Cancer is a leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. Lung cancer is one of the most common solid tumors and a leading cause of cancer-related deaths worldwide. Annually, approximately 2.09 million patients are diagnosed with lung cancer of whom 1.76 million die of lung cancer [1]. In Myanmar, where lung cancer is the leading cause of cancer death, the mortality rate of lung cancer is 16.6% in men and boys and 13.8% in women and girls [2].
Adenocarcinoma is one of the most common histological subtypes of non-small-cell lung carcinoma (NSCLC). Molecular profiling of tumor samples from patients with NSCLC has identified driver variants that may contribute to early carcinogenesis in >80% of cases of adenocarcinoma, including epidermal growth factor receptor (
Targeted EGFR-tyrosine kinase inhibitor (TKI) first-line treatment for sensitizing
Adenocarcinoma histology, female sex, never-smoking status, and Asian ethnicity have been considered the most important factors associated with
The prevalence of
The aim of the present study was to determine the
It is hoped that this study will fill the missing gap in knowledge of such patients to enable those with adenocarcinoma of the lung to benefit from EGFR-TKI therapy, which significantly prolongs the survival of patients. Therefore, it is of great importance to determine the prevalence and frequency of
All biopsy specimens from various hospitals and clinics were received at the Innovative Diagnostics laboratory, Victoria Hospital in 10% neutral-buffered formalin (NBF). Tissue was processed automatically to yield hematoxylin and eosin (HE) sections. Various histological types of lung carcinoma were categorized according to the morphology on the HE sections. After the present study was approved by the Ethics Review Committee on Medical Research Involving Human Subjects, Department of Medical Research, Ministry of Health and Sports, The Government of the Republic of the Union of Myanmar (approval No. Ethics/DMR/2018/051), the investigators randomly selected cases of carcinoma within the study period and contacted the patients or their family members. After describing the research protocol and project, we obtained 66 participants during a 6-month period from March 2018 to August 2018. Informed consent was documented on the consent forms provided, and the proforma were filled. The study was conducted in accordance with the principles of the contemporary revision of the Declaration of Helsinki. Victoria Hospital provided permission to conduct this research. Here we report results using the STrengthening the REporting of Genetic Association Studies (STREGA) reporting guidelines [8].
A panel of monoclonal antibodies and special stains was used as required in all 66 cases of lung cancer. The panel of the antibodies included those against TTF1, P40, synaptophysin, napsin A, and CK7. Of 66 patients with lung cancer, 40 were established as having primary adenocarcinoma of the lung. The patients with confirmed cases of adenocarcinoma proceeded to molecular testing for
The cobas EGFR Mutation Test is a real-time PCR test for the qualitative detection and identification of variants in exons 18, 19, 20, and 21 of the gene for EGFR in DNA derived from tumor tissue. The cobas EGFR Mutation Test consists of manual specimen preparation using the cobas DNA Sample Preparation Kit followed by amplification/detection on the cobas z 480 analyzer using a cobas EGFR Mutation Test kit. A single run could include from 1 to 30 specimens and 2 controls per 96 microwell plate. The turnaround time for one test run is about 8 h. The system can detect 42 variants.
In the manual sample preparation, initially unmounted 5 μm sections of formalin-fixed paraffin-embedded tissue (FFPET) was deparaffinized using xylene and ethanol. DNA was prepared from the deparaffinized tissue in microcentrifuge tubes by adding DNA tissue lysis buffer and proteinase K, with one extra tube prepared as a negative control. DNA was then isolated with protein binding buffers, isopropanol, wash buffers, and elution buffer. The concentration of DNA in the isolated stock solution was quantified using a Nano-drop 2000 spectrophotometer and an average of 2 consistent readings was calculated. The minimum DNA stock concentration required was 2 ng/μL. If the DNA stock concentration is exactly 2 ng/μL, it can be used without dilution. For concentrations of 2–36 ng/μL and >36 ng/μL, the stock was diluted with DNA Specimen Diluent by calculating with the equations provided. Consequently, working EGFR master mixes (MMX-1–3), mutant control, and negative control were prepared. Next, the controls and the DNA specimen diluents were transferred into a defined microwell plate to which the working MMX-1–3 had already been added in the areas of the plate specified by Roche Diagnostics. Finally, the plates were covered and sealed with sealing foil from the kit and PCR was conducted using a cobas z 480 real-time PCR analyzer (Roche Diagnostics). The results were generated automatically by the cobas system [9].
