Introduction
Breast cancer (BC) is the most common malignancy affecting western women. About 5% to 10% of all BC cases are due to inheritance of a susceptibility allele, consistent with transmission in an autosomal dominant fashion, and a substantial proportion of these are due to germline mutations of the two major highly penetrant cancer susceptibility genes,
BRCA1 (OMIM, 113705; GenBank, U14680.1) [
1,
2] and
BRCA2 (OMIM, 600185; GenBank, U43746.1) [
3‐
5]. Hereditary BC is characterized by an early age of onset, high incidence of bilateral disease and frequent association with ovarian cancer (OC). An increased incidence of other malignancies, such as colorectal, prostate and pancreatic cancer is also observed among
BRCA1/2 mutation carriers [
6‐
8]. The proportion of described mutations in
BRCA1 relative to
BRCA2 varies between populations. With the exception of a strong
BRCA2 founder effect in Iceland [
9], however,
BRCA1 mutations are generally more frequently reported. In the majority (>80%) of families with BC and OC, the diseases are linked to the
BRCA1 gene. Conversely, in the majority (>75%) of families with male and female BC, the disease is linked to
BRCA2. Among families with female BC only, proportions of diseases due to mutations in
BRCA1,
BRCA2 and other genes are similar [
10].
A large number of distinct mutations, polymorphisms and genetic variants of uncertain significance in the
BRCA1 and
BRCA2 genes is described in the Breast Cancer Information Core Database (BIC Database) [
11]. The majority of mutations known to be disease causing result in a truncated protein due to frameshift, nonsense or splice site alterations. The spectrum of mutations varies between populations, with some showing a high frequency of unique mutations, for example in Italy [
12,
13], whereas a small number of founder mutations is more common in other ethnic groups. Notably, a single founder mutation in
BRCA2 (c.771_775del5; commonly referred to as 999del5) accounts for the majority of hereditary cancer cases in Iceland [
9], and three ancestral mutations (c.68_69delAG and c.5266dupC in
BRCA1 and c.5946delT in
BRCA2; 185delAG, 5382insC and 6174delT, respectively) were identified in the vast majority of families with a history of BC and OC in Ashkenazi Jews [
14]. Population specific mutations have also been described in the Netherlands [
15], Sweden [
16], France [
17], Spain [
18] and other countries [
19]. Two
BRCA1 founder mutations, c.5266dupC and c.181T>G (300T>G), occur most frequently in countries of Central and Eastern Europe [
20‐
25], including the Czech Republic [
26].
The aim of this study was to estimate the incidence, spectrum and possible clustering of disease phenotypes associated with BRCA1 and BRCA2 mutations in the Prague area and Central Bohemia. The analysis was performed in families with a history of BC/OC and in high-risk patients not selected on the basis of their family history of cancer.
Results
Mutation analysis was performed in 96 women from BC/OC families and in 55 non-familial patients. Analysis revealed 44 pathogenic cancer predisposing mutations, 6 of which have been previously reported elsewhere [
27]. Within 151 analyzed individuals, 35 (23.2%) carried a
BRCA1 mutation and 9 (6.0%) a
BRCA2 mutation.
The
BRCA1 mutations comprised 13 distinct alterations distributed widely across the coding sequence of the gene (Table
1). Twelve gene alterations caused a premature protein termination: eight were frame-shift alterations, with the majority of small deletions and insertions occurring in stretches of mononucleotide or dinucleotide repeats, and four were nonsense mutations. The c.181T>G mutation leading to a substitution of conserved cysteine 61 with glycine (p.Cys61Gly) in the RING finger domain of the BRCA1 protein was the only missense mutation identified in the gene.
