as fibristal) and 1 patient with oral contraceptive medication before surgery. None of the patients other than LMS cases had a recurrence (Table 1).
Smooth muscle cell positivity was both nuclear and cytoplasmic (the latter not recorded) with the mild to moderate intensity (Fig. 1). A wide range of reactivity was observed among the cases of LM, LBN, and STUMP, ranging from <1% to 65% nuclear positivity. Overall, diffuse high percentage positivity was not observed in any of the categories. LMS cases in our cohort consistently showed low rate of positivity. Details about range and average percentage of nuclear positivity are summarized in Table 2. The sensitivity and specificity of cyclin D1 <5% for diagnosis of LMS in our study population was 100% and 52%, respectively (Table 7).
Smooth muscle cell positivity in our patient population was almost exclusively nuclear ranging from mild to strong intensity (Fig. 2). Details about range and mean of positivity are summarized in Table 3. In most LM, LBN, and LMS cases, the positivity was of weak to moderate intensity with patchy distribution. Diffuse (>80%) and strong positivity was observed in 1 case of LBN (1/10) and 1 case of STUMP (1/12). Two other STUMP cases (2/12) had over 70% positivity of mostly weak (1/12) or patchy weak/strong (1/12) intensity. Importantly, 12 of 13 cases of LMS (92%) showed diffuse (>80%) and strong nuclear positivity for MCM2. The only LMS case that was patchy positive for MCM2, showed some epithelioid features. The sensitivity and specificity of MCM2 >80% for diagnosis of LMS in our study population was 92% and 94%, respectively (Table 7).
Smooth muscle cell positivity was both nuclear and cytoplasmic (the latter not recorded) with the mild to strong intensity. Details about range and mean of positivity are summarized in Table 4. Although the staining ratio in LM, LBN, and STUMP was variable, all LM and STUMP cases had <50% reactivity. Interestingly, in 30% (3/10) of LBNs, >50% positivity was observed that was diffuse in one LBN case (>90%). Although most cases of LMS showed diffuse and strong positivity for p16 (10/13), 3 cases had <10% staining. Notably, these 3 cases all showed focal epithelioid morphology. The sensitivity and specificity of p16 >80% for diagnosis of LMS in our study population was 77% and 97%, respectively (Table 7).
Majority of our cases showed <10% nuclear positivity. p53 null phenotype was not observed in any of the cases. Two LBNs demonstrated 20% to 30% reactivity (2/10) and 38% of LMS cases showed >50% positivity. The highest rate of labeling was observed in 2 LMS cases each with >80% positivity. More details about p53 immunoreactivity are summarized in Table 5.
Ki67 range and mean of nuclear positivity in different categories are listed in Table 6. The range of positivity was <1% for LMs, <1% to 8% in LBNs and 1% to 15% in the STUMP category. Among the nonmalignant categories, only 2 of the STUMP cases (2/13) had 10% and above positivity (1 case with 10% and 1 with 15%). Most LMSs had 10% and above nuclear reactivity with Ki67 (Table 6). The sensitivity and specificity of Ki67≥10% for the diagnosis of LMS was 92% and 100%, respectively.
Specificity and sensitivity of MCM2, cyclin D1, and p16 for diagnosis of LMS are listed in Table 7. MCM2 >80% has a high sensitivity and specificity in diagnosing LMS. The only case for which MCM2 >80% is not sensitive enough to include as LMS, is a case of spindle cell LMS with focal epithelioid histologic features. The same case also shows a low p16 staining.
Using combined “MCM2 strong and diffuse (>80% of cells)” AND “p16 strong and diffuse (>80% of cells)” improves specificity. On the other hand if “MCM2 strong and diffuse >80%” OR “p16 strong and diffuse >80%” is considered, diagnostic sensitivity will be increased. In other words, p16>80% lacks sensitivity in our cohort and parallel testing with MCM2 can improve the overall sensitivity. Combination of p16 and MCM2 also demonstrates a high specificity.
Clinical, pathologic and staining patterns of all LMS cases are outlined in Table 8. Although overall survival time is not available for all our LMS patients, at least 54% (7/13) of patients had <2-year disease-free survival. Four cases had >5 years of overall survival. Interestingly, the LMSs in 2 of the patients with >5-year overall survival had lower than 10% positivity for p16 (Table 8). These cases presented at low stage (pT2a and pT1a) and showed diffuse and strong MCM2 positivity.
Our cohort also included a spindle cell LMS with focal epithelioid features that showed relatively low p16 and MCM2 positivity. This case presented at pT3b and was assumed palliative 2 mo after the initial diagnosis (case #10 at Table 8).
In the 32 cases included in nonmalignant categories, 2 cases showed strong and diffuse MCM2 staining and one case showed high p16 staining. No patient in the nonmalignant categories showed any recurrence in 3 to 5 yr of follow-up.
In this study, we described the reactivity profile of SMTs with 2 proliferation markers cyclin D1 and MCM2 in relation to the previously described markers p53, p16, and Ki67. Although a wide range of positivity is observed in LM, LBN, and STUMP cases, LMS cases consistently show low percentage of cyclin D1 and high percentage of MCM2 reactivity.
Despite the recent advances in our understanding of the molecular basis of smooth muscle malignant transformation, current day-to-day diagnostic challenges remain similar to the past. Excluding the diagnosis of LMS remains challenging in cases with borderline features. The most widely used ancillary tests that aid in the diagnosis remain to be Ki67 and p16.
However, diffuse p16 positivity is not entirely reliable in determining the nature of the neoplasm. First, significant increase in the expression of P16 (>50%) can be observed in atypical LMs, reported previously 9,34 and is consistent with our results. Also, in a fraction of cases, there is diffuse cytoplasmic staining of the p16, which makes evaluation of the nuclear positivity more challenging.
Cyclin D1 expression has been previously studied in uterine and nonuterine SMTs (please refer the Introduction section). Our results suggest that most uterine smooth muscle neoplasms demonstrate low labeling with cyclin D1 with low to moderate intensity. A wider range of positivity was observed in nonmalignant SMT compared to LMSs. Overall, the diagnostic value of low cyclin D1 level in LMS suffers from a low specificity (52%).
To our knowledge, this is the first description of MCM2 expression in uterine leiomyosarcomas. Yoshida et al. 35 in 2001 described increased MCM2 expression in high-grade leiomyosarcomas of soft tissue origin in comparison with their low grade counterparts. In our cohort, the sensitivity and specificity of strong and diffuse MCM2 for the diagnosis of LMS were 92% and 94%, respectively. In addition, we found that cleaner, exclusively nuclear MCM2 stain makes the interpretation easier. Although a fair number of our STUMP cases (5/12) showed >50% positivity, the intensity of stain is not diffusely strong in most cases.
The low number of cases with increased P53 expression in our study is consistent with the previous reports showing a limited mutation rate 9,33. We did not observe any p53 null phenotype amongst the LMS cases. While p53 expression >50% was not observed in LM, LBN, and STUMP, it was present in 38% of LMS cases, in line with previous studies 34.
In summary, our data suggests that MCM2 can be valuable in the differential diagnosis of LMS. Compared with p16, MCM2 proves to be more sensitive with comparable specificity. A combination of MCM2 with either p16 or Ki67 improves the specificity to 100% in our small study set. A larger sample size is required to validate these findings.
It is also important to note that our data is limited to the spindle cell LMS. Our LMS cases with focal epithelioid features did not follow the same trend in terms of p16 and MCM2 positivity. Further studies are needed to introduce the MCM2 staining signature in myxoid and epithelioid LMSs.
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