the ncic clinical trials group ma.17 study was a prospective , randomized , double - blind , placebo - controlled , phase iii trial of letrozole as extended adjuvant therapy for 5 years in postmenopausal breast cancer patients who remained disease - free after having completed approximately 5 years of standard adjuvant tamoxifen treatment ( figure 1a ) .
a total of 5157 eligible postmenopausal women with hormone receptor  positive ( er+ and/or pr+ ) tumors were randomly assigned to receive either letrozole or placebo after completing approximately 5 years of standard adjuvant tamoxifen therapy . in accordance with trial protocol ,
the ma.17 trial was unblinded in 2003 after the first interim analysis ( median follow - up of 30 months ) demonstrated a statistically significant disease - free survival benefit and a trend toward survival advantage in patients who received letrozole .
patients on the placebo arm were offered letrozole for a planned period of 5 years . the overall ma.17 trial design ( a ) , and the sample consort of the ma.17 biomarker study ( b ) . because of the practical obstacles to retrospectively obtain primary tumor blocks from ma.17 participants , a nested case - control design using all available recurrent case subjects with a planned ratio of 1:2 was used to study the ability of h / i to predict late recurrence and the benefit from extended therapy with letrozole .
case subjects included patients with local , regional , or distant recurrence for which primary tumor tissue blocks were available .
for each case subject , two matched control subjects were selected in which the control subject had been recurrence - free for a period of time longer than the case subject .
all patients with disease recurrence and with available formalin - fixed paraffin - embedded ( ffpe ) tumor tissue blocks were included in this study .
detailed methods for case - control selection are described in supplementary methods ( available online ) .
the prespecified objectives of this study were to determine whether h / i groups [ based on a prespecified and validated cut point ( 1113 ) ] would predict late disease recurrence and responsiveness / benefit from extended letrozole therapy after 5 years tamoxifen treatment .
the primary endpoint was any breast cancer recurrence , including breast , chest wall , nodal , or metastatic site . informed consent for the use of tumor specimens for correlative science studies were obtained from each subject .
the massachusetts general hospital institutional review board approved the use of the ma.17 tumor samples for this study .
tumor size and grade , lymph node status , and er and pr status were assessed using local institutional pathology laboratory criteria .
central tumor grade was assessed using the elston and ellis modified version of the bloom and richardson method by a single subspecialty - trained breast pathologist ( d.c .
central review for er , pr , and her-2 expression by immunohistochemistry ( ihc ) and her gene amplification by fluorescence in situ hybridization was scored by two pathologists ( d.c . sgroi and a.k .
bhan ) using two 1.0-mm tissue microarray cores using previously described methods summarized in the supplementary material ( available online ) ( 15,16 ) .
gene expression analysis of formalin - fixed paraffin - embedded specimens was performed blinded to clinical outcome by biotheranostics inc , as previously described ( 1113 ) .
briefly , hematoxylin and eosin  stained slides were reviewed to guide manual microdissection for tumor enrichment if necessary .
after rna extraction from three to six 8-m unstained tissue sections and dnase treatment , total rna content was measured and the absence of dna contamination verified as previously described ( 11,13 ) .
total rna was reverse transcribed , and the resulting cdna was preamplified by performing eight rounds of pcr using the preamp master mix kit ( applied biosystems , foster city , ca ) before being analyzed by taqman rt - pcr .
high and low h / i groups were determined using a prespecified cutpoint ( 0.06 ) that was determined and validated in our previous studies ( 1113 ) .
reportable results were generated using rna derived from all 249 formalin - fixed paraffin - embedded tumor samples submitted for testing . because of the nature of matched case - control study , unadjusted conditional logistic regression that takes matching set into consideration was used to examine whether h / i was prognostic of late disease recurrence and whether h / i predicts benefit from extended letrozole therapy by including an interaction term between the treatment and predefined h / i groups in the model and testing with statistical contrasts ( 17 ) .
conditional logistic regression was also performed in an adjusted analysis that also included the following prespecified covariates : age , tumor size , tumor grade , lymph node status , and er , pr , and her2 status .
similarly , the predictive analyses in clinically relevant patient subgroups was performed by conditional logistic regressions with a three - way interaction term that included the clinical variable of interest in addition to the treatment and h / i groups .
odds ratios ( ors ) and the 95% confidence intervals ( cis ) were estimated from conditional logistic regressions .