Table 1
Pathogenic germline BRCA1 mutations in breast and ovarian cancer patients from the Prague area
F-24 | 2 | c.187_188delAG | c.68_69delAG | p.Glu23fsX39 | Sequencing | 1 | 35 | Colon (54) |
F-111 | 5 | c.300T>G | c.181T>G | p.Cys61Gly | Sequencing | 3 | 42 | Stomach (51) |
F-126 | 5 | c.300T>G | c.181T>G | p.Cys61Gly | Sequencing | 2 | 45 | - |
F-252 | 5 | c.300T>G | c.181T>G | p.Cys61Gly | Sequencing | 1 | 29 | - |
F-43 | 11 | c.1135delA | c.1016delA | p.Lys339fsX340 | PTT | 2 | 41 | Colon (50), lung (64) |
F-361 | 11 | c.1246delA | c.1127delA | p.Asn376fsX393 | PTT | 1 | 37 | Ovarian (52, 54, 55) |
F-21 | 11 | c.1806C>T | c.1687C>T | p.Gln563X | PTT | 1 (1) | 46 | Ovarian (43), melanoma (53) |
F-397 | 11 | c.1866A>Ta | c.1747A>Ta | p.Lys583Xa | PTT | - | - | Ovarian (39, 43) |
F-249 | 11 | c.2382G>T | c.2263G>T | p.Glu755X | PTT | 4 | 53 | Ovarian (41, 54) |
F-61 | 11 | c.2530_2531delAG | c.2411_2412delAG | p.Gln804fsX808 | PTT | 3 | 49 | - |
F-80 | 11 | c.3450C>T | c.3331C>T | p.Gln1111X | PTT | 2 | 52 | - |
F-305 | 11 | c.3819_3823del5 | c.3700_3704del5 | p.Val1234fsX1241 | PTT | 1 | 38 | Leukemia (67), lung (65) |
F-337 | 11 | c.3819_3823del5 | c.3700_3704del5 | p.Val1234fsX1241 | PTT | 3 | 44 | - |
F-347 | 11 | c.3819_3823del5 | c.3700_3704del5 | p.Val1234fsX1241 | PTT | 2 | 42 | - |
F-390 | 11 | c.3819_3823del5 | c.3700_3704del5 | p.Val1234fsX1241 | PTT | 1 | 42 | Lung (56), kidney (65) |
F-164 | 11 | c.3875_3878delGTCT | c.3756_3759delGTCT | p.Leu1252fsX1262 | PTT | 2 (1) | 42 | Ovarian (40, 43), stomach (?) |
F-245 | 12 | c.4284_4285delAG | c.4165_4166delAG | p.Ser1389fsX | HDA | 2 | 38 | Ovarian (44, 50), kidney (75) |
F-15 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 1 | 32 | - |
F-75 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 1 | 44 | Ovarian (?, ?) |
F-152 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 2 | 58 | Ovarian (72) |
F-185 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 | 51 | Colon (?) |
F-187 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 | 48 | Ovarian (56) |
F-194 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 4 | 43 | Ovarian (52) |
F-201 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 2 | 41 | - |
F-239 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 | 46 | Ovarian (41), stomach (?) |
F-243 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 (1) | 42 | - |
F-261 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 2 | 29 | - |
F-265 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 (1) | 52 | Ovarian (?) |
F-273 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 | 36 | Ovarian (42), colon (51, 56) |
F-331 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 1 (1) | 31 | Uterus (60), colon (64) |
F-342 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 2 | 50 | Ovarian (41), colon (?) |
F-368 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 4 (2) | 37 | Ovarian (40), kidney 78) |
F-370 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 2 | 49 | Ovarian (55) |
F-385 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 1 | 52 | Ovarian (61), colon (83), melanoma (79) |
F-387 | 20 | c.5385dupC | c.5266dupC | p.Gln1756fsX1829 | Sequencing | 3 (1) | 36 | - |
Of the 10 BRCA1 mutations observed only once in our series, the c.1747A>T nonsense mutation is a novel gene alteration (not reported to the BIC by June 2004) found in family 397 with two cases of OC diagnosed at the ages of 39 and 43. Four additional mutations (c.1016delA, c.3331C>T, c.1127delA and c.2263G>T) belong to rare gene alterations (with one to four entries in the BIC database), whereas the others (c.68_69delAG, c.1687C>T, c.2411_2412delAG, c.3756_3759delGTCT and c.4165_4166delAG) occur frequently in various European regions, including Central Europe.
