a study data set of 817 cases with available oncotype dx test results ( cases from 2004 to 2009 , from magee - womens hospital of the university of pittsburgh medical center ) was used to build the prediction models .
all cases included in the study were sent for oncotype dx testing owing to clinical requests received by the department of pathology . tumor grading information ( including the nottingham grade , score , and the individual components of grading ) , tumor size , semiquantitative immunohistochemical results for er , pr , her2 , and ki-67 were available from pathology reports . at our institution ,
er and pr results are reported using a semiquantitative immunohistochemical score ( commonly known as 
h - score ' ) , which details the percentage of positive cells showing none , weak , moderate , or strong staining .
the score is given as the sum of the percent staining multiplied by an ordinal value corresponding to the intensity level ( 0 : none ; 1 + : weak ; 2 : moderate ; 3 + : strong ) .
the resulting score ranges from 0 ( no staining in the tumor ) to 300 ( diffuse intense staining ) .
her2 semiquantitated immunohistochemical results were reported according to the college of american pathologist / american society of clinical oncology ( cap / asco ) guidelines .
immunohistochemical scores of 0 and 1 + were considered a negative result and a score of 3 + was considered a positive result . the final her2 status on her2 2 + immunohistochemical cases was determined based on the fish result and classified as negative ( negative for amplification , ie her2:cep17 ratio of < 1.8 ) , positive ( positive for unequivocal amplification , her2:cep17 ratio of > 2.2 ) , or equivocal ( her2:cep17 ratio of 1.82.2 ) .
statistical analyses were performed using sas version 9.2 ( sas institute , cary , nc , usa ) .
a significance level was set at 0.05 and all p - values reported were two - sided .
multiple linear regression analysis was performed to model the prediction of the oncotype dx recurrence score by nottingham score ( range 39 ) , ki-67 labeling index ( 0100 ) , tumor size ( in cm ) , h - scores ( range : 0300 ) for er and pr , and her2 status ( negative , equivocal , or positive ) .
the first regression model included all available parameters ( including ki-67 index ) for prediction of oncotype dx recurrence score .
the third regression model included only semiquantitative immunohistochemical expression level for er , pr , her2 , and ki-67 .
ki-67 was available in less than half of the study data set cases , as it has only been routinely used at our institution since 2007 . using the estimated coefficients derived by the three regression models , three equations were created and were labeled as new magee equations .
the new magee equations were tested on a validation set of 255 cases ( cases from our institution , sent for oncotype dx testing from 2010 until early 2011 ) .
in addition , our previously published equation ( original magee equation ) was also applied to the validation set cases .
the original magee equation is represented as follows : recurrence score=13.424 + 5.420*(nuclear grade)+5.538*(mitotic count)0.045*(er h - score)0.030*(pr h - score)+9.486*(0 for negative / equivocal and 1 for her2 positive ) .
once the recurrence score was estimated using the four equations in microsoft excel worksheets ( see supplementary data ) , the cases were categorized as low risk , intermediate risk , and high risk , using the same cutoffs as the actual recurrence score .
concordance statistics were performed to compare the actual oncotype dx recurrence score category to the estimated / predicted recurrence scores category derived from each equation .
based on data availability , the linear regression analyses on study cases resulted in three new magee equations and are represented below : new magee equation 1 : recurrence score=15.31385+nottingham score*1.4055+erihc*(0.01924)+prihc*(0.02925)+(0 for her2 negative ,
new magee equation 2 : recurrence score=18.8042+nottingham score*2.34123+erihc*(0.03749)+prihc*(0.03065)+(0 for her2 negative , 1.82921 for equivocal , 11.51378 for her2 positive)+tumor size*0.04267 .
new magee equation 3 : recurrence score=24.30812+erihc*(0.02177)+prihc*(0.02884)+(0 for her2 negative , 1.46495 for equivocal , 12.75525 for her2 positive)+ki-67 * 0.18649 .
