Myanmar Health Sciences Research Journal
Original Articles :
Myanamr Health Research Registration 2020; 32(2): 166-172.
DOI: DOI: https://doi.org/10.34299/mhsrj.00998

Role of Immunofluorescence in Detecting HER2/neu Status of Breast Carcinoma

Ohnmar Kyaw, Aye Aye Lwin, Moh Moh Htun, Swe Zin Myint, Myint Myint Nyein, Khin Saw Aye

Myanmar Health Sciences Research Journal, 2020; 32(2):166-172

ABSTRACT

Breast carcinoma becomes the third most common female cancers in Myanmar. HER2/neu, one of the prognostic markers of breast carcinoma, triggers cell proliferation, differentiation and inhibits apoptosis and is associated with worse prognosis. Targeted therapy against HER2/neu, trastuzumab (Herceptin), significantly increases disease-free intervals and overall survivals in both early stage and metastatic breast carcinomas. And so, an appropriate HER2/neu evaluation becomes important for the proper identification of patients eligible for treatment with anti-HER2 targeted therapy. The present study was aimed to assess HER2/neu status in 94 Myanmar breast carcinoma patients attending the Yangon General Hospital from June 2016 to May 2017 by immunofluorescence and its expression was compared with immunohistochemistry (IHC) to determine the accuracy of immunofluorescence. The mean age of the patients was 52.23±11.82 years (26-87 years) and 60% of cases were B-R grade 2 (moderately differentiated) according to the Bloom-Richardson System. HER2/neuexpression was compared between two different methods. Immunofluorescence detected higher expression of HER2/neu in score +1 and score +3 but less expression in score +2 (less equivocal cases than routine IHC). There was significant statistical association between HER2/neupositivity and histological grade of breast carcinoma (pvalue=0.000) in both methods. Concordance rate between IHC and immunofluorescence was 88.3% (95% CI). The present study showed that immunofluorescence was a highly reproducible and very robust method with good concordance with routine IHC. The results highlighted the accurate assessment of HER2/neu status is a critical issue in selecting breast carcinoma patients that might benefit from targeted therapy, trastuzumab.


RESULT
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A total of ninety-four FFPE sections of invasive ductal carcinoma of breast were collected from the Pathology Department, Yangon General Hospital. The mean age of the patients was 52.23±11.82 years


Table 1. Age, BR grade, HER2/neuIHC and IF scores of invasive ductal carcinoma of breast

Total no of breast cancer
patients (n=94)

No

%

Age group (years)

 

 

20-29

2

2.1

30-39

7

7.4

40-49

30

31.9

50-59

27

28.7

≥60

28

29.9

B-R grade

 

 

Grade 1

14

15

Grade 2

56

60

Grade 3

24

25

HER2/neu IHC score

 

0

1

1.1

1

13

13.8

2

51

54.3

3

29

30.8

HER2/neu IF score

 

0

1

1.1

1

16

17.0

2

32

34.0

3

45

47.9

B-R=Bloom-Richardson, IHC=Immunohistochemistry, IF=Immunofluorescence (26-87 years) and the age distribution of the patients is described in Table 1.According to histological grade, most cases (60%) were B-R grade 2 (moderately differentiated) according to Bloom-Richardson Histological Grade System.17 Higher BR grading was strongly associated with HER2/neu positivity (p=0.000) in both IHC and Immunofluorescence (Fig.1,2).

 

A=H & E Stain x 200    

C=IF (score +1) x 200

B=IHC (score +1) x 200

D=IF (score +1) x 400




Fig. 1.Invasive ductalcarcinoma of breast B-R grade 1

 

A=H & E Stain x 200    

C=IF (score +3) x 200

B=IHC (score +3) x 200

D=IF (score +3) x 400







Fig. 2.     Invasive ductal carcinoma of breast B-R grade 3

 

Then, HER2/neu scores of immunohistochemistry and immunofluorescence was compared. For IHC, one (1/94) case of invasive ductal carcinoma of breast was scored as 0(1.1%), 13(13/94) cases were scored as +1(13.8%), 51(51/94) cases were scored as +2(54.3%) and 29(29/94) cases were scored as +3(30.9%). The highest immunoexpression of HER2/neu by IHC  was +2 score.

