progress in immunotherapy has provided novel insights into the roles of tumor - infiltrating lymphocytes ( tils ) in cancer .
the prognostic significance of tils has been determined in breast cancers , particularly human epidermal growth factor receptor 2 ( her2)positive breast cancers and triple - negative breast cancers .
the prognosis of her2-positive breast cancer has dramatically improved because of introduction of trastuzumab , a humanized monoclonal antibody targeting the extracellular domain of her2 .
immune - mediated mechanisms may play a role in treatment with trastuzumab . in the adjuvant
setting , a high level of tils is associated with better response to trastuzumab , and an abundant number of tils is an independent prognostic factor in her2-positive breast cancer .
based on this background , a group of investigators around the world convened to discuss the parameters and methodological issues associated with evaluating tils in breast cancers   and released recommendations for their evaluation .
they recommended that all mononuclear cells , including lymphocytes and plasma cells in intratumoral stroma , be scored as a percentage of the area occupied over the total intratumoral stromal area .
the innate immune system lies at the frontline of the cellular response to pathogen infection .
pattern recognition receptors sense microbial products , including rna produced during viral infection , and interferon ( ifn ) expression is then upregulated , resulting in stimulation of the transcription of various ifn - stimulated genes ( isgs ) , including the myxovirus resistance a ( mxa ) gene , through phosphorylation of signal transducer and activator of transcription 1 ( stat1 ) .
in addition to its function in innate immunity , isgs are known to be broadly expressed in various types of tumors , and ifns are known to participate in receptor - activated signaling pathways associated with the pathophysiology of malignancies .
additionally , some studies have shown that mxa may have a tumor - suppressive function .
expression of the mxa gene is regulated by ifns , but not by other cytokines . moreover
, mxa gene expression is not induced directly by viral infection , but is dependent on ifn signaling , making mxa an excellent marker for ifn action .
activation of the ifn pathway is a common outcome of damage - associated molecular pattern ( damp ) signaling .
a recent report demonstrated that high cytoplasmic expression of high mobility group ( hmg ) b1 and hmgn1 , which are damps associated with innate immunity in the extracellular environment , is associated with a high level of tils in her2-positive breast cancers .
we hypothesized that mxa , an effective marker of ifn signaling that is an important component of innate immunity , may play a role in tils in her2-positive breast cancers . in this study , we analyzed the expression of mxa protein in her2-positive breast cancers and the relationship between the expression level of mxa and various histopathologic variables , including til levels .
a total of 168 patients with her2-positive breast cancer who underwent surgery for primary breast cancer between 2011 and 2013 at pusan national university hospital were included .
expression statuses of estrogen receptor ( er ) , progesterone receptor ( pr ) , and her2 were evaluated for all samples , and immunohistochemistry for these proteins was performed at the time of diagnosis .
exemption from informed consent after de - identification of the patients information was approved by the institutional review board of pusan national university hospital .
hematoxylin and eosin ( h&e)-stained slides were reviewed by two pathologists ( a.k . and j.y.k . ) .
histopathologic factors included histologic grade , nuclear grade , necrosis , microcalcification , lymphovascular invasion , lymph node metastasis , tils level , tertiary lymphoid structures ( tlss ) around the ductal carcinoma in situ ( dcis ) , and tls around the invasive component .
tils levels were evaluated based on the recommendation by the international tils working group , and tils level was calculated according to the percentage of stroma in the invasive area ( fig .
tls is a lymph - node like structure characterized by ectopic aggregation of lymphoid cells with specialized high endothelial venules .
lymphoid accumulation containing vessels with high endothelial venule features ( plump and cuboidal endothelial cells ) was considered to be the tls ( fig .
we evaluated the ducts involved by dcis with surrounding tlss as a percentage of total ducts involved by dcis and the amount of tlss as a percentage of the total circumference of the invasive edge . to conduct statistical analysis ,
the tils levels were subdivided into three categories (  10% , 20%-60% , and > 60% ) or two categories (  60% and > 60% ) , as appropriate .
the level of tls abundance was divided into two subgroups ( low and high ) based on the mean value . to define the representative tumor area ,
each sample was arrayed with a tissue cylinder 2 mm in diameter using a tissue arraying instrument . to minimize tissue loss and the effects of tumor heterogeneity ,
tissue sections were stained with an automated immunohistochemical staining device ( benchmark xt , ventana medical systems , tucson , az ) .
briefly , 3-m - thick paraffin sections were deparaffinized in ez prep using an autostainer . for antigen retrieval , cell conditioner 1 (
ph 8.4 buffer ) or cell conditioner 2 ( ph 6.0 buffer ) was used .
