These authors have contributed equally to this work
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It is often difficult to histologically differentiate among endometrial dedifferentiated carcinoma (DC), endometrioid carcinoma (EC), serous carcinoma (SC), and carcinosarcoma (CS) due to the presence of solid components. In this study, we aimed to categorize these carcinomas according to The Cancer Genome Atlas (TCGA) classification using a small custom-made cancer genome panel (56 genes and 17 microsatellite regions) for integrated molecular diagnosis. A total of 36 endometrial cancer cases with solid components were assessed using IHC, next-generation sequencing (NGS), and the custom-made panel. Among 19 EC cases, six were categorized as MMR-deficient (MMR-d) and eight were classified as having a nonspecific molecular profile. Three EC cases were classified as
Dedifferentiated carcinoma (DC) is a rare endometrial cancer accounting for 2% of all endometrial cancers. It is composed of well differentiated endometrioid carcinoma (EC) and undifferentiated carcinoma [
Next-generation sequencing (NGS) has recently become a standard procedure for cancer genomic analysis [
All patients were registered at the Clinical Research of Cancer Gene Panel Analysis of Gynecologic Cancers Study, which was conducted from January 2019 to October 2021 at the Kagoshima University Hospital. The clinical samples used in this study were approved by the Ethics Committees for Clinical and Epidemiologic Research at Kagoshima University (approval number: 180215) and written informed consent was obtained from all participants. Among the 155 cases entered, 36 cases, including 16 G2 and 3 G3 EC cases, 3 DC cases, 4 CS cases, 9 SC cases, and 1 large cell neuroendocrine carcinoma (LCNEC) case with areas of solid proliferation were included in this study. Clear cell carcinoma (CCC) is considerably less frequent in the Gynecologic Cancers Study in our hospital, therefore, CCC cases were excluded from this study. No patients with undifferentiated carcinoma were included.
Resected tissues obtained by hysterectomy were fixed with 10% neutral phosphate-buffered formalin, routinely processed for paraffin embedding, and sectioned for hematoxylin and eosin (HE) staining, IHC, and NGS. Pathological diagnoses were made according to the World Health Organization (WHO) classification system [
The dedifferentiated parts of DC are positive for epithelial marker expression in scattered cells, and mesenchymal elements of CS are weakly positive or negative for these markers; hence, diffuse expression of keratins (AE1/AE3 and CAM5.2) and EMA was used to differentiate endometrial cancers [
The antibodies used for IHC are listed in Online Resource 1. MMR-proficient (MMR-p) was defined as positive nuclear staining for all MMR proteins (MLH1, PMS2, MSH2, and MSH6). MMR-d was defined as the complete absence of nuclear staining for any MMR proteins [
A cancer panel was redesigned by making a minor modification of the previous panel [
Sequence data were annotated as previously described using the Qiagen Web Portal service (
To calculate the copy number (CN) from the baseline data used for counting correction per amplicon, the number of reads sequenced in each amplicon was counted, and the reads per million (RPM) value was determined. The RPM coefficient of variation (CV), mean, and median value per amplicon in at least 100 FFPE samples were calculated. The RPM median of the amplicons with a CV < 0.34 and mean > 10 was set as the baseline. The number of reads sequenced in each 56‐panel amplicon was counted in the sample to calculate the CN of each sample. The baseline ratios {log2 ratio [ = log2 (sample RPM/baseline RPM median)]} in amplicons that satisfied the conditions of CV < 0.34 and mean >10 were counted, and the overall SD and median value of the log2 ratio for each gene was calculated. The genes with a log2 ratio median value >2 SD were categorized as amplified, while the genes with a log2 ratio median value <−2 SD were categorized as gene loss [
Missense mutations with more than 10% variant allele frequency, including nonsynonymous mutations and internal deletions, were counted as somatic mutations. The TMB was calculated as the number of single nucleotide variants million per base pairs (Mbp) of the DNA sequence [
All values were expressed as the mean ± standard error. Significant differences were identified using student’s or Welch’s
The IHC results regarding the MMR proteins, p53, ARID1A, PTEN, WT-1, ER, CycD1, and vimentin are summarized in
Summary for histological diagnosis, expression of MMR proteins and p53, and genomic profile in 36 cases.