For Immunofluorescence, one (1/94) case of invasive ductal carcinoma of breast (1.1%) was scored as 0, 16(16/94) cases were scored as +1(17%), 32 cases (32/94) were scored as +2 (34%) and 45(45/94) cases were scored as +3. The highest immunoexpression of ER2/neu by Immunofluorescence was +3 score. Immunofluorescence detected higherexpression of HER2/neu in score +1 and score +3 but less expression in score +2 (less equivocal cases than routine IHC) (p=0.000).

 

Table2. Determination of the accuracy of immunofluorescence in HER2/neu expression of invasive ductal carcinoma of breast

Immunofluorescence

IHC

Total

+

-

  Positive

73

4

77

  Negative

7

10

17

Total

80

14

94

IHC=Immunohistochemistry

Among ninety-four cases of invasive ductal carcinoma of breast, sensitivity of Immunofluorescence was 91.25% (83.02%-95.7%) and specificity was 71.43% (45.35%-88.28%). Positive predictive value was 94.81% (87.39%-97.96%) and negative predictive value was 58.82% (36.01%-78.39%). Concordance rate between IHC and Immunofluorescence was 88.3% (80.25%-93.34%)(95% CI) (Table 2).

 


INTRODUCTION
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Cancer rates are expected to grow worldwide in recent years and cancer burden rises to 18.1 million new cases and 9.6 million cancer deaths in 2018.1 Breast carcinoma is one of the most common malignant tumors and the leading cause of cancer death in women, accounting for 2.09 million cases in 2018.1 Though public awareness of screening mammography and annual clinical breast examination is increasing, breast carcinoma plays the top priority in both developed and developing countries worldwide. Approximately 70% of cancer deaths occur in low- and middle-income countries.1 In Asian countries, the incidence of breast carcinoma is lower than the Western countries but the mortality-to-incidence ratio is higher in Asian countries because breast carcinoma presents at a younger age and a later stage because of low screening rates.Breast carcinoma incidence rates have been steadily increasing in all Asian countries, with an annual percentage increase of between 1% and 3%.2

The highest incidence rate of breast carcinoma in Asia occurs in Singapore (incidence rate 65.7 per 100,000 population) which is followed by South Korea (incidence rate 52.1 per 100,000 population) and
Japan (incidence rate 51.5 per 100,000 population).3

In Myanmar, breast carcinoma is the third most common female cancers (incidence rate 22.1 per 100,000 population) accounting for 11.8% of all cancer cases.4 According to the cancer registry from Medical and Radiation Oncology Unit, Yangon General Hospital, breast carcinoma is the most common cancer in women and number of cases of breast carcinoma is increasing by year.Human epidermal growth factor receptor 2, HER2/neu, encoded by ERB B2 gene, is a member of human epidermal growth factor family.Binding to the specific ligand, it triggers cell proliferation, differentiation, and inhibits apoptosis and is associated with worse prognosis.5HER2/neu is expressed at low levels in a variety of normal epithelia, including breast duct epithelium6 and is amplified and over expressed in 20-30% of invasive breast carcinomas.7 HER2/neu over-expression is also found in ovarian, stomach and uterine carcinomas.8,9

Histological types, grade and HER2/neuprotein over-expression are some factors contributing to the prognosis of breast carcinoma. Concerning histological typing, invasive ductal carcinoma carries worse prognosis than other special types. Histological grade is also associated with the aggressiveness of tumor. HER2/neuprotein over-expression is associated with poor prognosis, higher recurrence and reduced survival rate.10

Tratstuzumab (Herceptin), a humanized monoclonal antibody against the HER2/neureceptor, is a form of target therapy that is effective for breast cancers that are HER2 positive. Herceptin works by attaching itself to the HER2 receptor on the surface of breast cancer cells and blocking them from receiving growth signals to slow or stop the growth of the breast carcinoma.11Trastuzumab significantly increases disease-free intervals and overall survivals in both early stage and metastatic breast carcinomas and can also be used in other HER2/neu positive adenocarcinomas.Since the costs for trastuzumab therapy are high and side effects are significant, accurate selection of eligible patients for this therapy is crucial.

In Myanmar, there have been many research papers regarding the role of HER2/neu in breast carcinoma12-14by immunohistochemistry (IHC) method. Immunohistochemistry is an excellent technique widely used in tumor diagnosis. Routine IHC is a relatively inexpensive and easy to perform method for most pathology laboratories in Myanmar.