Three recurrent mutations were found in 25 (71.4%) of the 35 women with detected alterations in BRCA1. The mutation c.5266dupC (5382insC) was a highly prominent mutation detected in 18 patients, which accounted for 51.4% of all identified alterations in BRCA1. The mutation c.3700_3704del5 found in four families was the second most commonly identified alteration, which contributed to 11.4% of mutations detected in BRCA1. The mutation c.181T>G (300T>G; p.C61G) identified in three families comprised 8.6% of detected mutations.
The
BRCA2 mutations included eight different gene abnormalities (Table
2). All alterations were localized to exon 11 and led to a truncated protein product: five were frameshift alterations and three were nonsense mutations. The mutation c.3939delC is a novel frameshift mutation, which results in a termination of translation at codon 1313. This mutation was detected in family 348 with two cases of OC diagnosed at the ages of 46 and 58. Both the c.3076A>T nonsense mutation (occurring in conjunction with the c.3075G>T missense mutation) and the frameshift mutation c.5238dupT belong to rare, infrequently reported gene alterations. Other identified mutations (c.2808_2811delACAA, c.3975_3978dupTGCT, c.5645C>A and c.5682C>G) occur frequently throughout Western Europe.
Table 2
Pathogenic germline BRCA2 mutations in breast and ovarian cancer patients from the Prague area
F-263 | 11 | c.3036_3039delACAA | c.2808_2811delACAA | p.Lys936fsX958 | PTT | 3 (1) | 41 | - |
F-279 | 11 | c.3303G>T; 3304A>T | c.3075G>T; 3076A>T | p.Lys1025Asn; p.Lys1026X | PTT | 1 | 50 | Ovarian (47, 50) |
F-348 | 11 | c.4167delCa | c.3939delCa | p.Tyr1313fsX | PTT | - | - | Ovarian (46, 58), lung (?) |
F-237 | 11 | c.4203_4206dupTGCT | c.3975_3978dupTGCT | p.Ala1327fsX1331 | PTT | 2 | 38 | Stomach (?) |
F-298 | 11 | c.5466dupT | c.5238dupT | pAsn1747fsX | PTT | 1 | 41 | Ovarian (59) |
F-304 | 11 | c.5873C>A | c.5645C>A | p.Ser1882X | PTT | 3 | 61 | Ovarian (49), colon (67) |
F-49 | 11 | c.5910C>G | c.5682C>G | p.Tyr1894X | PTT | 2 (1) | 45 | Lung (?) |
F-67 | 11 | c. 5991dupTa | c.5763dupTa | p.Ala1922fsX1923a | PTT | 1 | 48 | Ovarian (49), esophagus (?) |
F-327 | 11 | c. 5991dupTa | c.5763dupTa | p.Ala1922fsX1923a | PTT | 1 | 32 | - |
Recurrent mutations represented over two-thirds of all the BRCA1 mutations identified in this series. By contrast, the alterations in BRCA2 were mostly unique. The c.5763dupT mutation, which causes a premature termination of translation at codon 1923, appeared in two families and was the only recurrent alteration of the BRCA2 gene identified in this study. The mutation was first detected in family 67 with breast and ovarian cancer diagnosed before the age of 50. In the second unrelated family (F-327), only one affected woman who developed BC at the age of 32 was found. This gene alteration has since been reported once in Western Europe, as indicated in the BIC database.
Table
3 shows the prevalence of mutations in BC/OC families and in non-familial risk patients who had no reported family history of cancer. Pathogenic mutations were revealed in 35 (36.5%) of the 96 analyzed families. The incidence of mutations differed significantly between HBC (14/60; 23.3%) and HBOC (19/32; 59.4%) families (p = 0.0006). Two mutations were found in four HOC families.