IBM SPSS Statistics for Windows (version 25.0) was used for all analyses. The absolute and relative frequencies of the quantitative variables were calculated as percentages. We conducted a univariate analysis of categorical factors using a χ2 test or Fisher exact test as appropriate, with no correction for multiple comparisons.
The histological types of lung carcinomas were categorized by using a panel of antibodies. Of 66 cases of lung carcinoma, there were 40 cases (61%) of adenocarcinoma. In order of frequency, this was followed by squamous cell carcinoma 16 cases (24%), neuroendocrine carcinomas 6 cases (9%), undifferentiated carcinoma 1 case (2%), adenosquamous carcinoma 1 case (2%), small-cell anaplastic carcinoma 1 case (2%), and pleomorphic sarcoma 1 case (2%).
Using a panel of antibodies, the adenocarcinoma cases showed 85% immunoreactivity with TTF1, 87% with napsin A, and 95% with CK7 (
Immunoreactivity for a panel of antibodies in various lung carcinoma. The panel included antibodies to thyroid transcription factor-1 (TTF1; orange), P40 (yellow), synaptophysin (green), napsin A (blue), and cytokeratin-7 (CK7; brown). Ab, antibody; CA, carcinoma.
Of the 40 cases of confirmed adenocarcinoma of the lung,
Among the 15 cases of
Frequency of epidermal growth factor receptor sequence variation types by sex in 40 cases of confirmed adenocarcinoma of the lung. Female patients (light gray) male patients (dark gray).
All the cases of variants were found in patients with Bamar ethnicity. The most distinctive finding was seen in relation to the smoking history. Of 15 patients with a variant, 10 were never-smokers and a significant association was found (
Association between
Present | 7 | 2 (29%) | 5 (71%) | 0.69 |
Absent | 33 | 13 (39%) | 20 (61%) | |
<40 years | 1 | 1 (100%) | 0 (0%) | 0.35 |
40–59 years | 11 | 5 (45%) | 6 (55%) | |
≥60 years | 28 | 9 (32%) | 19 (68%) | |
Male | 19 | 5 (26%) | 14 (74%) | 0.17 |
Female | 21 | 10 (48%) | 11 (52%) | |
Bamar | 38 | 15 (39%) | 23 (61%) | 0.52 |
Kayin | 2 | 0 (0.0%) | 2 (100%) | |
Unemployed | 18 | 9 (50%) | 9 (50%) | 0.60 |
Service and sale workers | 7 | 1 (14%) | 6 (86%) | |
Officers/managers | 5 | 1 (20%) | 4 (80%) | |
Professionals | 3 | 1 (33%) | 2 (67%) | |
Armed forces occupations | 3 | 2 (67%) | 1 (33%) | |
Technicians | 1 | 0 (0%) | 1 (100%) | |
Transporters | 2 | 1 (50%) | 1 (50%) | |
Agricultural, forestry, and fishery workers | 1 | 0 (0%) | 1 (100%) | |
Regular smokers | 4 | 0 (0%) | 4 (100%) | 0.008* |
Ex-smokers | 21 | 5 (24%) | 16 (76%) | |
Never-smokers | 15 | 10 (67%) | 5 (33%) | |
Exposure present | 9 | 1 (11%) | 8 (89%) | 0.12 |
No exposure | 31 | 14 (45%) | 17 (55%) |
Of the 40 cases of adenocarcinoma of the lung,
The most frequent variant types found in this study were exon 19 deletion and exon 21 L858 R substitution each accounting for 6 cases (40%). This finding was similar to that in a study in mainland China that found the most common variants to be exon 19 deletion and L858R substitutions [11]. In that study,
One case of exon 20 S7681 insertion was found in our study. There were also cases of double variation, i.e., 1 case of exon 21 L858R and exon 18 G719X, and 1 case of exon 20 S7681 and exon 18 G719X. Both cases of double variants in our study were in female patients. In a study in China, double variants were also found to be more common in female patients [15]. The oncogenic potential of the less common variants and double variants is largely unknown, and there is a suggestion that these types confer resistance to therapy such as gefitinib [14].
In our patients with
There were altogether 38 patients from the Bamar ethnic group with cases of adenocarcinoma and
There was a higher frequency of
Limitations of our study include the limitations of the EGFR Mutation Test [9] and the small sample size compared with other studies. The logical validity of χ2 tests is limited by sample size. Nevertheless, we were able to obtain sufficient data to present a preliminary study of
Adenocarcinoma dominates the histological types of lung cancer of the patients from Myanmar whom we tested.