Case no. | Age | Histological diagnosis | MMR | p53 | MSI | TMB | SNV and delins mutations | TCGA type | ||||||||
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1 | 60 | EC (G2) | pro. | wt | Low | High | NSMP | |||||||||
2 | 54 | EC (G1) | pro. | wt | Low | U-high | p.Val63Met | c.2389G>T | p.Val411Leu | POLEmut | ||||||
EC (G2) | Low | U-high | c.2389G>T | p.Val411Leu | ||||||||||||
3 | 67 | EC (G2) | pro. | wt | Low | Low | NSMP | |||||||||
4 | 68 | EC (G2) | def. | wt | High | High | MMR-d | |||||||||
5 | 63 | EC (G2) | pro. | wt | Low | Low | NSMP | |||||||||
6 | 64 | EC (G3) | def. | wt | High | High | p.Arg202Cys | MMR-d | ||||||||
7 | 59 | EC (G2) | def. | wt | High | High | p.Pro991Leu | MMR-d | ||||||||
8 | 55 | EC (G2) | pro. | wt | Low | Low | NSMP | |||||||||
9 | 60 | EC (G2) | def. | wt | High | High | p.Phe22fs | MMR-d | ||||||||
10 | 61 | EC (G2) | pro. | wt | Low | Low | NSMP | |||||||||
11 | 78 | EC (G3) | def. | wt | High | U-high | p.Arg175His | p.Met491fs | MMR-d | |||||||
12 | 60 | EC (G2) | pro. | wt | Low | Low | NSMP | |||||||||
13 | 55 | EC (G2) | pro. | mt | Low | U-high | p.Arg213* | p.Arg107Trp |
p.Asp758Asn | p.Arg298Gln |
p.Pro286Ser |
p.Ser579Asn |
POLEmut | |||
14 | 65 | EC (G2) | gld. | pro. | mt | Low | Low | p.Arg273Cys | p53mut | |||||||
solid | pro. | mt | Low | Low | ||||||||||||
15 | 68 | EC (G2) | gld. | pro. | wt | Low | Low | c.903+2T>A | NSMP | |||||||
solid | pro. | wt | Low | High | c.903+2T>A | |||||||||||
16 | 55 | EC (G3) | pro. | mt | Low | Low | c.993+1G>T | p53mut | ||||||||
17 | 73 | EC (G2) | gld. | pro. | wt | Low | Low | NSMP | ||||||||
solid | pro. | wt | Low | Low | ||||||||||||
18 | 53 | EC (G2) | gld. | def. | wt | High | Low | p.Glu613_Phe614del | p.Arg678Trp | MMR-d | ||||||
solid | def. | wt | High | U-high | p.Glu613_Phe614del | c.3582+5C>T | p.Arg678Trp | |||||||||
19 | 56 | EC (G2) | gld. | pro. | wt | Low | U-high | p.Glu483* | p.Arg1201Gln |
p.Pro286Arg | POLEmut | |||||
solid | pro. | wt | Low | U-high | p.Glu483* | p.Arg240Gln |
p.Pro286Arg | |||||||||
20 | 51 | DC | EC (G1) | High | High | p.Asp289dup | MMR-d | |||||||||
De | def. | wt | High | High | p.Ser388Phe | p.Asp289dup | p.Ala288Val | |||||||||
21 | 57 | DC | EC (G3) | def. | wt | High | High | p.Lys373fs | p.Ala288Val |
MMR-d | ||||||
EC (G 1) | High | High | p.Asn287fs | p.Tyr581His | p.Trp371Cys | |||||||||||
De | High | High | p.Lys373fs | p.Ala69Thr | p.Tyr581His | p.Trp371Cys | ||||||||||
22 | 71 | DC | EC (G1) | High | High | p.Ser577Ser |
MMR-d | |||||||||
De | def. | wt | High | High | p.Ser577Ser |
p.Asp1013fs | ||||||||||
23 | 64 | SC | pro. | mt | Low | Low | p.Arg249Ser | p53mut | ||||||||
24 | 67 | SC | pro. | mt | Low | Low | p.Gly245Ser |
p53mut | ||||||||
25 | 57 | SC | pro. | mt | Low | Low | p.Arg248Gln | p53mut | ||||||||
26 | 63 | SC | Ad | pro. | mt | Low | Low | p.Ile255Ser | p53mut | |||||||
Spn | Low | Low | p.Ile255Ser | |||||||||||||
27 | 72 | SC | def. | mt | High | U-high | p.Arg306* | p.Arg352His |
p53mut/ MMR-d | |||||||
28 | 75 | SC | pro. | mt | Low | Low | p.Ser241Phe | p53mut | ||||||||
29 | 70 | SC | pro. | mt | Low | Low | p.Gln144* | p53mut | ||||||||
30 | 65 | SC | pro. | mt | Low | Low | c.817C>T | p53mut | ||||||||
31 | 77 | SC | pro. | mt | Low | Low | p.Ala276Gly | p53mut | ||||||||