the absolute recurrence - free survival ( rfs ) at 5 years and the corresponding 95% ci were estimated using methods developed by langholz and borgan ( 18 ) for nested case - control study by taking into consideration the number of patients at risk from the entire ma.17 trial at the time of each recurrence in the case - control study .
statistical significance of the interaction between extended letrozole treatment and h / i index as a continuous variable was assessed by a likelihood ratio test that compared a reduced model without h / i index by treatment interaction with the competing full model , which included the interaction .
linearity of h / i index was tested by a likelihood ratio test that compared a model including the h / i index transformed by restricted cubic spline ( 19 ) with a model including only the linear h / i index .
if a significant nonlinearity existed , then all analyses involving continuous h / i were based on h / i index transformed by restricted cubic spline .
all p values were two - sided , and a p value less than .05 was considered statistically significant .
all analyses were performed using r statistical package ( version 2.12.2 , http://www.r-project.org ) , except for absolute risk estimation for which sas software ( sas institute inc , cary , nc ) was used .
the ncic clinical trials group ma.17 study was a prospective , randomized , double - blind , placebo - controlled , phase iii trial of letrozole as extended adjuvant therapy for 5 years in postmenopausal breast cancer patients who remained disease - free after having completed approximately 5 years of standard adjuvant tamoxifen treatment ( figure 1a ) .
a total of 5157 eligible postmenopausal women with hormone receptor  positive ( er+ and/or pr+ ) tumors were randomly assigned to receive either letrozole or placebo after completing approximately 5 years of standard adjuvant tamoxifen therapy . in accordance with trial protocol ,
the ma.17 trial was unblinded in 2003 after the first interim analysis ( median follow - up of 30 months ) demonstrated a statistically significant disease - free survival benefit and a trend toward survival advantage in patients who received letrozole .
patients on the placebo arm were offered letrozole for a planned period of 5 years . the overall ma.17 trial design ( a ) , and the sample consort of the ma.17 biomarker study ( b ) . because of the practical obstacles to retrospectively obtain primary tumor blocks from ma.17 participants , a nested case - control design using all available recurrent case subjects with a planned ratio of 1:2 was used to study the ability of h / i to predict late recurrence and the benefit from extended therapy with letrozole .
case subjects included patients with local , regional , or distant recurrence for which primary tumor tissue blocks were available .
for each case subject , two matched control subjects were selected in which the control subject had been recurrence - free for a period of time longer than the case subject .
all patients with disease recurrence and with available formalin - fixed paraffin - embedded ( ffpe ) tumor tissue blocks were included in this study .
detailed methods for case - control selection are described in supplementary methods ( available online ) .
the prespecified objectives of this study were to determine whether h / i groups [ based on a prespecified and validated cut point ( 1113 ) ] would predict late disease recurrence and responsiveness / benefit from extended letrozole therapy after 5 years tamoxifen treatment .
the primary endpoint was any breast cancer recurrence , including breast , chest wall , nodal , or metastatic site . informed consent for the use of tumor specimens for correlative science studies were obtained from each subject .
the massachusetts general hospital institutional review board approved the use of the ma.17 tumor samples for this study .
tumor size and grade , lymph node status , and er and pr status were assessed using local institutional pathology laboratory criteria .
central tumor grade was assessed using the elston and ellis modified version of the bloom and richardson method by a single subspecialty - trained breast pathologist ( d.c .
central review for er , pr , and her-2 expression by immunohistochemistry ( ihc ) and her gene amplification by fluorescence in situ hybridization was scored by two pathologists ( d.c . sgroi and a.k .
bhan ) using two 1.0-mm tissue microarray cores using previously described methods summarized in the supplementary material ( available online ) ( 15,16 ) .
gene expression analysis of formalin - fixed paraffin - embedded specimens was performed blinded to clinical outcome by biotheranostics inc , as previously described ( 1113 ) .
briefly , hematoxylin and eosin  stained slides were reviewed to guide manual microdissection for tumor enrichment if necessary .
after rna extraction from three to six 8-m unstained tissue sections and dnase treatment , total rna content was measured and the absence of dna contamination verified as previously described ( 11,13 ) .
total rna was reverse transcribed , and the resulting cdna was preamplified by performing eight rounds of pcr using the preamp master mix kit ( applied biosystems , foster city , ca ) before being analyzed by taqman rt - pcr .
high and low h / i groups were determined using a prespecified cutpoint ( 0.06 ) that was determined and validated in our previous studies ( 1113 ) .