Table 3
Frequencies of BRCA1 and BRCA2 mutations in relation to the classification of patients and families
Cases without a family history of breast and ovarian cancer | | | | |
Breast cancer before 36 | 31 | 3 (9.7) | 1 (3.2) | 4 (12.9) |
Bilateral breast cancer before 51 | 3 | 1 (33.3) | - | 1 (33.3) |
Breast and ovarian cancer | 7 | 1 (14.3) | - | 1 (14.3) |
Medullary breast carcinoma | 14 | 3 (21.4) | - | 3 (21.4) |
Breast cancer families (HBC) | | | | |
2 breast cancer cases | 25 | 5 (20.0) | 2 (8.0) | 7 (28.0) |
≥3 breast cancer cases | 35 | 6 (17.1) | 1 (2.9) | 7 (20.0) |
Total | 60 | 11 (18.3) | 3 (5.0) | 14 (23.3) |
Breast and ovarian cancer families (HBOC) | | | | |
1 breast cancer and 1 ovarian cancer | 5 | 1 (20.0) | 2 (40.0) | 3 (60.0) |
≥3 breast and ovarian cancer cases | 27 | 14 (51.9) | 2 (7.4) | 16 (59.3) |
Total | 32 | 15 (46.9) | 4 (12.5) | 19 (59.4) |
Ovarian cancer families (HOC) | | | | |
≥2 ovarian cancer cases | 4 | 1 (25.0) | 1 (25.0) | 2 (50.0) |
Non-familial patients included a group of 31 women diagnosed with BC between ages 22 years and 35 years without history of BC or OC in their family. Pathogenic germline mutations in predisposing genes were detected in four (12.9%) women. Three mutations were identified in BRCA1 (c.68_69delAG, c.181T>G and c.5266dupC) and one in BRCA2 (c.5763dupT). Screening of seven patients with both breast and ovarian cancer and no family history identified one mutation (14.3%) in the BRCA1 gene (c.5266dupC). The mutation c.5266dupC was also detected in a group of three patients (33.3%) with bilateral BC.
A high incidence of medullary carcinoma has been reported among women with
BRCA1-associated BC [
34]. We performed the analysis of
BRCA1 and
BRCA2 genes in 14 women with this histological tumor subtype and found three truncating mutations (21.4%) in exon 11 of the
BRCA1 gene. The c.3331C>T nonsense mutation was detected once, whereas the c.3700_3704del5 frameshift mutation was identified in two cases. No alteration in the
BRCA2 gene was found among these analyzed patients.
Discussion
In our study, 35 mutations in the BRCA1 and BRCA2 genes were detected in 96 BC/OC families. In addition, we found four pathogenic mutations in patients with early onset BC, one mutation in a case of a bilateral BC, one mutation in a woman with both BC and OC, and three mutations in women with a medullary breast carcinoma. The majority of mutations identified in our study lead to protein truncations. Although short coding exons of both BRCA1 and BRCA2 were analyzed by either direct sequencing or heteroduplex analysis, only the BRCA1 c.181T>G (c.300T>G; p.C61G) missense mutation and BRCA2 c.3075G>T (p.K1025N) missense alteration (present in conjunction with the K1026X nonsense mutation) of unknown clinical significance were observed. One novel mutation was found in BRCA1 (c.1747A>T); two novel mutations were identified in BRCA2 (c.3939delC and c.5763dupT).
The prevalence of inherited
BRCA1/2 mutations observed in different studies varies according to the ethnic origin of analyzed individuals, criteria of selection for genetic testing and techniques used for mutational screening. Although the techniques we applied in our study compromised our ability to detect missense mutations in regions of the
BRCA1/2 genes screened only by PTT, at present the majority of reported missense alterations are difficult to interpret with respect to potential clinical significance, as the effect of the amino acid substitution on protein function is not yet well understood. Conversely, most protein truncating
BRCA1/2 mutations are presumed to be pathogenic, thereby permitting women harboring such mutations to be provided appropriate clinical counseling and management. Our study may not have detected large genomic rearrangements encompassing regions outside the primer sets used for RT-PCR amplification. On the other hand, in the absence of rapid degradation of aberrant transcripts by nonsense-mediated mRNA decay [
35], any rearrangements encompassing the exons within the regions amplified from cDNA would have been observed as aberrantly sized PCR products, as has also been found in other studies [
36]. Further, the prevalence of genomic rearrangements varies between populations. To our knowledge, large deletions and rearrangements in
BRCA1/2 genes have not been reported in countries of Central and Eastern Europe. In our series, analysis of truncated RT-PCR products and sequencing of corresponding genomic fragments identified one case with the rearrangement that involved exons 21 and 22 of the
BRCA1 gene (Zikan, unpublished results). Despite the limitations of our approach, the prevalence of mutations in our group of high-risk families was comparable to that observed in Central Europe [
21,
23,
26]. Further, the distribution of germline
BRCA1/2 mutations in our high-risk families is consistent with a higher prevalence in the context of OC [
10]; mutations were detected significantly more frequently in HBOC and HOC families than in HBC families (58.3% of 36 versus 23.3% of 60; p = 0.0006).