the original and new magee equations were then applied to each of the 255 validation set cases to estimate the recurrence score . using this data set ,
the mean ( median ) recurrence score for actual oncotype dx was 20 ( 19 ) compared with 17.8 ( 16.3 ) for original magee equation , 20 ( 19.1 ) for new magee equation 1 , 19.9 ( 19.6 ) for new magee equation 2 , and 19.5 ( 18.5 ) for new magee equation 3 ( table 1 ) .
the concordance between actual oncotype dx recurrence score and the estimated recurrence score calculated from magee equations with respect to categorization ranged from 54.3 to 59.4% ( tables 2 , 3 , 4 , 5 ) .
the pearson 's correlation coefficient ( figure 1 ) between estimated and actual recurrence score was similar for each of the equations ( 0.60404 , 0.61661 , 0.60386 , and 0.59407 for original magee equation , new magee equation 1 , new magee equation 2 , and new magee equation 3 , respectively ) . with the exclusion of the intermediate - risk categories for both the actual recurrence score and estimated recurrence score , the concordance for each equation increased to more than 95% , reflecting the very low two - step discordance ( concordance 96.9% ( 95/98 ) , 100% ( 76/76 ) , 98.6% ( 75/76 ) , and 98.7% ( 79/80 ) for original magee equation , new magee equation 1 , new magee equation 2 , and new magee equation 3 , respectively ) .
when the estimated recurrence score fell in the intermediate category with any of the equations , the actual recurrence score was either intermediate or low in more than 80% of the cases ( tables 2 , 3 , 4 , 5 ) . with original magee equation ,
93 cases were estimated to be intermediate recurrence score ( table 2 ) . of these , 75 ( 81% ) remained either low or intermediate with actual oncotype dx recurrence score .
the remaining 18 cases ( 19% ) that were determined to be high on actual oncotype dx recurrence score had a median score of 36 compared with the estimated median score of 26 .
with new magee equation 1 , 122 cases were estimated to be intermediate recurrence score ( table 3 ) .
of these , 104 ( 85% ) remained either low or intermediate with actual oncotype dx recurrence score .
the remaining 18 cases ( 15% ) that were determined to be high on actual oncotype dx recurrence score had a median score of 33 compared with the estimated median score of 25 . with new magee equation 2 ,
of these , 121 ( 85% ) remained either low or intermediate with actual oncotype dx recurrence score .
the remaining 21 cases ( 15% ) that were determined to be high on actual oncotype dx recurrence score had a median score of 35 compared with the estimated median score of 26 . with new magee equation 3 ,
of these , 101 ( 85% ) remained either low or intermediate with actual oncotype dx recurrence score .
the remaining 18 cases ( 15% ) that were determined to be high on actual oncotype dx recurrence score had a median score of 33 compared with the estimated median score of 23 .
oncotype dx test is currently widely used by clinicians in the united states . despite the known pitfalls of oncotype dx assay in evaluation of single gene status ,
this is somewhat expected as many of the genes analyzed in the assay have individually been shown to have prognostic and predictive value .
although the seminal paper was reported to show benefit of 21 gene / oncotype dx assay over individual standard histopathologic parameters , there are dearth of studies that have compared the combined prognostic / predictive power of standard histology and immunohistochemical markers with oncotype dx assay .
recently , the study by cuzick et al provide evidence that standard clinical and pathologic parameters can be even better than oncotype dx assay for prognostication .
this study was undertaken to estimate the oncotype dx recurrence score using standard histologic and immunohistologic findings that are routinely reported .
the concordance between the estimated recurrence score category and the oncotype dx recurrence score category ranged from 55 to 60% .
the two - step discordance ( meaning an estimated recurrence score that is high , when the oncotype dx recurrence score is low , and vice versa ) was either not present ( for new magee equation 1 ) or negligible ( for the other equations ) .
one can therefore conclude that if the estimated recurrence score is clearly in the high or low categories , it is predictive of the oncotype dx recurrence score category with > 95% certainty . even in cases where the estimated recurrence score category predicted an intermediate category , the results were clinically useful .
an estimated recurrence score ( using new magee equations ) in the intermediate category means that the actual oncotype dx recurrence score category will be either intermediate or low approximately 85% of the time .
the remainder 15% cases in the validation set that were determined to be high risk by actual oncotype dx recurrence score had median score close to the 31 point cutoff .
these results are important as the national surgical adjuvant breast and bowel project - b20 trial showed that cases with intermediate recurrence score derive minimal or no benefit with adjuvant chemotherapy
. therefore , majority of the patients with estimated recurrence score in the intermediate category are unlikely to derive large benefit from chemotherapy .
the cases that have estimated recurrence score at the higher end of intermediate category may be considered for chemotherapy based on individual benefit and risk .
these equations provide clinically useful information , using data from standard pathologic reporting of breast cancer . of our four equations , new magee equation 2 had the highest concordance for categorization ( 60% ) , and includes nottingham score , er h - score , pr h - score , her2 status , and tumor size ( and does not include ki-67 ) .
this may indicate some confounding results from the equations that use ki-67 ( new magee equations 1 and 3 ) .
given that all of the equations utilize either the nottingham score or the mitotic count component of the nottingham score , the actual mitotic activity is either similar or better predictor of tumor proliferation than ki-67 immunohistochemistry .
the previous studies have shown the importance of ki-67 in determining the oncotype dx recurrence score , but ki-67 alone appears to be inferior to combined histologic and immunophenotypic data . although the overall correlation between estimated and actual recurrence score was good , it was not perfect .
first , the estimated recurrence score measures the morphologic and immunophenotypic data and actual recurrence score measures gene expression levels and some variability is expected if two different parameters are measured . secondly , interobserver variability for grading and semiquantifying immunohistochemical results among pathologists can lead to under- or overestimation of recurrence score .
last , but not the least , there may be characteristics intrinsic to the oncotype dx assay that can alter the recurrence score .
one major issue is the possible effect of various benign breast components on recurrence score . despite gross macrodissection or even microdissection of tumor tissue for polymerase chain reaction analysis , many non - invasive tumor tissue components ( such as stromal fibroblasts , adipose tissue , lymphocytes , macrophages , normal ducts and lobules , in situ carcinoma , epithelial proliferation , etc )
can be admixed during mrna extraction and may alter the recurrence score by at least a few points .
recently , acs et al demonstrated the impact of mitotically active cellular stroma on oncotype dx recurrence score .
their results suggested that increased stromal cellularity and/or associated inflammatory cells in low - grade invasive breast cancer may contribute to apparent increase in recurrence score .
apart from tumor macrodissection / microdissection , reproducibility of the assay between the same tumor block and in between different tumor blocks of the same tumor has not been extensively studied .
cronin et al reported the analytical validation of the oncotype dx assay , but did not address the reproducibility of recurrence score on re - extracted mrna from the same block or difference in recurrence score values between different blocks of the same tumor .
it is also possible that variation in tissue handling and fixation can alter recurrence score , but there is no published data on this subject .
delay to formalin fixation can not only alter immunohistochemical results and mitotic counts by a pathologist but can also alter mrna expression level of many genes . owing to the variability of all of these factors , it is not surprising that there is less than perfect correlation between oncotype dx recurrence score and the magee equations that we have created for estimating recurrence score .
decades of prior work has shown the importance of tumor grade , tumor size , lymph node status , hormone receptors and her2 status in determining patient prognosis and prediction of response to hormonal therapy and systemic chemotherapy .
our equations use all of these factors and we have demonstrated that they can be useful in estimating the recurrence score .
risk prediction models such as the st gallen , adjuvant ! online , and the nottingham prognostic index use some of these factors to predict outcome , and have been used by clinicians to guide therapy .
the st gallen system does not include her2 status in prognostication ( although accepted her2-positive status to assign trastuzumab in 2007 ) and owing to overlapping features in some cases , risk categorization could be difficult .
online does not include her2 status in the equation and also does not account for tumor hormone receptor content ( quantification ) for prognostication and prediction of chemotherapy effectiveness .
the oncotype dx include expression levels of genes that are critical in determining prognosis and effectiveness of chemotherapy , but this test also has several shortcomings as discussed above .
therefore , all of these predictive models and assays should be considered as estimates and clinicians should use all available information rather than result of one assay .
our study reinforces the findings in our pilot study , and prior studies that show correlation between standard pathology findings and the oncotype dx recurrence score . in a study of 77 cases , tang and
co - workers showed the correlation between aggressive morphologic features and lack of pr expression with high oncotype dx recurrence score .
the importance of pr semiquantitative score was also shown by clark et al , who identified an inverse relationship between pr expression level and oncotype dx recurrence score , which was independent of tumor grade . recently
, one large study has shown the prognostic significance of semiquantitative er , pr , her2 , and ki-67 immunohistochemical results to be similar to the oncotype dx recurrence score .
the study also showed that combining the immunohistochemical results with clinical parameters provided superior prognostic information than oncotype dx alone .
in another large recent study , tang et al integrated the recurrence score with clinical and pathologic factors and reported a better assessment of distant recurrence .
the follow - up time for patients in our study ( the 255 validation set cases from 2010 to 2011 ) is short . by following clinical outcomes ,
we hope to verify the utility of our findings , and reinforce the importance of standard morphoimmunohistologic variables .
the equations we have created are user - friendly and available free of any cost to the user . by using the microsoft excel worksheets we have provided ( see supplementary data ) , pathologists and oncologists
can easily calculate estimated recurrence score themselves and compare the result to the actual recurrence score .
if an estimated recurrence score falls clearly in the high - risk or low - risk category , then oncologists should not expect a dramatically different result from oncotype dx .
in these cases , the use of oncotype dx may be avoided . in cases where oncotype dx is performed
, a result dramatically different from the estimated recurrence score should be thoroughly investigated by the pathologist in concert with the clinical team , to ensure that the oncotype dx result is accurate in the sense that it is representative of the tumor that is properly microdissected .
 oncotype dx is a commercial assay frequently used for making chemotherapy decisions in estrogen receptor ( er)-positive breast cancers . 
 the result is reported as a recurrence score ranging from 0 to 100 , divided into low - risk ( < 18 ) , intermediate - risk ( 1830 ) , and high - risk ( 31 ) categories . 
 our pilot study showed that recurrence score can be predicted by an equation incorporating standard morphoimmunohistologic variables ( referred to as original magee equation ) . using a data set of 817 cases , we formulated three additional equations ( referred to as new magee equations 1 , 2 , and 3 ) to predict the recurrence score category for an independent set of 255 cases . 
 the concordance between the risk category of oncotype dx and our equations was 54.3% , 55.8% , 59.4% , and 54.4% for original magee equation , new magee equations 1 , 2 , and 3 , respectively . 
 when the intermediate category was eliminated , the concordance increased to 96.9% , 100% , 98.6% , and 98.7% for original magee equation , new magee equations 1 , 2 , and 3 , respectively . 
 even when the estimated recurrence score fell in the intermediate category with any of the equations , the actual recurrence score was either intermediate or low in more than 80% of the cases . 
 any of the four equations can be used to estimate the recurrence score depending on available data . 
 if the estimated recurrence score is clearly high or low , the oncologists should not expect a dramatically different result from oncotype dx , and the oncotype dx test may not be needed . 
 conversely , an oncotype dx result that is dramatically different from what is expected based on standard morphoimmunohistologic variables should be thoroughly investigated . 
Materials and methods
Results
Discussion
Supplementary Material