Research Article ISSN 2639-8478

Research Article Cancer Science & Research

Immunohistochemical and Molecular Evaluation of Oncoprotein HER-2 in

Women's Breast Cancer in The Republic of Congo
Anicet Luc Magloire Boumba1,3,4*, Dimitry Moudiongui Mboungou Malanda1,2, Fabien Gaël Mouamaba1,2,
Exaucé Céleste Abena Gueyan1, Fidèle Détila Mabouene1,2, Donatien Moukassa1,3 and Jean Félix Peko1,2

Faculty des Sciences de la Santé, University Marien Ngouabi,

1

BP : 69, Brazzaville, Congo.

Service Anatomie et Cytologie Pathologiques,

2
Centre
Hospitalier et Universitaire de Brazzaville (CHUB). *
Correspondence:
Dr Boumba Anicet Luc Magloire, Faculty des Sciences de
3
Zone de recherche Pointe-Noire, Institut National de Recherche la Santé, University Marien Ngouabi, BP: 69, Brazzaville,
en Sciences de la Santé (IRSSA). République du Congo, Tel: +242 05 660 10 40.

Laboratoire d’Analyses Médicales et Morphologiques, Hôspital

4 Received: 07 August 2021; Accepted: 01 October 2021
Général de Loandjili de Pointe-Noire (HGL).

Service Anatomie et Cytologie Pathologiques, Hôspital Général

5

Edith Lucie Bongo Ondimba d’Oyo (HGELBO).

Citation: Boumba ALM, Malanda DMM, Mouamaba FG, et al. Immunohistochemical and Molecular Evaluation of Oncoprotein
HER-2 in Women's Breast Cancer in The Republic of Congo. Cancer Sci Res. 2021; 4(3): 1-9.

ABSTRACT
Introduction: Breast cancer is a heterogeneous disease with a variety of morphological and molecular
characteristics impacting treatment response. This study aimed to evaluate by immunohistochemistry and RT-PCR
the overexpression of HER2 in breast cancer in women in the Republic of Congo.

Materials and Methods: We conducted an 8-month cross-sectional descriptive study. 25 paraffin biopsies of breast
cancer cases in patients diagnosed at the University Hospital of Brazzaville were collected. Epidemiological,
clinical, histological, immunohistochemical and molecular aspects were studied.

Results: The mean age of the patients was 49.64 ± 13.20 years (31-80 years). 60% of the patients had a right
localization of the tumor. 76% of the patients had an invasive nonspecific type carcinoma. The T4b N1a M0 stage
was predominant, representing 56% of the study population. SBR histopronostic grade 1 was represented by 60% of
patients. Estrogen and progesterone receptors were positive in the range of 45% and 60%, respectively. The HER2
oncoprotein was positive in 12% (3/25) of 25 cases for IHC. The luminal group was in the majority with 32%.
Molecular analysis of the HER2 gene by RT-PCR revealed over expression in 60% (15/25) of cases, 3 of which were
already positive for IHC. With the "AmoyDx® HER2 Mutation Detection Kit", 12 mutations were identified, 10 of
which involved exon 20, ie 83.33% and 2 mutations with exon 19, ie 16.67% of cases. The correlation of the over
expression of the HER2 gene showed a statistically significant difference between the two techniques, p <0.00003.

Conclusion: HER2 is known as a prognostic and predictive marker in breast cancer, making this receptor a
valuable therapeutic target. However, its highlighting by IHC remains cumbersome and subject to false negatives.
Hence molecular analysis could play a crucial role in decision-making when implementing targeted breast cancer
therapies in Congo.

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Keywords at the same time. The overexpression of HER2 is currently
HER2, immunohistochemistry, qRT-PCR, Breast cancer, Republic defined according to the scoring system of the ASCO/CAP 2014
of Congo. guidelines to achieve reproducible dosing performance [10,16-
19]. Several studies have been carried out around the world and
Introduction in Africa studying different aspects including epidemiological,
Female breast cancer has now surpassed lung cancer as the leading diagnostic and therapeutic. However, almost no studies have
cause of global cancer incidence in 2020, with around 2.3 million looked at immunohistochemical and molecular approaches to
new cases, or 11.7% of all cancer cases. It is the fifth leading cause HER2 oncoprotein in breast cancer in Congo. The objective of this
of cancer death in the world, with 685,000 deaths (1). In Congo, work was to evaluate the overexpression of oncoprotein HER-2
it is the first of all cancers according to the latest estimates of by IHC and molecular biology in order to contribute to a better
GLOBOCAN 2020 [1]. It is a real public health problem affecting therapeutic management of breast cancer patients in Congo.
both sexes with a large predominance of women in the order of
99% of cases [2]. Breast cancer is a heterogeneous disease with Materials and Methods
a variety of morphological and molecular characteristics and It was a cross-sectional descriptive study with retrospective data
response to treatment and clinical outcome. It is divided into five collection, which took place from April 1 to November 1, 2020, a
molecular subtypes: luminal A, luminal B, basal type and normal period of 8 months.
type, human epidermal growth factor (HER2) positive receptor
2. The latter, is localized in chromosome 17 and encodes for a The study population involved 25 breast biopsies and operating
protein of 185 kPa that functions as a transmembran growth factor parts included in paraffin blocks with available and interpretable
receptor. it is a cell proliferation marker with high metastatic clinical data, and with a confirmed diagnosis of breast cancer after
potential [3,4]. Tumours that overexpress this gene have a poor triple reading. The paraffin blocks were identified on the basis of
prognosis. Its evaluation involves immunohistochemistry and the selection criteria in the Department of Pathological Cytological
currently molecular biology techniques to better study its profile Anatomy, Brazzaville University Hospital Center from January
and thus institute the new therapeutic approach which is none 2019 to March 2020.
other than the indicated targeted therapies (Trastuzumab) [5,6].
This study was conducted successively in Brazzaville (Brazzaville
The relationship between the overexpression of HER2 and the University Hospital) for the selection of samples, in Oyo in the
evolution of breast cancers on the one hand, and the new therapeutic department of the basin (Edith Lucie BONGO ONDIMBA
possibilities by anti-HER2 antibodies on the other hand have General Hospital) for the immunohistochemistry analysis (IHC)
made the HER2 status a commonly sought-after element currently and in Pointe Noire (Marie Madeleine GOMBES Laboratory) for
in breast cancers. Knowledge of HER2 status is essential for the molecular analysis. This work has been approved by the Committee
selection of breast cancer patients for treatment with trastuzumab on Ethical Research in Health Science (CESRB) according to
(Herceptin®). According to clinical data, her2-targeted therapy Opinion No. 061.
significantly improves the survival of breast cancer patients with
overexpression of HER2 [7-9]. However, recent data suggest the Technical support of the samples
presence of oncogenic mutations in HER2 affects the technical and Histopathology
clinical outcomes of the IHC in patients with HER2-positive breast All breast tissues were fixed in 10% neutral buffered formalin
cancer [10]. Some authors attributed somatic mutations to HER2, and included in paraffin under standard conditions. Sections of 4
a role in resistance to anti-HER2 treatment because differential μm were performed at the microtome for each of the 25 blocks of
regulation of HER2 was observed in patients [10-13]. paraffin breast to examine microscopically the presence of more
than 80% of breast cancer cells. The tissue ribbons were spread
out on the door blades objects for hematxyline and eosin (H&E)
A reliable assessment of HER2 status is essential for a selection
staining. The resulting slides were deparaffin in three toluene baths
of all patients to benefit from targeted therapy. Many tests are
for 5 minutes each. Then they were rehydrated with a series of
available to assess HER2 status. The two most commonly used alcohol baths to decreasing degrees (100%, 95% and 80%), each
tests are immunohistochemistry (IHC) and fluorescence in situ for 3 minutes. The cuts were then stained with hematxyline for 5
hybridization (FISH) [8,10,14]. The determination of the HER-2 minutes, rinsed gently in tap water for 10 min, then colored with
status can be carried out either by the search for amplification of eosin for about 1 minute. The blades were subsequently dehydrated
the gene or by that of the overexpression of the protein encoded with an increasing series of alcohol (80%, 95% and 100% ethanol).
by this gene. Several methods can be used, only morphological After immersion for 5 min in a toluene bath, they were mounted
methods are currently the subject of recommendations by with slats covering objects using an assembly medium (Eukkit).
international bodies [9,10,15]. In addition, PCR-based methods This phase was carried out in the Laboratory of Pathological
have become important in clinical analyses, especially quantitative Cytology Anatomy at the Brazzaville University Hospital.
PCR methods such as real-time polymerase chain reaction (PCR),
which is based on the detection of DNA amplification. Real- The paraffin blocks diagnosed with breast cancer were all sent
time PCR is cost-effective and many samples can be analyzed to the Morphological and Molecular Analysis Laboratory of the

Cancer Sci Res, 2021 Volume 4 | Issue 3 | 2 of 9

Edith Lucie BONGO ONDIMBA General Hospital for the manual DNA extraction
immunostaining technique. Clinical data including age, stage, Sections of 5 μM were made using the Leica microtome from the
histological grade and histological type were collected on the paraffin blocks and put in the eppendorf tubes.
survey sheets. The histological classification was carried out using
the Nottingham classification system and staging according to the Dewaxing was achieved by adding 1 ml of xylene to each
8th edition of the 2017 AJCC classification [11,20]. eppendorfs tube. After vortexing for 30 seconds, the tubes were
incubated at room temperature for 15 min and then centrifuged at
Immunohistochemistry 6000 rpm at 15°C for 5 minutes. After removal of the supernatant,
Immunohistochemistry procedures examining hormone receptor 1mL of ethanol 70% was added, voratexed, incubated for 15 min at room
labeling (RO for estrogen receptor and RP for progesterone temperature and then centrifuged for 5 min at 6000 rpm at 15°C.
receptor) and her-2 expression were performed manually.
The operation was repeated 2 times. Finally the pellet was dried
The technology used was the "Ultra vision quanto system" using at room temperature and then washed with PBS (saline phosphate
horseradish peroxidase (HRP) and Diaminobenzydine (DAB). buffer) two (2) times. The samples were re-suspended in 180 .mu.l
The following standard practices were performed: To analyze of lysis buffer to which 20 .mu.l of proteinase K was added. After
the labeling profile of hormone receptors and the expression of incubation at 56°C for one hour, the extraction continued with the
Her-2 with super Frost plus blades, each was deparaffin in xylene, kit "ReliaPrepTM gDNA Tissue Miniprep System (Promega)"
rehydrated in alcohol baths to decreasing degrees (100°, 95°,70°) following the manufacturer's instructions. The elution was done
and boiled for 45 minutes (in a water bath set at 96°) in 10 mM with 50 .mu.l of elution solution and the collected DNA was
citrate buffer (pH 6.0) for the restoration of antigenic sites. immediately stored at -20°C before use. The concentration of
the extract was determined using Qubit® 3.0 (life technologies,
Then the slides were incubated for 35 minutes at 37°C with Invitrogen).
primary monoclonal antibodies (See Table 1 below) using manual
Molecular analysis by real-time PCR of the HER-2 gene
mode. Thermo Scientist's manual Ultra vision detection system
Her2/neu gene expression was analyzed using the "AmoyDx®
uses an indirect biotin-avidine system with a universal biotinylated
HER2 Mutation Detection Kit (Amoy Diagnostics Co., Ltd.
immunoglobulin secondary antibody. The slides were incubated
Xiamen, China)". The kit uses refractory mutation system
for 5 minutes with 3,3'-diaminobenzidine (DAB) until appropriate
amplification technology (ARMS) that includes specific primers
brown staining.
and fluorescent probes to detect gene mutations in the HER-2 gene
by real-time PCR. During nucleic acid amplification, the targeted
Then the blades were counter-stained with hematxyline before mutant DNA is matched with the bases at the 3 ' end of the primer,
assembly. The staining procedures were carried out in accordance amplified selectively, then the mutant amplicon is detected by
with the manufacturer's recommendations. Negative and positive fluorescent probes labeled with FAM. This kit is composed of 6
control slides were included in each test. The samples were reaction mixtures and positive control. Table I shows the primer
interpreted according to the guidelines of the American Society and probe concentrations used. The master mix and primers /
of Clinical Oncology / College of American Pathology (ASCO probes were combined with 0.3 .3 μL of MATRIX DNA in a final
/ CAP): negative (0, 1+), weakly positive (2+) and strongly volume of 35.3 .mu.l (Table 2). The amplification conditions were
positive (3+), with a threshold of more than 10% of tumor cells reported in Table 3.
that must have a homogeneous and dark circumferential pattern
(mesh) to call the results 3+, HER2 positive [17]. The results of Triple reactions were performed and the mean cycle cut-off value
estrogen receptors (ER) and progesterone (PR) were interpreted (Ct) was used to determine the relative amount of PCR product.
in accordance with the guidelines recommended by asco/CAP The hybridization probes used for quantification were based on
[9,14,18,19]. primers and probes described in Table 4.

Table 1: Contents of the kit according to the Master Mix.

Tube No Contents Main ingredient Quantity Fluorescent Ssignal
1 HER2 Reaction Mix (1) Primers, probes, Mg2+, dNTPS 550 µL/tube × 1 FAM, HEX/VIC
2 HER2 Reaction Mix (2) Primers, probes, Mg2+, dNTPS 550 µL/tube × 1 FAM, HEX/VIC
3 HER2 Reaction Mix (3) Primers, probes, Mg2+, dNTPS 550 µL/tube × 1 FAM, HEX/VIC
4 HER2 Reaction Mix (4) Amorces, sondes, Mg2+, dNTPS 550 µL/tube × 1 FAM, HEX/VIC
5 HER2 Reaction Mix (5) Primers, probes, Mg2+, dNTPS 550 µL/tube × 1 FAM, HEX/VIC
6 External control reaction mixture HER2 Primers, probes, Mg2+, dNTPS 550 µL/tube × 1 FAM
Taq DNA Polymerase, Uracil-N-
/ Enzyme mixture HER2 30 µL/tube × 1 /
Glycosylase
/ Positive control HER2 plasmid DNA 250 µL/tube × 1 /

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Table 2: HER2 Master Mix.
Content Volume per test
Reaction mixture 35 μL
HER2 enzyme mixture 0.3 µL
Total volume 35.3 μL

Table 3: Represents cyclic parameters.

Cycles Temperature Time Data collection
93°C 25s /
31 60°C 35s FAM et HEX/VIC
72°C 20s /

Table 4: HER2 Mutations detected by the kit.

Reagents Exon Mutation Base change
A775_G776insYVMA 2325_2326 ins12 (TACGTGATGGCT)
HER2 Reaction mixture (1) 20 A775_G776insYVMA 2324_2325 ins12 (ATACGTGATGGC)
M774_A775insAYVM 2322_2323 ins12 (GCATACGTGATG)
G776> VC 2326_2327 ins3 (TGT)
G776> VC 2326_2327 ins3 (TTT)
HER2 Reaction mixture (2) 20
G776R 2326G> C
G776C 2326G> T
P780_Y781insGSP 2339_2340 ins9 (TGGCTCCCC)
P780_Y781insGSP 2339_2340 ins9 (GGGCTCCCC)
HER2 Reaction mixture (3) 20 2340_2341 ins9 (GGCTCCCCA)
P780_Y781insGSP
P780_Y781insGSP 2339_2340 ins9 (CGGCTCCCC)
HER2 Reaction mixture (4) 20 V777L 2329G> T

HER2 Reaction mixture (5) 19 L755P 2263_2264 TT> CC

Statistical analysis Molecular profile of the HER-2 gene

Qualitative variables were expressed as a percentage. The All 25 samples were analyzed. The expression of the HER2 gene
qualitative data was compared with McNemar's Fisher Exact test, was observed in 15 of the 25 samples or 60%. Of the 15 samples
at the risk α = 5%. detected positive, 3 were also tested by the IHC technique.
Analysis of the 12 samples exclusively positive for RT-PCR
Results showed mutations that were described in Table 6.
Clinico-pathological features
All the clinico-pathological characteristics are reported in Table 5.
Molecular and immunohistochemical correlations of HER-2
Since the expression of the HER-2 gene is crucial in the
The average age of the study population was 49.64 ± 13.20 years
with extremes ranging from 31 to 80 years. The majority age group management of cases of breast cancer, its expression gives the
of the study population was between 36 and 45 years of age, or 32%. possibility of prescribing Herceptin as an important molecular in
the target therapy of this cancer. Table VII reports the bivariate
The right tumor localization was the most represented in our analysis between the results of IHC and RT-PCR in the detection
study with 15 patients or 60%. Invasive carcinoma was the most of oncoprotein HER-2. A statistically significant difference was
frequent with 76% for 19 patients. The T4b N1a M0 stage was observed for the detection of her-2 oncoprotein expression between
in the majority representing 56% of the study population. Histo- the IHC technique and RT-PCR, p<0.00003.
prognostic grade 1 of SBR was represented by 60% of the patients.
Estrogen receptors were negative in the order of 56% and positive
in the order of 44%. Progesterone receptors were positive in 40%
Discussion
of cases. HER2 oncoprotein was positive with 12%. The luminal Oncoprotein HER-2 is recognized as one of the biomarkers of
group was in the majority with 32%. cell proliferation with high metastatic potential and very poor
prognosis. His systematic research in breast cancer offers new
Immunohistochemical profile (IHC) of oncoprotein HER-2 therapeutic possibilities thanks to the discovery of new molecules
A total of 25 samples were immunohistochemically analyzed for such as Trastuzumab. Thanks to the IHC, the overexpression of
her-2 oncoprotein. Three (03) of the 25 expressed oncoprotein HER-2 makes it possible to define the HER-2 status in breast
HER-2 or 12% positivity. cancers.

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Table 5: Clinico-pathological characteristics.
Variables Effectif (n) Percentage (%)
Age groups
Average age ± SD 49,64 ± 13,20
Extremes of age 31 - 80 years
≤ 35 years 5 20
36-45 8 32
46-55 7 28
56-65 2 8
≥ 66 yeans 3 12
Tumor localization
Right breast 15 60
Left breast 10 40
Histological types
Nonspecific infiltrating ductal carcinoma 19 76
Invasive lobular carcinoma 3 12
Medullary carcinoma 1 4
Mixed invasive carcinoma 1 4
Mucinous carcinoma 1 4
TNM Classification
T0N0M0 1 4
T3N0M0 4 16
T3N1aM0 2 8
T4bN0M0 4 16
T4bN1aM0 14 56
Histo-prognostic grade of SBR
Grade 1 15 60
Grade 2 8 32
Grade 3 2 8
Immunohistochemical aspect
RO+ 11 44
RO- 14 56
RP+ 10 40
RP- 15 60
HER2+ (2+ ; 3+) 3 12
HER2- 22 88
Molecular profile
RT-PCR of the HER2 gene
HER2+ Gene 15 60
HER2- Gene 10 40
Molecular classification
Luminal A 8 32
Luminal B 8 32
HER2 positive 3 12
Triple négative 6 24

Table 6: Distribution of the 12 MUTATIONS of the HER2 gene.

Number of
Reaction mixture Exon Mutations Genes mutations
Samples
HER2 Mix (1) 20 A775_G776insYVMA 2325_2326ins12 (TACGTGATGGCT) 3
G776 > VC 2326_2327 ins3 (TGT)
HER2 Mix (2) 20 2
G776 > VC 2326_2327 ins3 (TTT)
P780_Y781insGSP 2339_2340 ins9 (TGGCTCCCC)
HER2 Mix (3) 20 2339_2340 ins9 (GGGCTCCCC) 2
P780_Y781insGSP
HER2 Mix (4) 20 V777L 2329G > T 3
HER2 Mix (5) 19 L755P 2263_2264 TT > CC 2

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Table 7: expression of oncoprotein HER-2 by IHC and RT-PCR. was obtained against 12% by the IHC. These observations confirm
RT-PCR Gene HER-2 the results obtained at the IHC in particular the low positivity and
IHC HER-2 Total P-value
Positive n (%) Négative n (%) show the importance of molecular biology in the expression of
Positive 3 (20) 0 (00) 3 0,00003 her-2 gene in breast cancer in Congo.
Négative 12 (80) 10 (100) 22 (χ2=12.25)
Total 15 (100) 10 (100) 25 Moreover, this divergence of the overexpression of HER-2
Kappa coefficient = 0.16, poor agreement between the two tests. observed can also be explained by the tumor heterogeneity of
breast cancer and the HER2 gene described in the literature which
However, this technique is still used empirically in resource- is defined by the coexistence, within the same carcinomatous
constrained countries such as Congo, leading to high proportions lesion, of several subpopulations of tumor cells with a different
of false negatives. Molecular biology thus gives another alternative status of the HER2 gene. According to studies, it is found in 11 to
in the analysis of the expression of this oncoprotein, especially 40% of breast cancers, [23,50]. There are currently 2 types, a form
with the appearance of mutations at the level of the epitopes called "cluster", characterized by the presence of a population
essential for the IHC technique [7-10]. It is with this in mind that of contiguous cells with amplification of HER2 grouped in a
this work has made it possible to evaluate the expression of the specific geographical area within the tumor, well separated from
HER-2 oncoprotein by RT-PCR in a population of Congolese tumor cells without amplification of HER2, and a form, called
women with breast cancer. "mosaic", characterized by the presence of cells with amplification
of HER2 scattered within a majority population of cells without
In connection with the expression of oncoprotein HER-2, the IHC amplification of HER2 [23]. Intra tumor heterogeneity may
detected 3 positive cases out of 25 or 12% of the samples analyzed. explain the discrepancies observed regarding the status of HER2
These data are lower than those reported by Moudiongui et al. [20] between the biopsy and the operating room or between 2 different
in another study in Brazzaville which found a positivity of the blocks of the same operating room. It can also explain the inter-
oncoprotein HER2 at 37.5%. Bel Charlyne in France in 2019 [21] observer discrepancies in HIS, in the establishment of the average
reported the presence of HER2 in 68% cases. count of HER2 signals per nucleus or the RATIO HER2 / CEP17,
depending on the part of the tumor analyzed. Little is known about
The low percentage of HER-2 positivity in our study population the prognostic significance of this phenomenon and the sensitivity
suggests two possible reasons : (i) either they are true negatives, the of different clones of the same tumor to anti-HER2 therapies.
conditions have been well met from the sampling to the realization Intra-tumor heterogeneity is believed to be associated with a high
of the IHC technique; ii) either they are false negatives because histological grade and polysomy of chromosome 17. A study
of the IHC technique which is still widely practiced empirically also showed that if less than 30% of tumor cells were amplified
in our country or, biopsies once taken, are often put in unbuffered in a tumor, this had no prognostic impact [24]. Our results are
formalin and spend more than 72 hours without being analyzed, discordant from those of Lehmann et al. [25] in England who rather
which can strongly impact the epitopes resulting in these false demonstrated in 2011 a strong convergence in the order of 97%
negatives , or somatic mutations widely described at some of the between the IHC and the PCR in real time. Bel Charlyne in France
exons, leading to a change in conformation of the HER-2 protein in 2019 [21] demonstrated a passage of 68% of the oncoprotein
and during the antigen-antibody reactions of the IHC inducing HER2 to the IHC to 92% of the HER2 gene to molecular biology,
false negatives [10]. an average of 24% more positivity, this result is close to ours. Two
other studies were carried out by Rosa et al. [26] and Olsson et al.
However, HER-2 positive patients are eligible for her-2 [27] the concordance between IHC and q RT-PCR was observed
antibody targeted therapy for example, although the presence in 59 cases out of 75 (78.7%). In the second, there was an 86%
of overexpression of HER2 in breast cancers is a sign of poor match rate between real-time PCR and IHC. Although there are
prognosis because the tumors are more aggressive, have greater several techniques to determine the expression of the HER2/neu
metastatic potential and are less sensitive to hormonal treatments gene; q RT-PCR is more convenient, easier and faster than IHC,
and/or chemotherapy [22]. FISH and CISH.

Molecular amplification of the HER2 gene was observed in 15 As for the kit used, it made it possible to describe the mutations of
cases out of 25 or 60% of the samples analyzed. These results this gene (HER2) in other types of cancer including lung cancer.
made it possible to highlight a great divergence from those Indeed, in several studies conducted in China using the same kit,
observed at the IHC which reported on the same population as conducted respectively by Li X and al in 2016 [28]; Wang et al. in
12% of positivity. In addition, 12 or 80% of PCR-positive cases 2017 [29] and Ma Y et al. in 2020 [30] studied the 13 mutations in
were negative for HCI. With a kappa at 0.16 reflecting a poor the gene during lung cancer.
concordance between these two techniques in the overexpression
of HER-2, the low positivity observed at the IHC would reflect According to existing data, the probability of HER-2 mutations
false negatives due to several causes mentioned above. The use of is 1.67% in breast cancer, 1 - 4% in lung cancer and 2.9% in
a mutation detection kit identified a high proportion of mutations in colorectal cancer [31-33]. The gene encoding HER-2 is located
our study population. Indeed, 60% detection of HER2 by RT-PCR in chromosome 17 and encodes an 185 kPa protein that functions

Cancer Sci Res, 2021 Volume 4 | Issue 3 | 6 of 9

as a transmembran growth factor receptor [34]. The intracellular Limitations of the study: In addition to the small size of our
domain of HER-2 contains about 500 residues and is composed of study population, the correlation of these two variables made
three parts: a cytoplasmic membrane juxta linker, a tyrosine kinase it possible to note that 12 samples previously negative to the
domain (TK) and a carboxyl-terminal tail [35,36]. IHC, after realization of RT-PCR by the kit "AmoyDx® HER2
Mutation Detection Kit" became positive with a statistically
In our study all mutations were observed in the Tyrosine Kinase significant difference p =0.00003. The discrepancy between these
(TK) domain: 83.3% of the mutations concerned exon 20 and methodologies could include inter-observer errors, due to the
16.7% concerned exon 19. These results are in line with the data subjectivity of IHC interpretation and RT-PCR analysis, which can
of the literature related to mutations of the HER2 gene which are cause deviations in particular in the initial cycles, which depend
mainly localized in the three exons [18,19,37] of the TK domain not only on the melting temperature of the amplicon, but also on
[3,10,38] and are encoded by dna sequences in exons 18 - 23 the behavior of the amplicon [57]. Using PCR-based methods, the
[39]. Mutations in the HER-2 kinase domain can be classified as expression of a specific tumor gene or tissue and the presence of
follows: missense point mutations, small insertions in the frame or genetic abnormalities can be detected in a clinical sample with
duplications that occur almost in exon 20 and in frame releases. higher sensitivity (one malignant cell out of 106-107 normal
Among these mutations, inframe insertions or duplications in exon cells) than that of other techniques such as optical microscopy
20 are the most frequently encountered types of mutations [40,41]. (one malignant cell out of 102-103 normal cells). Using RT-PCR,
The publication of next-generation sequencing data indicated that nucleic acid molecules can be amplified 1010 times [58].
somatic mutations in HER2 are present in about 2-5% of primary
breast cancers [42-47]. Most HER2 somatic mutations have been Nistor et al. [59] conclude from their results, obtained from HER2
reported in her2 breast cancers with negative amplification [48,49]. gene amplification, that real-time PCR combined with the IHC
These mutations are proportionally more frequent in lobular cancers approach for determining HER2 status in breast cancer patients
than ductal cancers. They mainly concern the extracellular domain may be an effective and efficient strategy, but HER2 detection
of the HER2 gene (exon 8, residues 309-310) in 20% of cases and using qPCR was more accurate and reproducible compared to HCI.
the tyrosine kinase domain (exons 19-20, between residues 755-
781) in 68% of cases. Most mutations in the HER2 gene have been Conclusion
detected in exons 19 and 20 of the tyrosine kinase domain (TK), This study focused on the great biological heterogeneity of HER2
at the level of the C- α position of the protein helix [49]. Several in breast cancer and the need to implement reproducible and
authors propose that mutations in this area could be an alternative sensitive tests such as RT-PCR to measure the low expression of
mechanism to her2 activation and effective sensitivity to anti- HER2 observed with the IHC technique.
HER2 therapy, as a mechanism of acquired resistance to this
form of therapy. The TK mutations described to date in HER2+ Amplification of the HER2 gene, is still considered a major
breast cancer promote the activation of protein functionality and mechanism of HER2-induced tumorigenesis and is used as the
increase the oncogenicity of HER2, in addition to inducing the main predictive biomarker to identify patients who could benefit
phosphorylation of other cell signaling proteins [49,27,50]. Some from treatment with anti-HER2 agents.
of these mutations are activators (HER2 G309A, D769H, D769Y,
V777L, P780-Y781insGSP, V842I, R896C, G309E and S310F), Thus, the IHC should be considered as an initial screening strategy
others would confer resistance to lapatinib (HER2 L755S), still to distinguish between positive and negative cases of HER2
others would be of unknown significance. These findings may amplification; on the other hand, molecular biology through RT-
explain the clinical response to anti-Her2 therapies in some PCR must remain by far the examination of choice for determining
patients, whose tumours are commonly considered Her2-negative the profile of the HER2 gene, in order to effectively improve the
by the IHC and HIS [9]. therapeutic and personalized management of breast cancer patients.

In addition, some authors have attributed somatic mutations References

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