reportable results were generated using rna derived from all 249 formalin - fixed paraffin - embedded tumor samples submitted for testing .
because of the nature of matched case - control study , unadjusted conditional logistic regression that takes matching set into consideration was used to examine whether h / i was prognostic of late disease recurrence and whether h / i predicts benefit from extended letrozole therapy by including an interaction term between the treatment and predefined h / i groups in the model and testing with statistical contrasts ( 17 ) .
conditional logistic regression was also performed in an adjusted analysis that also included the following prespecified covariates : age , tumor size , tumor grade , lymph node status , and er , pr , and her2 status .
similarly , the predictive analyses in clinically relevant patient subgroups was performed by conditional logistic regressions with a three - way interaction term that included the clinical variable of interest in addition to the treatment and h / i groups .
odds ratios ( ors ) and the 95% confidence intervals ( cis ) were estimated from conditional logistic regressions . the absolute recurrence - free survival ( rfs ) at 5 years and the corresponding 95% ci were estimated using methods developed by langholz and borgan ( 18 ) for nested case - control study by taking into consideration the number of patients at risk from the entire ma.17 trial at the time of each recurrence in the case - control study .
statistical significance of the interaction between extended letrozole treatment and h / i index as a continuous variable was assessed by a likelihood ratio test that compared a reduced model without h / i index by treatment interaction with the competing full model , which included the interaction .
linearity of h / i index was tested by a likelihood ratio test that compared a model including the h / i index transformed by restricted cubic spline ( 19 ) with a model including only the linear h / i index .
if a significant nonlinearity existed , then all analyses involving continuous h / i were based on h / i index transformed by restricted cubic spline .
all p values were two - sided , and a p value less than .05 was considered statistically significant .
all analyses were performed using r statistical package ( version 2.12.2 , http://www.r-project.org ) , except for absolute risk estimation for which sas software ( sas institute inc , cary , nc ) was used .
tumor samples from 100 patients with disease recurrence were obtained retrospectively and matched to 200 patients without disease recurrence . because the predefined cutpoint of h / i was previously tested with samples associated with patient outcome defined as local , regional , or distant metastasis ( 1113 ) , patients with contralateral and unknown recurrences were removed .
therefore our final cohort consisted of 83 patients with local , regional , or distant recurrence matched with 166 patients without recurrence ( figure 1b ) .
a post hoc power analysis indicated that with this sample size , the power to detect an odds ratio of 3 for comparing risk of late recurrence without extended letrozole vs that with letrozole in the h / i - high group was 82% at a 5% significance level .
the majority of patients was aged greater than 50 years with t1/t2 stage disease , and a larger proportion was node - positive and did not receive adjuvant chemotherapy . within unmatched variables , there was a statistically significant difference between the number of case subjects and control subjects for letrozole vs placebo and high vs low h / i groups ( table 1 ) .
case subjects and control subjects are similar to the overall ma.17 study population with respect to key clinical parameters with the exception of nodal status and radiation therapy ( supplementary table 1 , available online ) .
er = estrogen receptor ; h / i = hoxb13/il17br ; pr = progesterone receptor .
 p values were calculated using the fisher exact test , except for  type of surgery ,  for which binomial proportional test was used . 
high and low h / i groups were determined using a prespecified cutpoint ( 0.06 ) that was determined and validated in our previous studies ( 1113 ) .
the observed clinical benefit from letrozole within different patient subgroups was similar between the case - control study and the entire study population reported in the ma.17 trial ( table 2 ) .
the benefit was statistically significant in both studies in all subgroups except within the lymph node  positive subgroup for the case - control , which may be because of small sample size for the relatively smaller size of the benefit ( vs lymph node negative ) ( table 2 ) .
for example , the odds ratio estimated by conditional logistic regression with an interaction between clinical factors and the treatment in this case - control study and the hazard ratio estimated from the overall ma.17 trial as in published literature ( 6,20 ) were as follows : 0.20 and 0.45 for node negative , 0.63 and 0.61 for node positive , 0.44 and 0.73 for aged greater or equal to 50 years , 0.42 and 0.58 for hormone receptor positive , and 0.45 and 0.49 for er+/pr+ ( table 2 ) .
comparison of clinical benefit from extended letrozole therapy in patient subgroups of the nested case - control study and the overall ma.17 trial * * odds ratio ( or ) and 95% confidence interval ( ci ) were calculated from conditional logistic regression .