Immunofluorescence is a type of IHC using fluorescent-labeled antibody. Immunofluorescence can be used in tissue sections (especially in frozen sections) and cultured cell line to detect antigen of interest and can also be used to detect microorganisms.15Immunofluorescence labeling is not often used for formalin-fixed paraffin-embedded tissue (FFPE) sections because of auto-fluorescence which was usually found in FFPE sections.16

This study aimed to evaluate the accurate assessment of HER2/neu status by Immunofluorescence method in FFPE tissues of invasive ductal carcinoma of breast of Myanmar patients and to evaluate the sensitivity and specificity of immunofluorescence compared to routine IHC.


SUPPLEMENTARY MATERIAL
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A cross-sectional descriptive study was carried out from June 2016 to May 2017 and a total of 94 histologically proved cases of invasive ductal carcinomas of breast of Myanmar patients were included in thisstudy. The haematoxylinand eosin sections from each block were reidentified and were graded histologically according to Nottingham Modification of the Bloom-Richardson system.17 Scoring of HER2/neu status was done according to guidelines in American Society of Clinical Oncology and the College of American Pathologists (ASCO/ACP).18

Specimen preparation

All specimens were prepared to preserve the tissue for immunohistochemical staining. Standard methods of tissue processing were used for all specimens. Tissues were prepared in formalin-based fixatives and will be routinely processed and paraffin-embedded.

Four micrometer sections of breast tissues were cut and affixed onto silane-coated glass slides and pre-warmed at 60oC for  30 minutes. For removal of paraffin, the slides were immersed in xylene for 5 minutes 3 times, followed by graded ethanol for 3 minutes 4 times (in 100%, 90%, 70%, 50% ethanol, respectively). Then, the slides were rinsed under gently running water and phosphate buffered saline (PBS).

Antigen retrieval

The slides were placed in a jar containing sodium citrate buffer (pH 6) and placed in the microwave for 10 minutes. And then, the slides were allowed to gradually cool in the same buffer after heating.

Immunohistochemistry procedure

The tissue sections were rinsed with distilled water (DW) for 5 minutes 3 times and then with PBS for 5 minutes once and then tissues were blocked with 3% H2O2-methanol  for 15 minutes at room temperature for endogenous peroxidase ablation. Then, the tissue sections were rinsed with PBS for
5 minutes 3 times and wiped off excess buffer and a circle was drawn around the tissue with a PAP pen. Slides were blocked by incubation in normal goat serum for  30 minutes at room temperature.

After discarding the blocking serum,
the diluted primary antibody (HER2 monoclonal primary antibody, Rabbit IgG, Clone EP3, BioGenex) were dropped on the sections and incubated for 2 hours in the moist chamber at 4oC in the refrigerator. Then, the slides were rinsed in PBS for 5 minutes 3 times. HER2 polyclonal secondary antibody (Super sensitive TM IHC detection system, Biogenex) was added and then the slides were incubated in the moist chamber for an hour. After rinsing in PBS for 5 minutes 3times, the slides were colorized with 3,3’-diaminobenzidin (DAB), kept at room temperature without light for 15 minutes and finished coloration with the distilled water. The sections were counter-stained with Mayer’s haematoxylin solution for 2 minutes, thoroughly rinsed with DW, dehydrated to xylene and permanently mounted. Positive and negative control slides were run in parallel with each batch of slides undergoing IHC procedures.Membrane staining pattern and intensity were evaluated according to guidelines in American Society of Clinical Oncology and the College of American Pathologists (ASCO/ACP).18

Immunofluorescence procedure

Immunofluorescence staining on paraffin sections was carried out with the same steps as IHC procedures on paraffin sections, but anti-rabbit secondary antibody, Alexa Flour 488 conjugate (Thermo-Fisher Scientific, Cat No.A 11008) was used . Then, the slides were counterstained with DAPI (4’,6’-Diamidino-2-phenylindole, dihydrochloride) (Thermo-Fisher Scientific) for nuclear staining. Then, the sections were covered with Anti-Fade reagents (PermaFlour Aqueous mounting medium, Thermo-Fisher Scientific) and observed with fluorescent microscope (Zeiss Axio Imager M2) under appropriate wavelength filters. During preparation, the slides must be kept away from direct light to prevent color fading.The immunofluorescent slides should be stored in -20oC freezer before evaluation to keep thequality of slides.In immunofluorescence, membrane staining pattern and intensity of fluorescent staining were evaluated. Alexa Flour 488 produced green fluorescent light around cell membrane while DAPI produced light-blue color for nuclear staining.