Interestingly, both
BRCA1 and
BRCA2 mutationswere more prevalent in families with OC (Table
3). Of the 27
BRCA1 mutations detected in high-risk families, 11 were present in HBC families (18.3%), whereas 16 were identified in HBOC and HOC families (44.4%). Of the eight mutations detected in
BRCA2, three were present in HBC families (5%), relative to five in HBOC and HOC families (13.9%). This observation is in contrast to observations in larger series of examined families, where the risk of OC was significantly greater among women with
BRCA1 mutations compared to women with
BRCA2 mutations [
10,
37]. The higher prevalence of
BRCA2 mutations among families with OC in our study may be due to the preponderance of mutations identified in the ovarian cancer cluster region, and lend support to the increased risk of OC suggested to be conferred by mutations within this region relative to other
BRCA2 mutations [
38,
39].
Gayther
et al. [
40] have reported that mutations in the 3' third of the
BRCA1 gene are associated with a lower proportion of ovarian cancer. The border for this phenotype correlation was located at exon 13, between codons 1435 and 1443. Further studies provided proof for this genotype-phenotype correlation [
16,
41], although other authors failed to replicate this observation for
BRCA1 mutations [
17,
20]. Despite the higher occurrence of ovarian cancer in high-risk families with mutations located in exons 2 to 12, the relative frequency (12/37 cancer cases; 32.4%) did not differ significantly in our study from the frequency of ovarian cancer (11/51 cancer cases; 21.6%) in patients with mutations in exons 14 to 24 (p = 0.25).
The surprising finding of our investigation was a high predominance of recurrent mutations in the
BRCA1 gene, which contributed to a substantial proportion of hereditary BC and OC cases. The three repeatedly occurring mutations in
BRCA1 were detected in more than 56% of women with identified alterations in
BRCA1/2 genes and in more than 71% of women with alterations in
BRCA1. The most frequent mutation was c.5266dupC (5382insC) found in 15 (15.6%) of 96 analyzed BC/OC families and in 18 (40.9%) of 44
BRCA1/2 mutation positive patients. The occurrence of this mutation is comparable to that found in Polish [
22,
23,
25,
42] and Russian [
20] populations, but significantly higher than that described in Germany [
43] and Austria [
21]. The c.5266dupC mutation is the most prevalent
BRCA1 alteration in Europe and a geographic distribution of this mutation is consistent with its Baltic origin [
19]. The c.5266dupC allele also occurs at a high frequency (0.11%) in the Ashkenazi Jewish population [
14], although the 18 Czech patients carrying this gene defect did not report an Ashkenazi Jewish heritage. The other two recurrent mutations detected in our group of patients, c.3700_3704del5 and c.181T>G (300T>G), also belong to gene alterations that have been repeatedly detected in the Central European population [
23‐
26].
The
BRCA1 c.5266dupC and c.181T>G mutations are prevalent in Poland and in the Czech Republic, although spectra of mutations display significant differences in these countries [
26,
42]. The mutation c.4034delA (4153delA), contributing to 9.8% of mutations identified in
BRCA1 in Poland [
42], did not occur either in our or in Moravian families [
26]. We identified 10 unique mutations in
BRCA1, which suggests that the spectrum of alterations in this gene is more heterogeneous than that reported in Poland [
42].
