الفهرس 
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Abstract
Breast cancer is the leading cause of cancer deaths in developing countries and the
second in developed ones among females according to GLOBOCAN 2012. It is a complex
and heterogeneous disease. Many factors play important roles in prognosis and clinical
management decisions, including: patient‘s age, tumor size, grade, stage and molecular
subtypes.
Molecular analysis of cancer is gaining enormous focus and awaits a fundamental
role in clinical decision making on a wider scale in the future. Several guidelines have
already adopted molecular profiling of breast tumors using multigene panel assays to aid in
staging and selecting patients that can forgo chemotherapeutic treatment. Although
treatment modalities for breast cancer have evolved in the past few years, yet, survival
rates in advanced breast cancer are still modest.
Epigenetic modification plays an important role in the pathogenesis of cancer and
responsiveness to therapeutic regimens. selective CpG island methylation is an important
epigenetic modification that occurs at genes promoters involved in DNA repair and tumor
suppression leading to their silencing and subsequently enhancement of tumor growth.
MGMT is a direct DNA repair gene that is ubiquitously expressed. It is responsible
for the removal of alkyl adducts from O6 position on guanine induced by alkylating agents
(such as cyclophosphamide) used as chemotherapy. MGMT promoter methylation had
been correlated with low expression of its protein and was found to have a predictive value
for gliomas response to alkylating agent, tenozolomide and in diffuse large B-cell
lymphoma patients treated with regimens including cyclophosphamide.
ABCG2, an ATP binding cassette membrane transporter, or breast cancer resistance
protein (BCRP) is responsible for the export of compounds outside the cell. Different
molecules have been identified as substrates for it including: doxorubicin, 5‘fluorouracil
and paclitaxel (Taxane). It’s over expression had been linked to chemoresistance as well.
Our work aimed at studying the predictive role of MGMT and ABCG2 promoter
methylation in the response of breast cancer patients receiving doxorubicincyclophosphamide
regimens.
Forty three female patients with advanced breast cancer (stage III and IV) were
included in our study assessed before receiving chemotherapeutic drugs: AC, FAC and
Taxane and after the completion of regimens. Response was successfully assessed in forty
two patients after AC (29 patients) and FAC (13 patients). Twenty eight patients only
could be assessed after Taxane therapy. They were sub grouped according to response into
responders: patients who achieved complete or partial response and non-responders:
patients who had a stable or progressive disease course according to RECIST criteria
version 1.1.
Responders to AC and FAC were 38.1% (16/42) [AC:14/16 and FAC:2/16], while,
non- responders were 61.9% (26/42) [AC:15/26 and FAC:11/26], the remaining patient
was deceased after two cycles of AC therefore, response could not be assessed. On the
other hand, responders to Taxane were 35.7% (10/28), while non-responders were 64.3% (18/28), the rest of the patients could not be assessed for response. Response was assessed
after completion of course (AC followed by Taxane) in 20 cases and response was seen in
50% of the patients, on the other hand, only 25% of patients that received FAC followed
by Taxane had response.
Age of the included cases at presentation ranged from 30-74 years with a mean age
of 48. IDC type was predominant in our study accounting for 93% (41/43) of the cases.
Molecular subtypes were evaluated using immunohistochemistry assay and grouped into
luminal (A and B), triple negative (TN) and HER2 expressing tumor. Cases with luminal
subtype were 72.1% (31/43), TN subtype 16.3% (7/43) and HER2 expressing subtype
11.6% (5/43).
Tumor grading was applied to tumor tissue sample and scored according to
Nottingham grading system. Tumors assessed in our study were grade 2 and 3 only, where
grade 2 accounted for the majority of cases; 67.4% (29/43) and grade 3 were 16.3% (7/43).
We could not retrieve data about grade for the remaining 7 cases. Staging of patients was
done at presentation according to the American Joint Committee on Cancer (AJCC) 8th
manual of cancer staging. 65.1% (28/43) of the cases presented with stage III while 34.9%
(15/43) presented with stage IV in our study.
In our study, there was no significant association between age and response to any
regimen/drug (AC-FAC-Taxane) alone or combined. Although, lower tumor grade and
stage were observed in relatively younger age groups in our study, we did not find
significant association between age and those factors. Molecular subtype was also not
associated with stage, age, grade or response.
Most patients with stage III at presentation had grade 2 tumors (22/25). However, no
statistically significant association was present between grade of tumor and clinical stage
of patients at presentation.
We also examined the predictive effect of grade and molecular subtypes on response
to doxorubicin- cyclophosphamide based regimen, we could not find any association
between them and response to different regimens/drugs (AC-FAC-Taxane) alone or
combined.
Stage III in our study was statistically associated with response to different
regimens/drugs (AC-FAC-Taxane) alone and combined; OR 95%CI 7(1.32-37.15) for
association with response to AC and FAC regimen combined, OR 95%CI 14.1(1.46-137.3)
for association with response to Taxane, OR 95%CI 8.3(1.34-51.67) for association with
response to AC and FAC followed by Taxane and OR 95%CI 13.5 (1.2-152.2) for
association with response to AC followed by Taxane.
In our study we conducted the methylation status analysis of MGMT and ABCG2
genes promoters using methyl specific PCR (MSP) technique. Methylation analysis of
MGMT and ABCG2 genes were performed on 41 formalin fixed paraffin embedded breast
cancer tissues and 2 slide stained fine needle aspiration samples. MSP was done and
products were successfully obtained in 100% of cases for both genes. MGMT promoter
was found to be methylated in 39.5% (17/43) of the cases. ABCG2 promoter was
methylated in only 14% (6/43) of cases. No association was observed between MGMT and /or ABCG2 methylation and age, molecular subtype, grade and stage. Moreover, no
correlation was found between the methylation of both genes.
Although there was lack of association of each gene with response to therapy when
assessed separately, we found a significant association of unmethylation of both genes
combined with response to AC followed by Taxane therapy.
Based on our work the following could be concluded:
1) Stage is the most important independent factor for prediction of response to
doxorubicin-cyclophosphamide based therapy.
2) Methylation event of the promoters of MGMT and ABCG2 combined could be a
potential bad predictive factor for response to AC/Taxane therapy and might be
considered as a predictive biomarker prior to administration of that regimen.
3) Classical prognostic factors such as grade, age and molecular subtypes were not
predictive of response to doxorubicin-cyclophosphamide based therapy.