Statistical analysis

The data were collected in Microsoft excel and were analyzed in Statistical Package for Social Science Study for Windows version 16 (SPSS version 16). Simple descriptive analysis for each single variable was done and the data were summarized in pie chart, frequency tables and the proportion and percentage. P values less than 0.05 were considered statistically significant.

Ethical considerations

The study was approved by Research and Ethics Review Committee of University of Medicine (1).


DISCUSSION
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In this study, HER2/neustatus in invasive ductal carcinoma of breast was compared by two different methods (IHC and immunofluorescence) with the same primary antibody after the staining conditions were optimized.The results showed that the detection rate of IHC and immunofluorescence in breast tissues is mostly depending on the staining system. In IHC,an enzyme, horseradish peroxidase (HRP) converts a soluble substrate including 3,3’-diaminobenzidin (DAB) into an insoluble colored product at the antigen site. In preparation of DAB, it must be ensured that pH of the solution should be buffered around 7.2. A pH below 7.0 will result in weak DAB intensity and a pH above 7.6 will result in a high level of background stain. So DAB must be prepared just before staining with an appropriate pH range. In immunofluorescence, fluorescent labeled antibody indirectly bind to the antigen of interest and the background staining is much more reduced than that of routine IHC because no extra staining is needed in immunofluorescence.

In this study, HER2/neu expression was higher than that of the other studies done in Myanmar12-14 (30.9% by IHC and 47.9% by immunofluorescence). The reasons may be due to changing genetic predisposition or the changing effect of the environmental factors19 or the changing guidelines in American Society of Clinical Oncology and the College of American Pathologists (ASCO/ACP).18

In 2013, the ASCO/ACP guideline on scoring of HER2/neu has changed. In 2013 guideline, >10% of invasive tumor cells with uniform intense membrane staining was scored as +3 while >30% of tumor cells in 2007 guideline. For score +2, >10% of invasive tumor cells must be stained incompletely/weakly or <10% of tumor cells must have intense membrane staining in 2013 guideline while weak or incomplete membrane staining in 10% - 30% of tumor cells in 2007 guideline. And so, HER2 expression showed higher positivity rate using this 2103 guideline than that using 2007 guideline.

Immunofluorescence is a type of IHC using fluorescent-labeled antibody. In Immunofluorescence, an antibody labeled with a fluorescent dye (fluorochrome) is used to combine with the specific antigen and
its fluorescence is detected by fluorescentmicroscopy. Immunofluorescence was rarely used in formalin-fixed paraffin-embedded sections (FFPE) because of auto-fluorescence which was usually found in FFPE sections.20 Higher resolution fluorescent microscope rather than epifluorescent microscope and new generation dyes must be used for focusing antigens in formalin-fixed paraffin-embedded tissue sections.21,22 In this study, the new generation AlexaFluore dye (Alexa 488) and higher resolution fluo-rescent microscope (Zeiss Axio Imager M2) were used for detecting HER2/neu in FFPE specimens of invasive ductal carcinoma of breast. Immunofluorescence was a highly reproducible and very robust method with good concordance with routine IHC.

In Myanmar, frozen sections were not widely used and so immunofluorescence on FFPE sections may be the useful alternative and could be applicable on FFPE sections. This method helped to bridge a gap between routine IHC of FFPE sections and immunofluorescence.

As a result, the potential of novel therapeutic option (anti-HER2 therapies) in breast cancer patients is becoming the standard of care in breast cancer and appropriate patient selection must be done by IHC or Immunofluorescence and Immunofluorescence will now be part of routine pathology in the very near future.



ACKNOWLEDGMENT
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 We would like to express our special thanks of gratitude to Dr Khin Saw Aye (Deputy Director-General, DMR) and Professor Myint Myint Nyein for their guidance and support in completing this research.
We would like to extend our gratitude to Professor Shigeru Okada, Dr. Moh Moh Htun(Director, DMR) and Dr. Kyaw Soe (Deputy Director, Pathology Research Division, DMR) for their conscientious support and encouragement to accomplish this work.


CONFLICT OF INTEREST
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The authors declare that they have no competing interests.

            



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