optimally formulated antibodies targeting er ( 1:200 , leica biosystems , newcastle upon tyne , uk ) , pr ( 1:200 , leica biosystems ) , and her-2/neu ( 1:8 , ventana medical systems ) were used , and an anti - mxa antibody ( 1:1,000 , ab95926 , abcam , cambridge , uk ) was also applied . a her2/cep17 chromosome dual - probe ( ventana medical systems )
er and pr positivity were defined as at least 1% positive staining in tumor nuclei .
tumors that were positive for er or pr were categorized as the hormone receptor ( hr)positive group .
positivity was defined as an immunohistochemical staining score of 3 or gene amplification as identified by sish .
tumors were classified into the following two subtypes according to hr ( er and pr ) expression status : hr+/her2 + and hr/her2 + .
the intensity of the staining and percentage of positive tumor cells were considered together for mxa .
the intensity of the immunohistochemistry was graded as 0 ( negative ) , 1 ( weak ) , 2 ( moderate ) , and 3 strong ( fig .
the immunoscore was generated by multiplying the staining intensity by the percentage of the positive tumor cells . finally , we subdivided the tumors into two groups ( low and high expression ) according to the mean value to enable statistical analysis .
chi - square tests , linear - by - linear association tests , fisher exact tests , and spearman rank correlation tests were used as appropriate .
all tests were two - sided , and differences with p - values of less than 0.05 were considered significant .
a total of 168 patients with her2-positive breast cancer who underwent surgery for primary breast cancer between 2011 and 2013 at pusan national university hospital were included .
expression statuses of estrogen receptor ( er ) , progesterone receptor ( pr ) , and her2 were evaluated for all samples , and immunohistochemistry for these proteins was performed at the time of diagnosis .
exemption from informed consent after de - identification of the patients information was approved by the institutional review board of pusan national university hospital .
hematoxylin and eosin ( h&e)-stained slides were reviewed by two pathologists ( a.k . and j.y.k . ) .
histopathologic factors included histologic grade , nuclear grade , necrosis , microcalcification , lymphovascular invasion , lymph node metastasis , tils level , tertiary lymphoid structures ( tlss ) around the ductal carcinoma in situ ( dcis ) , and tls around the invasive component .
tils levels were evaluated based on the recommendation by the international tils working group , and tils level was calculated according to the percentage of stroma in the invasive area ( fig .
tls is a lymph - node like structure characterized by ectopic aggregation of lymphoid cells with specialized high endothelial venules .
lymphoid accumulation containing vessels with high endothelial venule features ( plump and cuboidal endothelial cells ) was considered to be the tls ( fig .
we evaluated the ducts involved by dcis with surrounding tlss as a percentage of total ducts involved by dcis and the amount of tlss as a percentage of the total circumference of the invasive edge . to conduct statistical analysis ,
the tils levels were subdivided into three categories (  10% , 20%-60% , and > 60% ) or two categories (  60% and > 60% ) , as appropriate .
the level of tls abundance was divided into two subgroups ( low and high ) based on the mean value .
each sample was arrayed with a tissue cylinder 2 mm in diameter using a tissue arraying instrument . to minimize tissue loss and the effects of tumor heterogeneity ,
tissue sections were stained with an automated immunohistochemical staining device ( benchmark xt , ventana medical systems , tucson , az ) .
briefly , 3-m - thick paraffin sections were deparaffinized in ez prep using an autostainer . for antigen retrieval , cell conditioner 1 (
ph 8.4 buffer ) or cell conditioner 2 ( ph 6.0 buffer ) was used .
optimally formulated antibodies targeting er ( 1:200 , leica biosystems , newcastle upon tyne , uk ) , pr ( 1:200 , leica biosystems ) , and her-2/neu ( 1:8 , ventana medical systems ) were used , and an anti - mxa antibody ( 1:1,000 , ab95926 , abcam , cambridge , uk ) was also applied . a her2/cep17 chromosome dual - probe ( ventana medical systems )
er and pr positivity were defined as at least 1% positive staining in tumor nuclei .
tumors that were positive for er or pr were categorized as the hormone receptor ( hr)positive group .
positivity was defined as an immunohistochemical staining score of 3 or gene amplification as identified by sish .
tumors were classified into the following two subtypes according to hr ( er and pr ) expression status : hr+/her2 + and hr/her2 + .
the intensity of the staining and percentage of positive tumor cells were considered together for mxa .
the intensity of the immunohistochemistry was graded as 0 ( negative ) , 1 ( weak ) , 2 ( moderate ) , and 3 strong ( fig .
the immunoscore was generated by multiplying the staining intensity by the percentage of the positive tumor cells .
finally , we subdivided the tumors into two groups ( low and high expression ) according to the mean value to enable statistical analysis .
all statistical analyses were conducted using the spss ver . 22 ( ibm corp . , armonk , ny ) .
chi - square tests , linear - by - linear association tests , fisher exact tests , and spearman rank correlation tests were used as appropriate .
all tests were two - sided , and differences with p - values of less than 0.05 were considered significant .
the patients ranged in age from 23 to 85 years ( mean age , 52.62 years ) .
all included patients were female , and the cohort was composed of 93 cases of left breast cancer and 75 cases of right breast cancer .
ninety cases of hr+/her2 + tumors and 78 cases of hr/her2 + tumors were included .
the clinicopathological characteristics of her2 + breast cancers according to hr expression status are summarized in table 1 .
high histological grade , high nuclear grade , and high level of tils were significantly associated with hr - negative cases ( p < 0.001 for all comparisons ) .
in her2-positive breast cancers , tumor cells only showed mxa protein expression in the cytoplasm .
high expression of mxa protein was significantly associated with high histological grade ( p=0.023 ) and high level of tils ( p=0.002 ) .
the level of tils was divided into three categories ( 0%-10% , 20%-60% , and > 60% ) for comparisons with various histopathologic parameters ( table 3 ) .
the high level of tils was significantly correlated with high histological grade ( p=0.001 ) , absence of lymphovascular invasion ( p=0.007 ) , absence of lymph node metastasis ( p=0.007 ) , absence of hr expression ( p < 0.001 ) , abundant tlss around dcis ( p=0.018 ) , and abundant tlss around the invasive component ( p < 0.001 ) .
the level of tils was divided into two groups (  60% and > 60% ) for survival analysis .
a high level of tils was associated with better disease - free survival in total her2-positive breast cancers ( p=0.028 ) and particularly in hr/her2 + breast cancers ( p=0.019 ) ( fig .
however , the expression level of mxa was not a prognostic factor ( data not shown ) .
the patients ranged in age from 23 to 85 years ( mean age , 52.62 years ) .
all included patients were female , and the cohort was composed of 93 cases of left breast cancer and 75 cases of right breast cancer .
ninety cases of hr+/her2 + tumors and 78 cases of hr/her2 + tumors were included .
the clinicopathological characteristics of her2 + breast cancers according to hr expression status are summarized in table 1 .
high histological grade , high nuclear grade , and high level of tils were significantly associated with hr - negative cases ( p < 0.001 for all comparisons ) .
we analyzed mxa protein expression in breast tissue by immunohistochemistry . in her2-positive breast cancers ,
high expression of mxa protein was significantly associated with high histological grade ( p=0.023 ) and high level of tils ( p=0.002 ) .
the level of tils was divided into three categories ( 0%-10% , 20%-60% , and > 60% ) for comparisons with various histopathologic parameters ( table 3 ) .
the high level of tils was significantly correlated with high histological grade ( p=0.001 ) , absence of lymphovascular invasion ( p=0.007 ) , absence of lymph node metastasis ( p=0.007 ) , absence of hr expression ( p < 0.001 ) , abundant tlss around dcis ( p=0.018 ) , and abundant tlss around the invasive component ( p < 0.001 ) .
the level of tils was divided into two groups (  60% and > 60% ) for survival analysis .
a high level of tils was associated with better disease - free survival in total her2-positive breast cancers ( p=0.028 ) and particularly in hr/her2 + breast cancers ( p=0.019 ) ( fig .
however , the expression level of mxa was not a prognostic factor ( data not shown ) .
ifn genes are transcriptionally silent under normal conditions . production of ifns is transient and triggered with stimulation by pathogen - associated molecular patterns , which are conserved among microbial species , and by damps , which are endogenous molecules released from damaged or injured cells .
ifns directly affect t - cell activation as a signal 3 cytokine , which is a specific cytokine signal involved in differentiation of nave t cells into effector and memory t cells .
additionally , type i ifn can affect t - cell activation indirectly through mediation of the differentiation of monocytes into mature dendritic cells , which are important antigen - presenting cells , and enhancement of antigen presentation by major histocompatibility ( mhc ) class ii synthesis and antigen processing .
type i ifns can elevate the expression of tumor programmed cell death ligand 1 ( pd - l1 ) , and the ifn - driven elevation of pd - l1 is thought to interact with programmed death 1 on t cells , resulting in t - cell exhaustion .
moreover , one report demonstrated that high expression of isgs is associated with poor response to therapy and immune suppression .
expression of mxa in cancer has been reported in various types of organs . in head and neck squamous cell carcinoma , hypermethylation of mxa
was observed relative to lymphocytes from healthy individuals . in the human prostate carcinoma cell line ,
brown et al .   revealed that silencing of mxa expression enhances cell migration , invasion , and anchorage independent growth , which are the hallmarks of an aggressive tumor .