32 | 80 | CS | pro. | mt | Low | Low | p.Asn239Ser | p53mut | ||||||||
33 | 58 | CS | pro. | mt | Low | Low | p.Cys176Phe |
p53mut | ||||||||
34 | 63 | CS | Car | pro. | wt | Low | Low | NSMP | ||||||||
Src | Low | Low | ||||||||||||||
35 | 64 | CS | Car | def. | wt | High | High | MMR-d | ||||||||
Src | High | High | ||||||||||||||
36 | 63 | LCNEC | pro. | mt | Low | Low | p.Phe113Ser | p53mut |
EC, endometrioid carcinoma; gld., glandular part; solid, solid part; DC, dedifferentiated endometroid carcinoma; CS, carcinosarcoma; SC, serous carcinoma; LCNEC, large cell neuroendocrine carcinoma; De, dedifferentiated part; Ad, adenocarcinoma part; Car, carcinomatous part; Sar, sarcomatous part; Spn, spindle cell part; MMR, mismatch repair protein; MSI, macrisatellite instability; TMB, tumor mutation burden; CNA, copy number alteration; Amp, amplification; SNV, single nucleotide variant; U-high, ultra high; pro., proficient; def., deficient; wt, wild-type; mt, mutation; PLOEmut, POLE, mutation; MMR-d, MMR-deficient; NSMP, no specific molecular profile; p53mut, p53 mutation.
Representative histology and IHC of p53 and MMR proteins. Representative HE sections from G2 EC (no. 2) showing well differentiated glandular and less differentiated solid areas. A few p53-positive tumor cells are observed, indicating wt p53 expression. All four MMR proteins are diffusely positive. The dedifferentiated part of DC (no. 22) exhibits wt p53 expression pattern and loss of MLH1 and PMS2 expression. Stromal lymphocytes also show a positive reaction as an internal control. The sarcomatous element of CS (no. 34) shows diffuse staining for p53 and all four MMR proteins. SC (no. 29) shows complete loss of p53 expression in both glandular and solid elements. The MMR protein expression is well preserved. CS, carcinosarcoma; SC, serous carcinoma; HE, hematoxylin and eosin (Original magnification: ×200); IHC, immunohistochemistry. (Original magnification: p53 × 400, MMR ×200).
The TMB values of wildtype and mutated
Values of MSI and TMB in endometrial cancers.
Genomic profiles of 36 cases are shown in
MSI and TMB in each histological subtype.
n | MSI score | EC | CS | SC | |
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DC | 3 | 39.2 ± 5.5 |
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Ns |
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EC | 16 | 16.5 ± 5.0 | Ns | ns | |
CS | 4 | 20.5 ± 10.2 | ns | ||
SC | 9 | 7.9 ± 3.6 | |||
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DC | 3 | 68.7 ± 4.6 | ns |
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EC | 16 | 43.2 ± 6.9 | Ns |
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CS | 4 | 23.2 ± 11.6 | ns | ||
SC | 9 | 21.8 ± 9.9 |
The MSI, and TMB, scores are presented as mean ± standrad error. EC with POLE, mutation is excluded; MSI, microsatelliete instability; TMB, tumor mutation burden; EC, endometrioid carcinoma; CS, carcinosarcoma; SC, serous carcinoma; ns, not significant.
MSI and TMB in EC.
EC | n | MSI score | TMB score |
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MMR-p | 10 | 2.3 ± 0.6 | 26.3 ± 3.5 |
MMP-d | 6 | 40.4 ± 3.7 | 66.1 ± 12.8 |
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<0.0001 | 0.012 |
MSI, microsatellite instability; TMB, tumor mutation burden; EC, endometroid carcinoma; MMR-p, mismatch repair-proficient; MMR-d, mismatch repair-deficient.