er = estrogen receptor ; hr = hazard ratio ; pr = progesterone receptor . in the placebo arm , h
/ i status was statistically significantly associated with prognosis in the unadjusted model , as women in the high h / i group experienced substantially worse prognosis than those in the low h / i group ( or = 2.24 ; 95% ci = 1.09 to 4.61 ; p = .03 ) .
h / i status remained marginally statistically significant in the adjusted model ( or = 2.15 ; 95% ci = 1.00 to 4.64 ; p = .05 ) after adjusting for all clinicopathological factors examined ( age , tumor size , tumor grade , nodal status , er , pr , her2 ) ( supplementary table 2 ) . in the letrozole arm , however , h / i was not prognostic for late recurrence in neither the unadjusted nor the adjusted models ( p = .72 and .63 , respectively ) . in the unadjusted analysis , high h / i group was statistically significantly associated with patient benefit from letrozole ( or = 0.35 ; 95% ci = 0.16 to 0.75 ; p = .007 ) ( table 3 ) . in the adjusted model , which included all clinicopathological factors as covariates , high h
/ i remained statistically significantly associated with patient benefit from letrozole ( or = 0.33 ; 95% ci = 0.15 to 0.73 ; p = .006 ) ( table 3 ; figure 2 ) , which represented a 67% reduction in the risk of recurrence with extended letrozole treatment as compared with placebo .
unadjusted and adjusted analyses of treatment benefit by hoxb13/il17br ( h / i ) groups * odds ratio ( or ) , 95% confidence interval ( ci ) , and p values were calculated from conditional logistic regression .
er = estrogen receptor ; pr = progesterone receptor . forest plots showing odds ratio ( or ) for recurrence associated with clinico - pathological factors and treatment effect for each of the hoxb13/il17br ( h / i ) groups .
red color indicates h / i - high patients ; black color indicates h / i - low patients .
the absolute rfs at 4 years estimated from this case - control study was 86.6% ( 95% ci = 80.6% to 90.9% ) and 93.4% ( 95% ci = 90.2% to 95.6% ) for patients receiving placebo and letrozole , respectively .
this is consistent with the 4-year disease - free survival ( 87% and 93% for placebo and letrozole , respectively ) estimated from the parental ma.17 trial ( 2 ) for low h / i patients , the 5-year rfs was 87% ( 95% ci = 76.8% to 92.9% ) and 91% ( 95% ci = 83.1% to 95.3% ) in the placebo and letrozole group , respectively , showing a non - statistically significant reduction in the risk of recurrence of 4% ( p = .35 ) .
however , for high h / i patients , the 5-year rfs was 73% ( 95% ci = 56.6% to 84.1% ) and 89.5%
( 95% ci = 80.3% to 94.5% ) in the placebo and letrozole group , demonstrating a 16.5% reduction in the absolute risk of recurrence at 5 years ( p = .007 ) ( table 4 ) .
estimates of recurrence - free survival ( rfs ) at 5 years in patients who were treated with placebo or extended letrozole * rfss and 95% confidence intervals ( cis ) were calculated from conditional logistic regression using the method developed by langholz and borgan for nested case - control study .
h / i = hoxb13/il17br . to examine whether continuous er and pr measurements would affect the predictive performance of h / i , gene expression values of er and pr by rt - pcr were used in the adjusted model instead of binary immunohistochemistry er and pr values .
high h / i remained statistically significant with extended letrozole benefit ( or = 0.32 ; 95% ci = 0.14 to 0.72 ; p = .006 ) , whereas low h / i still showed no statistically significant benefit ( or = 0.59 ; 95% ci = 0.25 to 1.39 ; p = .23 ) .
linearity testing with a conditional logistic regression model indicated that there was a significant nonlinear relationship of h / i index as a continuous variable with recurrence ( p = .02 ) ; therefore h / i index was transformed by restricted cubic spline when assessing the statistical significance of the interaction between the treatment and continuous h / i index .
likelihood ratio test showed that the interaction between h / i and letrozole therapy was statistically significant ( p = 0.03 ) , adjusting for clinicopathological factors .
unplanned analyses were conducted to determine whether patient subgroups were differentially associated with the predictive performance of h / i by including a conditional logistic regression three - way interaction term between the clinical variable of interest , treatment , and h / i groups .
high h / i was statistically significantly predictive of letrozole benefit in our overall study population .