In contrast to the BRCA1 gene, there were few recurrent mutations within BRCA2. With the exception of the c.5763dupT mutation detected in two unrelated individuals, each alteration identified in BRCA2 was found in only one family.
In a set of pathogenic mutations in
BRCA1 identified in Brno (Moravia) [
26], the prevalence of the three most common mutations (c.5266dupC, c.3700_3704del5 and c.181T>G) was 37.3% (22/59), 13.6% (8/59) and 10.2% (6/59), respectively, whereas in our group of patients, the prevalence of these alterations was 51.4% (18/35), 11.4% (4/35) and 8.6% (3/35). Small variations in the mutation spectra observed in both studies may be caused by limited sample size and may also reflect differences in the groups of patients selected for genetic testing.
The occurrence of
BRCA2 mutations was higher in Moravia (33%; 29/88 of mutation positive patients) than in our study (20.5%; 9/44 of mutation positive patients) and the spectrum of genetic alterations was completely different [
26]. The c.7910_7914del5 (8138_8142del5) and c.8537_8538delAG (8765_8766deAG) alterations found in Brno in 7 (24.1%) of the 29 families with detected mutations in
BRCA2, were not present in our group of patients. On the contrary, the mutation c.5763dupT, the only recurrent alteration of the
BRCA2 gene identified in our study, was not found in Moravia. A high frequency of unique
BRCA2 mutations may be characteristic of the examined Prague area and Central Bohemia, although the examination of a larger group of families is required to obtain valid results.
The incidence of
BRCA1/2 mutations in a group of Czech women with early onset non-familial BC was 12.9% (4/31; Table
3), whichsuggests that the age at diagnosis in patients with a negative family history is an important indicator for the presence of a pathogenic mutation and lends support to the screening of
BRCA1/2 genes in patients with early onset disease. In contrast to our findings, only 2% of non-familial patients had pathogenic germline
BRCA1 and
BRCA2 mutations in a group of patients in Great Britain who were diagnosed with BC at the age of 30 years or younger [
44]. A similarly low frequency of mutations was found in non-familial patients in Spain and Iran [
45,
46]. In our study, all patients carried the most common, easily detectable mutations of
BRCA1 or
BRCA2 that prevail in this region and can be associated with early onset cancer. A larger set of patients with early onset BC is currently under investigation to determine the incidence of mutations in this risk group.
Medullary carcinoma of the breast is not common (2% to 3%) in patients with
BRCA2 mutations and those with no known germline gene alteration [
47]. In
BRCA1-related BC, the incidence of typical medullary carcinoma was 19% (6/32) in a French study [
34], 8% (4/49) in a Dutch study [
48], but 0% in a Swedish series of 40
BRCA1-associated tumors [
49]. In our study, one medullary carcinoma was identified in a group of 22 patients (4.5%) with
BRCA1-related BC (F-252), whereas no tumor of this histological type was found in seven women with
BRCA2-associated tumors. We did, however, find three germline
BRCA1 mutations in a set of 14 patients (21.4%) with medullary breast carcinoma selected for examination regardless of the family history. These women did not belong to high-risk families and did not fulfill common criteria for genetic testing. One patient (Table
1; F-80) with a medullary carcinoma at age 50 had a sister affected with BC at age 54 and the other two (Table
1; F-305 and F-390) with medullary carcinomas at ages 38 and 42, respectively, were without a family history of breast or ovarian cancer. Our results are in agreement with studies of Eisinger
et al. [
34] who tested 18 cases of medullary carcinoma for mutations in
BRCA1 gene and found two (11%) harboring
BRCA1 mutations. Interestingly, these cases did not belong to high-risk families either. Indication of cases with medullary carcinoma for
BRCA1 testing, regardless of the family history, may be helpful in mutation screening. Examination of a larger group of patients with this histological type of carcinoma is required to determine the importance of this morphological parameter more exactly.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PP and MZ contributed equally to this work.