quantitative proteome profiling showed that mxa has a potential role as a marker for lymph node metastasis in colorectal cancer .
mxa expression has been reported to be upregulated in cell line and xenograft models of hr - positive breast cancer with anti - estrogen resistance .
additionally , johansson et al .   showed that mxa expression was higher in a relapse group than in controls , but that it was not correlated with relapse - free survival and breast cancer - specific survival in er - positive breast cancer treated with adjuvant tamoxifen .
conversely , sistigu et al .   showed that mxa expression is positively associated with tumor grade , and that the type i ifn metagene centered around mxa can function as a biomarker for pathologic complete response to neoadjuvant anthracycline - based chemotherapy in breast cancer .
moreover , mxa expression predicts metastasis - free survival after adjuvant chemotherapy in the subset of patients of breast cancer with poor prognosis .
this study revealed the prognostic value of tils in her2-positive breast cancer and the correlation between mxa expression and til level .
it should be noted that this study had several limitations , including a relatively small cohort and a short follow - up period , which may not have been sufficient to draw conclusive results .
furthermore , multiple cell types in the tumor microenvironment can show high expression of isgs and affect the therapeutic response in breast cancer . in this study
, we only evaluated mxa expression in tumor cells due to constraints with tissue microarray sections .
overall , further studies of the effects of ifn signaling on tumor immunology in breast cancer are required to better elucidate the mechanism of tils and develop immunotherapies .
the prognostic value of til evaluation with h&e sections was assumed in this study , supporting the recommendations by an international tils working group 2014 .
several reports have indirectly supported the hypothesis that peripheral blood t and b lymphocytes are marshaled to tlss and that differentiated cells migrate to the tumor , where they develop into tils . in this study
, we showed that a high level of tils was significantly associated with the abundance of tlss around both the invasive component and dcis , which also indirectly supported the above hypothesis .
there are at least two subtypes of her2-positive breast cancers according to the cancer genome atlas network , the her2-enriched mrna subtype and luminal mrna subtype
. moreover , different clinical behaviors and histopathologic characteristics of hr+/her2 + breast cancers and hr/her2 + breast cancers have been reported . in this study
, we found that hr/her2 + breast cancers were significantly associated with a higher histologic grade , higher nuclear grade , and abundant amount of tils when compared to hr+/her2 + breast cancers , consistent with previous studies .
in summary , evaluation of tils with h&e section can provide prognostic information in routine practice . moreover , high expression of mxa in tumor cells was associated with a high level of tils in her2-positive breast cancers and a high level of tils was a prognostic factor for breast cancer .
 purposethe prognostic significance of tumor - infiltrating lymphocytes ( tils ) has been determined in breast cancers . 
 interferons can affect t - cell activity through direct and indirect mechanisms . 
 myxovirus resistance a ( mxa ) is an excellent marker of interferon activity . here , 
 we evaluated tils and mxa expression in human epidermal growth factor receptor 2 ( her2)positive breast cancers.materials and methodsninety cases of hormone receptor ( hr)+/her2 + tumors and 78 cases of hr/her2 + tumors were included . 
 the tils level was assessed using hematoxylin and eosin  stained full face sections , and mxa expressionwas evaluated by immunohistochemistrywith a tissue microarray.resultsmxa protein expression was significantly higher in tumors with high histologic grade ( p=0.023 ) and high levels of tils ( p=0.002 ) . 
 high levels of tils were correlated with high histological grade ( p=0.001 ) , negative lymphovascular invasion ( p=0.007 ) , negative lymph node metastasis ( p=0.007 ) , absence of hr expression ( p < 0.001 ) , abundant tertiary lymphoid structures ( tlss ) around ductal carcinoma in situ ( p=0.018 ) , and abundant tlss around the invasive component ( p < 0.001 ) . 
 high levels of tils were also associated with improved disease - free survival , particularly in hr/her2 + breast cancers . 
 however , mxa was not a prognostic factor.conclusionhigh expression of mxa in tumor cells was associated with high levels of tils in her2-positive breast cancers . 
 additionally , a high level of tils was a prognostic factor for breast cancer , whereas the level of mxa expression had no prognostic value . 
Introduction
Materials and Methods
1. Patient and tissue specimens
2. Histologic evaluation
3. Construction of tissue microarray blocks and immunohistochemical evaluation
4. Statistical analysis
Results
1. Clinicopathological characteristics of HER2-positive breast cancers
2. Expression of MxA protein in HER2-positive breast cancers
3. Prognostic significance of TILs and MxA protein expression
Discussion
Conclusion