analyses of subsets including nodal , her2 , er , and pr status are shown in supplementary table 3 and supplementary figure 1 ( available online ) .
tumor samples from 100 patients with disease recurrence were obtained retrospectively and matched to 200 patients without disease recurrence . because the predefined cutpoint of h / i was previously tested with samples associated with patient outcome defined as local , regional , or distant metastasis ( 1113 ) , patients with contralateral and unknown recurrences were removed .
therefore our final cohort consisted of 83 patients with local , regional , or distant recurrence matched with 166 patients without recurrence ( figure 1b ) .
a post hoc power analysis indicated that with this sample size , the power to detect an odds ratio of 3 for comparing risk of late recurrence without extended letrozole vs that with letrozole in the h / i - high group was 82% at a 5% significance level .
the majority of patients was aged greater than 50 years with t1/t2 stage disease , and a larger proportion was node - positive and did not receive adjuvant chemotherapy . within unmatched variables , there was a statistically significant difference between the number of case subjects and control subjects for letrozole vs placebo and high vs low h / i groups ( table 1 ) .
case subjects and control subjects are similar to the overall ma.17 study population with respect to key clinical parameters with the exception of nodal status and radiation therapy ( supplementary table 1 , available online ) .
er = estrogen receptor ; h / i = hoxb13/il17br ; pr = progesterone receptor .
 p values were calculated using the fisher exact test , except for  type of surgery ,  for which binomial proportional test was used . 
high and low h / i groups were determined using a prespecified cutpoint ( 0.06 ) that was determined and validated in our previous studies ( 1113 ) .
the observed clinical benefit from letrozole within different patient subgroups was similar between the case - control study and the entire study population reported in the ma.17 trial ( table 2 ) .
the benefit was statistically significant in both studies in all subgroups except within the lymph node  positive subgroup for the case - control , which may be because of small sample size for the relatively smaller size of the benefit ( vs lymph node negative ) ( table 2 ) .
for example , the odds ratio estimated by conditional logistic regression with an interaction between clinical factors and the treatment in this case - control study and the hazard ratio estimated from the overall ma.17 trial as in published literature ( 6,20 ) were as follows : 0.20 and 0.45 for node negative , 0.63 and 0.61 for node positive , 0.44 and 0.73 for aged greater or equal to 50 years , 0.42 and 0.58 for hormone receptor positive , and 0.45 and 0.49 for er+/pr+ ( table 2 ) .
comparison of clinical benefit from extended letrozole therapy in patient subgroups of the nested case - control study and the overall ma.17 trial * * odds ratio ( or ) and 95% confidence interval ( ci ) were calculated from conditional logistic regression .
in the placebo arm , h / i status was statistically significantly associated with prognosis in the unadjusted model , as women in the high h / i group experienced substantially worse prognosis than those in the low h / i group ( or = 2.24 ; 95% ci = 1.09 to 4.61 ; p = .03 ) .
h / i status remained marginally statistically significant in the adjusted model ( or = 2.15 ; 95% ci = 1.00 to 4.64 ; p = .05 ) after adjusting for all clinicopathological factors examined ( age , tumor size , tumor grade , nodal status , er , pr , her2 ) ( supplementary table 2 ) . in the letrozole arm , however , h / i was not prognostic for late recurrence in neither the unadjusted nor the adjusted models ( p = .72 and .63 , respectively ) .
in the unadjusted analysis , high h / i group was statistically significantly associated with patient benefit from letrozole ( or = 0.35 ; 95% ci = 0.16 to 0.75 ; p = .007 ) ( table 3 ) . in the adjusted model , which included all clinicopathological factors as covariates , high h
/ i remained statistically significantly associated with patient benefit from letrozole ( or = 0.33 ; 95% ci = 0.15 to 0.73 ; p = .006 ) ( table 3 ; figure 2 ) , which represented a 67% reduction in the risk of recurrence with extended letrozole treatment as compared with placebo .
unadjusted and adjusted analyses of treatment benefit by hoxb13/il17br ( h / i ) groups * odds ratio ( or ) , 95% confidence interval ( ci ) , and p values were calculated from conditional logistic regression .
er = estrogen receptor ; pr = progesterone receptor . forest plots showing odds ratio ( or ) for recurrence associated with clinico - pathological factors and treatment effect for each of the hoxb13/il17br ( h / i ) groups .
red color indicates h / i - high patients ; black color indicates h / i - low patients .
the absolute rfs at 4 years estimated from this case - control study was 86.6% ( 95% ci = 80.6% to 90.9% ) and 93.4% ( 95% ci = 90.2% to 95.6% ) for patients receiving placebo and letrozole , respectively .
this is consistent with the 4-year disease - free survival ( 87% and 93% for placebo and letrozole , respectively ) estimated from the parental ma.17 trial ( 2 ) for low h / i patients , the 5-year rfs was 87% ( 95% ci = 76.8% to 92.9% ) and 91% ( 95% ci = 83.1% to 95.3% ) in the placebo and letrozole group , respectively , showing a non - statistically significant reduction in the risk of recurrence of 4% ( p = .35 ) .
however , for high h / i patients , the 5-year rfs was 73% ( 95% ci = 56.6% to 84.1% ) and 89.5% ( 95% ci = 80.3% to 94.5% ) in the placebo and letrozole group , demonstrating a 16.5% reduction in the absolute risk of recurrence at 5 years ( p = .007 ) ( table 4 ) .
estimates of recurrence - free survival ( rfs ) at 5 years in patients who were treated with placebo or extended letrozole * rfss and 95% confidence intervals ( cis ) were calculated from conditional logistic regression using the method developed by langholz and borgan for nested case - control study .
to examine whether continuous er and pr measurements would affect the predictive performance of h / i , gene expression values of er and pr by rt - pcr were used in the adjusted model instead of binary immunohistochemistry er and pr values .
high h / i remained statistically significant with extended letrozole benefit ( or = 0.32 ; 95% ci = 0.14 to 0.72 ; p = .006 ) , whereas low h / i still showed no statistically significant benefit ( or = 0.59 ; 95% ci = 0.25 to 1.39 ; p = .23 ) .
linearity testing with a conditional logistic regression model indicated that there was a significant nonlinear relationship of h / i index as a continuous variable with recurrence ( p = .02 ) ; therefore h / i index was transformed by restricted cubic spline when assessing the statistical significance of the interaction between the treatment and continuous h / i index .
likelihood ratio test showed that the interaction between h / i and letrozole therapy was statistically significant ( p = 0.03 ) , adjusting for clinicopathological factors .
unplanned analyses were conducted to determine whether patient subgroups were differentially associated with the predictive performance of h / i by including a conditional logistic regression three - way interaction term between the clinical variable of interest , treatment , and h / i groups .
high h / i was statistically significantly predictive of letrozole benefit in our overall study population .
analyses of subsets including nodal , her2 , er , and pr status are shown in supplementary table 3 and supplementary figure 1 ( available online ) .
currently , for er - positive breast cancer patients , there are no established tests that predict treatment benefit from extended adjuvant endocrine therapy ( 15 ) . in this
prospectively defined , retrospective analysis of a nested case - controlled study of ma.17 , patients with high h / i  expressing tumors had a 67% reduction in relative risk of recurrence and 16.5% reduction in the absolute risk of recurrence at 5 years when taking letrozole , as compared with placebo .
notably , the strong consistency in the estimates of 4-year rfs in both treatment groups calculated from this case - control study vs those from the parental ma.17 study population contends that the case - control samples are representative of the entire ma.17 study population .
in addition , there was a statistically significant interaction between h / i as a continuous index and extended aromatase inhibitor ( ai ) treatment .
as a comparison , continuous er and pr values measured by rt - pcr had no statistically significant interaction with extended ai treatment ( p = .11 and .24 , respectively ) .
the prognostic performance of high h / i for late recurrence within the placebo group was offset by the benefit from letrozole therapy for patients with tumors expressing high h / i .
these data suggest that high h / i within the primary tumor stratified a group of patients that had a greater likelihood for benefit from extended adjuvant letrozole above the known residual benefit from 5 years of adjuvant tamoxifen therapy . in addition
, patients with high h / i in their primary tumor have a greater likelihood of experiencing a recurrence beyond 5 years of tamoxifen treatment if their adjuvant therapy is not extended with letrozole .
although mechanistically unclear , h / i has both prognostic and predictive performance similar to her2 in that higher expression of the biomarker suggests a worse prognosis for breast cancer patients yet indicates a favorable response to a given therapy .
as our retrospective case subject selection was based on available tissue blocks , the size of our case - control cohort is substantially smaller than the overall ma.17 trial , and this selection could have introduced bias . despite this ,
apart from fewer patients treated with adjuvant radiation therapy and a greater proportion with lymph node  positive disease , our cohort was similar to the overall ma.17 study population with respect to key clinical parameters ( supplementary table 1 , available online ) .
moreover , we showed similar benefit from letrozole treatment in our overall case - control cohort and within important subgroups in our study as compared with the overall ma.17 patient population .
the clinical applications of our findings apply to many women with er+ early breast cancer .
first , 5 years of adjuvant tamoxifen remains the standard adjuvant therapy for premenopausal women most of whom are menopausal after chemotherapy and 5 years of tamoxifen and are therefore potential candidates for extended ai therapy .
second , although upfront ai adjuvant therapy has become more common , 5 years of tamoxifen remains a common adjuvant therapy among postmenopausal women globally .
identifying which patients are vulnerable to a late recurrence and who among them will benefit from extended therapy is of substantial clinical value .
our biomarker should allow many women to avoid unnecessary treatment and for the focus to center on those in most need of therapy .
this , in turn , could improve compliance with medication and further improve outcomes ( 2124 ) . in conclusion ,
we have demonstrated in a prospective  retrospective analysis of the ma.17 trial that patients with primary tumors with a high h / i index who are disease - free after 4.5 to 6 years of adjuvant tamoxifen therapy receive benefit from extended adjuvant letrozole therapy .
performance of h / i in predicting treatment benefit from extended ai therapy for women who have used tamoxifen for less than 5 years and those who have taken upfront ai without any tamoxifen for 5 years awaits prospective  retrospective analyses of currently ongoing trials such as ma.17r , nsabp42 , and others ( 25,26 ) .
this work was supported by grants from the avon foundation ( to d.c.s . and p.e.g . ) ; the national institute of health ( r01ca112021 to d.c.s . ) ; the breast cancer foundation ( to d.c.s . and
; the department of defense breast cancer research program ( w81xwh-04 - 1 - 0606 to d.c.s . ) ; the nci spore in breast cancer at massachusetts general hospital ( to p.e.g . and d.c.s . ) ; and novartis .
 backgroundbiomarkers to optimize extended adjuvant endocrine therapy for women with estrogen receptor ( er)positive breast cancer are limited . the hoxb13/il17br ( h / i ) 
 biomarker predicts recurrence risk in er - positive , lymph node  negative breast cancer patients . 
 h / i was evaluated in ma.17 trial for prognostic performance for late recurrence and treatment benefit from extended adjuvant letrozole.methodsa prospective  retrospective , nested case - control design of 83 recurrences matched to 166 nonrecurrences from letrozole- and placebo - treated patients within ma.17 was conducted . 
 expression of h / i within primary tumors was determined by reverse - transcription polymerase chain reaction with a prespecified cutpoint . 
 the predictive ability of h / i for ascertaining benefit from letrozole was determined using multivariable conditional logistic regression including standard clinicopathological factors as covariates . 
 all statistical tests were two-sided.resultshigh h / i was statistically significantly associated with a decrease in late recurrence in patients receiving extended letrozole therapy ( odds ratio [ or ] = 0.35 ; 95% confidence interval [ ci ] = 0.16 to 0.75 ; p = .007 ) . in an adjusted model with standard clinicopathological factors , high h 
 / i remained statistically significantly associated with patient benefit from letrozole ( or = 0.33 ; 95% ci = 0.15 to 0.73 ; p = .006 ) . 
 reduction in the absolute risk of recurrence at 5 years was 16.5% for patients with high h / i ( p = .007 ) . 
 the interaction between h / i and letrozole treatment was statistically significant ( p = .03).conclusionsin the absence of extended letrozole therapy , high h / i identifies a subgroup of er - positive patients disease - free after 5 years of tamoxifen who are at risk for late recurrence . when extended endocrine therapy with letrozole is prescribed , high h / i predicts benefit from therapy and a decreased probability of late disease recurrence . 
Methods
Patients, Case-Control Selection, Study Design, and Endpoints
Sample Preparation and Pathological Evaluation
Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Analysis and Calculation of H/I
Statistical Analysis
Results
Study Design and Patient and Tumor Characteristics
Prognostication of Late Disease Recurrence by H/I
Predictive Performance of H/I for Benefit From Letrozole Treatment
Predictive Performance of H/I Within Clinically Relevant Subgroups
Discussion
Funding
Supplementary Material