Study selection
After removing the duplicates, we screened 651 documents, 649 of which came from electronic databases, and two from grey literature (Figure 1).(33, 34) Of them, 167 were assessed for eligibility in full-text in English, German, Chinese, Russian, and Turkish languages with the aid of the Google Translator tool, and finally, 65 were included in the review, as described. The main reasons for the exclusion were the inability to locate the article/unavailability of its full-text version (31), wrong outcomes (20), and duplicated reports (14).
Study Characteristics
Included studies were published between 1990 and 2020, 64 of them in English and one in Russian.(35) Twenty-six studies were case series, 26 were retrospective cohorts, six were cross-sectional studies, three were case-control studies, three were prospective cohorts, and one study was a mixed cohort (see Additional file 4).
As for their region of origin, 22 studies were from Europe, 18 were from Asia Pacific Region, 14 were from the Americas, ten were from South-Eastern Asia, and one study was from Eastern Mediterranean WHO Region (Additional file 5).
Outcomes, Assays, and Measurements
The 65 studies included in the review (N = 5237) were classified into three overlapping categories (Fig. 2, Table 1). The first subset comprised 62 studies that reported on the prevalence of HER2 overexpression by IHC (N = 5076). Fifty-four of them (N = 4399) reported on IHC only. In the remaining eight studies (N = 677) ISH was also performed in addition to IHC (N = 406).
Table 1. Main characteristics of included studies.
Author
|
Year of publication
|
Country
|
Histology
|
Method Used
|
ASCO/CAP compliance
|
N Analyzed IHC
|
% 3+ IHC
|
N Aalized by ISH
|
% Positive by ISH
|
Shi(36)
|
2020
|
China
|
Ns
|
IHC,FISH
|
Yes
|
209
|
18.2
|
209
|
6.2
|
Varshney(47)
|
2020
|
India
|
Sq, Ns
|
IHC
|
Yes
|
38
|
21.1
|
0
|
|
Wong(62)
|
2020
|
China
|
Ns
|
IHC
|
Yes
|
14
|
21.4
|
0
|
|
Nakamura(45)
|
2019
|
Japan
|
Ns
|
IHC, DISH
|
Yes
|
13
|
0
|
4
|
25.0**
|
Rahmani(69)
|
2018
|
Sudan
|
Sq, Ns
|
IHC
|
No
|
65
|
43.1
|
0
|
|
Kumari Mitra(34)
|
2018
|
India
|
Sq, Ns
|
IHC
|
Yes
|
30
|
10
|
0
|
|
Bajpai(50)
|
2017
|
India
|
Sq, Ns
|
IHC
|
Yes
|
43
|
4.7
|
0
|
|
Halle(51)
|
2017
|
Norway
|
Sq, Ns
|
IHC
|
Yes
|
292
|
20.8
|
0
|
|
Martinho(70)
|
2017
|
Brazil
|
Sq, Ns
|
IHC
|
No
|
170
|
53.5
|
0
|
|
Ueda(23)
|
2017
|
Japan
|
Ns
|
IHC
|
No
|
43
|
20.9
|
0
|
|
Xiang(44)
|
2017
|
China
|
NA
|
IHC, FISH
|
Yes
|
157
|
2.5
|
8
|
25.0**
|
Carleton(48)
|
2016
|
UK
|
Ns
|
IHC
|
Yes
|
26
|
3.8
|
0
|
|
Sarwade(33)
|
2016
|
India
|
Sq, Ns
|
IHC
|
Yes
|
41
|
7.3
|
0
|
|
Nimisha Sharma(52)
|
2016
|
India
|
Sq, Ns
|
IHC
|
Yes
|
25
|
4.0
|
0
|
|
Fukazawa(71)
|
2014
|
Brazil
|
Sq
|
IHC
|
No
|
179
|
16.2
|
0
|
|
Nishio(53)
|
2014
|
Japan
|
Sq, Ns
|
IHC
|
Yes
|
204
|
4.9
|
0
|
|
Vosmik(54)
|
2014
|
Czech Rep
|
Sq
|
IHC
|
Yes
|
70
|
0
|
0
|
|
Barbu(49)
|
2013
|
Romania
|
Ns
|
IHC
|
Yes
|
13
|
23.1
|
0
|
|
Conesa-Zamora(38)
|
2013
|
Spain
|
Sq
|
IHC, FISH
|
Yes
|
32
|
3.1
|
32
|
0
|
Khalimbekova(35)
|
2013
|
Russia
|
Clear cell
|
IHC
|
Yes
|
14
|
0
|
0
|
|
Ueno(43)
|
2013
|
Japan
|
Clear cell
|
IHC, FISH
|
Yes
|
13
|
23.1
|
8
|
12.5
|
Sukpan(72)
|
2011
|
Thailand
|
NE
|
IHC
|
No
|
100
|
2.0
|
0
|
|
Perez -Regadera(73)
|
2010
|
Spain
|
Sq, Ns
|
IHC
|
No
|
136
|
23.5
|
0
|
|
Gupta(55)
|
2009
|
India
|
Sq, Ns
|
IHC
|
Yes
|
65
|
27.6
|
0
|
|
Lesnikova(39)
|
2009
|
Denmark
|
Sq, Ns
|
IHC, CISH
|
Yes
|
136
|
0.7
|
136
|
3.7
|
Yamashita(74)
|
2009
|
Japan
|
Sq
|
IHC
|
No
|
57
|
24
|
0
|
|
Shen(56)
|
2008
|
China
|
Sq
|
IHC
|
Yes
|
53
|
0
|
0
|
|
Carreras(75)
|
2007
|
Spain
|
Sq
|
IHC
|
No
|
10
|
50
|
0
|
|
Panek(58)
|
2007
|
Poland
|
Sq, Ns
|
IHC
|
Yes
|
298
|
7.8
|
0
|
|
Protrka(76)
|
2007
|
Serbia
|
Sq
|
IHC
|
No
|
13
|
46.2
|
0
|
|
Fuchs(46)
|
2007
|
Germany
|
Sq
|
IHC, FISH
|
No
|
78
|
ND
|
ND
|
21.8*
|
Califano(77)
|
2006
|
Italy
|
Sq, Ns
|
IHC
|
No
|
65
|
0
|
0
|
|
Kuroda(25)
|
2006
|
Japan
|
Glassy cell
|
IHC
|
No
|
11
|
45.4
|
0
|
|
Ravazoula(78)
|
2006
|
Greece
|
Sq
|
IHC
|
No
|
42
|
19.0
|
0
|
|
Kim(57)
|
2005
|
Korea
|
Sq, Ns
|
IHC
|
Yes
|
258
|
0.4
|
0
|
|
Tangjitgamol(79)
|
2005
|
USA
|
NE
|
IHC
|
No
|
24
|
0
|
0
|
|
Chavez -Blanco(37)
|
2004
|
Mexico
|
Sq, Ns
|
IHC, FISH
|
Yes
|
35
|
2.9
|
4
|
0
|
Graflund(80)
|
2004
|
Sweden
|
Sq, Ns
|
IHC
|
No
|
172
|
5.2
|
0
|
|
Rosty(41)
|
2004
|
France
|
Sq, Ns
|
IHC, FISH
|
No
|
82
|
2.4
|
5
|
0
|
Bellone(59)
|
2003
|
USA
|
ND
|
IHC
|
Yes
|
10
|
20.0
|
0
|
|
Dellas(61)
|
2003
|
Switzerland
|
Ns
|
IHC
|
Yes
|
22
|
0
|
0
|
|
Heller(60)
|
2003
|
USA
|
Sq
|
IHC
|
Yes
|
24
|
0
|
0
|
|
Niibe(81)
|
2003
|
Japan
|
Sq
|
IHC
|
No
|
21
|
42.8
|
0
|
|
Kedzia(82)
|
2002
|
Poland
|
Sq
|
IHC
|
No
|
47
|
4.3
|
0
|
|
Lee(83)
|
2002
|
Korea
|
Ns
|
IHC
|
No
|
37
|
29.7
|
0
|
|
Bhadauria(84)
|
2001
|
India
|
Sq
|
IHC
|
No
|
50
|
26.0
|
0
|
|
Leung (26)
|
2001
|
China
|
Ns
|
IHC
|
No
|
78
|
87.2
|
0
|
|
Ngan(85)
|
2001
|
China
|
Sq
|
IHC
|
No
|
101
|
19.8**
|
0
|
|
Straughn(86)
|
2001
|
USA
|
NE
|
IHC
|
No
|
16
|
0
|
0
|
|
Chang(87)
|
1999
|
China
|
Sq
|
IHC
|
No
|
56
|
46.4
|
0
|
|
Kersemaekers(88)
|
1999
|
Netherlands
|
Sq, Ns
|
IHC
|
No
|
132
|
9.1
|
0
|
|
Laksmi(89)
|
1999
|
India
|
Sq
|
IHC
|
No
|
166
|
34.9
|
0
|
|
Mark(40)
|
1999
|
USA
|
Sq, Ns
|
FISH
|
No
|
0
|
|
23
|
8.7
|
Nevin(90)
|
1999
|
UK
|
Sq, Ns
|
IHC
|
No
|
126
|
38.1
|
0
|
|
Nishioka(91)
|
1999
|
UK
|
Sq, Ns
|
IHC
|
No
|
107
|
32.7
|
0
|
|
Sharma(42)
|
1999
|
India
|
Sq
|
FISH
|
No
|
0
|
|
60
|
36.6
|
Mandai(92)
|
1997
|
Japan
|
Sq, Ns
|
IHC
|
No
|
88
|
38.6
|
0
|
|
Ndubisi(93)
|
1997
|
USA
|
Sq, Ns
|
IHC
|
No
|
150
|
22.7
|
0
|
|
Kristensen(94)
|
1996
|
Norway
|
Glassy cell
|
IHC
|
No
|
132
|
12.1
|
0
|
|
Nakano(95)
|
1996
|
Japan
|
Sq
|
IHC
|
No
|
52
|
46.2
|
0
|
|
Costa(96)
|
1995
|
USA
|
Ns
|
IHC
|
No
|
82
|
39.0
|
0
|
|
Kihana(97)
|
1994
|
Japan
|
Ns
|
IHC
|
No
|
44
|
25.0
|
11
|
|
Oka(98)
|
1994
|
Japan
|
Sq, Ns
|
IHC
|
No
|
192
|
19.3
|
0
|
|
Hale(99)
|
1992
|
UK
|
Sq, Ns
|
IHC
|
No
|
62
|
38.7
|
0
|
|
Berchuck(100)
|
1990
|
USA
|
Sq, Ns
|
IHC
|
No
|
33
|
9.1
|
0
|
|
*Among all included patients. **Among cases 2+ by immunohistochemistry.
Abbreviations: USA = The United States of America, Czech Rep = The Czech Republic, UK = The United Kingdom of Great Britain and Northern Ireland, Sq = squamous, Nsq = non-squamous, NE = neuroendocrine, IHC = immunohistochemistry, FISH = fluorescence in situ hybridization, CISH = chromogenic in situ hybridization, DISH = dual in situ hybridization.
The second subset encompassed ten studies that reported the rates of HER2 amplification by ISH (N = 489).(36–45) Eight of them, besides, determined the HER2 overexpression by IHC and two studies (N = 83) used ISH as the only method.(40, 42) Among eight studies that reported results of both IHC and ISH, six studies (N = 394)(36–39, 41, 43) performed ISH in patients not previously selected by IHC and two studies (N = 170)(44, 45) only in those patients with equivocal results (2+) of IHC testing.
In the third subset, there was only one study (N = 78).(46) It reported the prevalence of the HER2 positivity by IHC tiebreaking the equivocal cases (2+) by ISH, but positivity rates by each method were not provided separately (Fig. 2). No study performed IHC in patients pre-selected by ISH.
Of 11 studies that assessed HER2 positivity using ISH, nine performed FISH, one study chromogenic ISH (CISH), and the other one dual ISH (DISH) technique (Additional file 6).
Of 62 studies reporting on HER2 positivity prevalence by IHC, 26 (41.9%) used the positivity definition and grading criteria compatible with ASCO/CAP 2007, 2013, or 2018 guidelines and have been classified as ASCO/CAP compliant, while the remaining 36 (58,1%) studies as ASCO/CAP non-compliant. (Fig. 3A, Table 1). Among ASCO/CAP compliant studies, five used the 30% positivity cut-off (ASCO/CAP 2007)(35, 43, 47–49) and 19 studies the 10% cut off (ASCO/CAP 2013 or 2018).(33, 34, 36, 37, 39, 44, 45, 50–62)
Nine out of 11 studies that used ISH followed ASCO/CAP compliant or slightly more stringent positivity criteria (see Additional file 6).
Subjects
The subjects’ median age was 49.0 years, (interquartile range [IQR] 45.0 – 51.0). Data on the tumor stage was available from 56 studies. Five studies included women with stage I only, 11 studies with stages I and II, two studies with stage III only. The remaining studies contained a mixture of several stages. Median HPV positivity rate was 76.5%, (IQR: 57.3% to 90.3%). For more detailed information on patients’ clinical features see Additional file 7.
Tumors and samples
In 14 studies (28.6%) the tissue for analyses was obtained by biopsy, in 25 (51.0%) by surgery, and the remaining ten studies (20.4%) used samples from either biopsy or surgery combined in varied proportions. Of them, the primary tumor was the exclusive sampling site in 41 (91.1%) of studies. In the residuary 4 (8.9%) studies, aside from the primary, the samples from nodes or either distant or local recurrences were included. No study provided sampling-to-fixation and fixation-to-assay times.
Data on histologic subtypes were available from 63 (96.9%) of studies. Eighteen studies (28.6%) included patients with squamous carcinoma, 18 (28.6%) non-squamous histology, and 27 (42.9%) studies with both.
Risk of bias assessment.
The risk of bias appraisal in studies is summarized in Additional file 8. In the tool utilized, the two domains of the potential bias most frequently involved were “outcomes measurements“(60%) and ”outcome assessment by two independent pathologists” (75%).
The funnel plots for publication bias assessment are shown in Additional files 3A, 3B, and 3C. No significant asymmetry has been detected neither in the entire set of included studies (IHC, p = 0.769; ISH p = 0.543) nor in the subset of ASCO/CAP compliant ones, p = 0.936, suggesting the absence of substantial bias.
Immunohistochemistry
Overall, the estimated pooled prevalence of HER2 overexpression was 17.0% (95% confidence interval [95%CI]: 11.7% to 23.0%), I2 = 96% (Additional file 9).
If only studies that used an ASCO/CAP compliant IHC method (26 studies, N=2135) were included, under the random-effects model the estimated pooled prevalence of HER2 overexpression was 5.7%, (CI 95%: 1.5% to 11.7%), I2 = 87% (Figure 3A).(33-39, 43-45, 47-62) In the subset of studies judged ASCO/CAP non-compliant (36 studies, N=2941), the estimated pooled prevalence of HER2 overexpression was 27.0%, (IC 95%: 19.8% to 34.8%), I2 = 96% (Figure 3A). In the mixed-effects model, the difference between these ASCO/CAP compliant and non-compliant subgroups was statistically significant (p < 0.001). The amount of heterogeneity accounted for (R2) has been estimated at 26.8%. Thus, for further analyses, we only considered the subgroup of ASCO/CAP compliant studies. As there was no statistically significant difference in the pooled prevalence of HER2 overexpression between subsets of studies that used ASCO/CAP 2007 and 2013/2018 cut-offs, p = 0.11 (not shown), we pooled them together for subsequent analyses.
As significant heterogeneity in the ASCO/CAP compliant subgroup persisted, we conducted influence and moderator analyses. Although two studies have been identified as outliers, they were not judged influential cases since their removal neither significantly shifted the summary proportion nor markedly reduced the heterogeneity (Additional file 10).(51, 55)
Moderator analyses
In the subgroup of the squamous histology (12 studies, N=1018) the estimated pooled prevalence of HER2 overexpression was 4.1% (CI 95%: 0.6% to 9.8 %), I2 = 91%, while in the non-squamous carcinoma studies (15 studies, N=467) it was 10.3% (CI 95%: 3.6 to 19.2 %), I2 = 73%. The differences were not statistically significant neither if all the studies were considered, p = 0.054, R2 = 11%, (Figure 4A), nor if the analysis was restrained to only those studies that included both histologic subtypes, p = 0.12, R2 = 5.6% (Figure 4B).
No statistically significant relationship has been observed between pooled HER2 overexpression rate and predictor variables geographic region (p = 0.40), primary antibody brand (p = 0.051), year of study publication (p = 0.067), study size (p = 0.871), and the proportion of the HPV positive tumors (p = 0.842). See Figures 5 - 7.
In situ Hybridization
Of 10 studies reporting on the proportion of HER2 positivity by ISH, two were excluded from the pooled analysis as they determined HER2 in patients with equivocal (2+) results of IHC.(44, 45) In the remaining eight studies of unselected patients, the estimated pooled prevalence of HER2 amplification was 5.9% (CI 95%: 1.9% to 11.3%), I2 = 84%. (Figure 3B)
In the subset of ASCO/CAP compliant studies the estimated pooled prevalence of HER2 amplification was 1.2% (CI 95% 0.0% to 5.8%) I2 = 0%.(36-39, 41, 43) compared to 24.9% (IC 95% 12.6% to 39.6%), I2 = 86%. among ASCO/CAP non-compliant ones (40, 42) The difference was statistically significant, p = 0.004.
Two studies reported ISH positivity rates among HER2 2+ tumors.(44, 45). Two out of eight (25%) and one out of four (25%) patients respectively were positive by ISH.
Special histologic subtypes
Varying degrees of HER2 overexpression have been observed in most histologic subtypes, except for mesonephric and perhaps neuroendocrine carcinomas (Figure 8, Additional file 11).
Micro-invasive carcinoma
Among 103 microinvasive carcinoma samples analyzed by Kim et al. using IHC, there was no HER2 positive case.(57)
Tumor stage and HER2 positivity prevalence
A meaningful and reliable analysis of the relationship between tumor stage and HER2 positivity prevalence was not possible to undertake as most of them included a mixture of several stages in varied, often unknown proportions not always providing the mapping of HER2 status to the tumor stage of the participants.
The main findings of the review are summarized in Table 2.
Table 2
Summary of the main findings of the review.
Grouping
|
Estimated HER2 Pooled Prevalence
(95% CI)
|
№ of participants
(studies)
|
P-value
|
Risk of bias*
|
All studies
|
|
|
|
|
IHC all studies
|
17.0%
(11.7–23.0)
|
5076
(62)
|
-
|
-
|
ISH all studies
|
5.9%
(1.9–11.3)
|
477
(8)
|
-
|
By ASCO/CAP guidelines compliance
|
|
|
|
|
IHC ASCO/CAP compliant
|
5.7%
(1.5–11.7)
|
2135
(26)
|
< 0.001
|
Low
|
IHC ASCO/CAP non-compliant
|
27.0%
(19.9–34.8)
|
2941
(36)
|
High
|
ISH ASCO/CAP compliant
|
1.2
(0.0-5.8)
|
394
(6)
|
0.004
|
Low
|
ISH ASCO/CAP non-compliant
|
24.9%
(12.6–39.6)
|
83
(2)
|
High
|
By histologic subtype
|
|
|
0.054
|
|
Squamous
|
4.1%
(0.6–9.8)
|
12
(1018)
|
Low
|
Non-squamous
|
10.3%
(3.6–19.2)
|
15
(467)
|
Low
|
By WHO geographic region
|
|
|
0.40
|
|
Southeast Asia
|
11.7
(4.3–21.8)
|
6
(242)
|
Low
|
Americas
|
3.4
(0.0-16.5)
|
4
(95)
|
Low
|
Europe
|
4.1
(0.5; 10.0)
|
8
(877)
|
Low
|
Pacific
|
4.0
(0.3; 10.2)
|
8
(921)
|
Low
|
Abbreviations: ASCO = American Society of Clinical Oncology, CAP = College of American Pathologists, WHO = World Health Organization, *According to the compliance with the ASCO/CAP guidelines. |