Among the four CS cases (no. 32–35), two contained mutations in
For the tumors composing of heterogenous elements, the genomic profile was evaluated separately for each element suing two or three FFPE sections in six cases of EC (no. 2, 14, 15, and 17–19), two of CS (nos. 34 and 35), one of SC (no. 26), and three of DC (nos. 20, 21, and 22). The genomic profiles were not exactly matched between the heterogenous elements, but were not so different as much as TCGA classification was revised (Online Resource 4).
The correlation between the genomic and IHC analyses of ARID1A and PTEN is presented in Online Resource 5. Most CS (3/4) and SC cases (8/9) exhibiting high ARID1A expression (>90% of the positive area) had no mutations. Fourteen EC cases (14/19) harbored frameshift, nonsense mutations or splice variants. Among these, eight cases exhibited a loss of ARID1A expression (<5% of the positive area). PTEN expression was lost in one CS case with gene alterations (1/4). Although SC cases did not display
Based on TCGA classification [
Distribution of TCGA classification*.
EC | DC | CS | SC | LCNEC | |
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POLEmut | 3 | 0 | 0 | 0 | 0 |
MMR-d | 6 | 3 | 1 | 1 | 0 |
p53mut | 2 | 0 | 2 | 8 | 1 |
NSMP | 8 | 0 | 1 | 0 | 0 |
36 | 19 | 3 | 4 | 9 | 1 |
*Case no. 27, which exhibits both p53mut and MMR-d features, is categorized as p53mut type. EC, endometrioid carcinoma; DC, dedifferentiated edometrioid carcinoma; CS, carcinosarcoma; SC, seous carcinoma; LCNEC, neuroendocrine carcinoma; PLOEmut, POLE, mutation; MMR-d, mismatch repair-deficient; NSMP, no specific molecular profile; p53mut, p53 mutation.
Our study indicated that NGS-based genomic analysis using the custom-made small panel could be used to evaluate TMB and MSI and for the detection of gene mutations, thus aiding in the categorization of endometrial cancers with solid proliferation according to TCGA classification.
DC comprises well differentiated EC and undifferentiated carcinoma. Therefore, it is often difficult to differentiate DC from CS and EC. Differential diagnosis by HE staining alone is challenging when CS tissues exhibiting only subtle spindle cells or ambiguous heterologous sarcomatous elements, such as chondrosarcoma and rhabdomyosarcoma, and less evident serous morphology are involved [
We found that DC cases typically harbored mutations in
Using IHC as a tool for p53 and MMR protein analysis may facilitate differential diagnosis of DC and CS, as demonstrated by a recent study [
EC comprises glandular and solid (>5%) areas diagnosed as G2 or G3 EC, based on the percentage of the solid components and nuclear atypia [
In accordance with previous studies [
Compared with IHC-based analyses of MMR proteins, characteristic IHC profiles are not known for being used for the differential diagnosis of
Overall,
As endometrial LCNEC also demonstrates undifferentiated solid elements, LCNEC should be considered in the differential diagnosis of endometrial cancer with solid proliferation. However, LCNEC diagnosis is not challenging due to its distinct IHC profile [
In summary, the feasibility of using a small NGS cancer panel to facilitate the molecular categorization of endometrial DC, SC, CS, and EC with solid proliferation was investigated. Genomic analyses that detect alterations in TMB, MSI, and gene mutations including
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
The studies involving human participants were reviewed and approved by The Ethics Committees for Clinical and Epidemiologic Research at the Kagoshima University. The patients/participants provided their written informed consent to participate in this study.
YK and IK summarized the pathological and genomic data; TA analyzed and interpreted the sequencing data; KT contributed as a pathologist; SHY, MK, and ST summarized the clinical data; IS, SN, and SEY contributed to genomic data analysis; AT and HK organized the study and wrote the article; and all authors read and approved the final manuscript.
SN and IS are employed by Mitsubishi Space Software.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors greatly appreciate the excellent technical assistance of Orie Iwaya and Mai Tokudome at the Department of Pathology, Kagoshima University Graduate School of Medical and Dental Sciences.
The Supplementary Material for this article can be found online at: