876
`
`3' Chit HIM 1995;48:876-373
`
`A “quickscore” method for
`immunohistochemical semiquantitation:
`validation for oestrogen receptor in breast
`carcinomas
`
`S Detre, G Saccani Jotti, M Dowsett
`
`Abstract
`increasingly
`hnmunohistochemistry is
`used in the assessment of markers for
`breast cancer prognosis. Semiquanu'tation
`is frequently desirable but, other than by
`the use of image analysis, the appmaches
`currently in use are cumbersome. The
`most common method used is the H-score
`which takes into consideration the staining
`intensity in conjunction with the per-
`centage ofcells staining positively in breast
`carcinoma tissue. A “quickscore” has been
`developed which dispenses with the need
`to count individual cells. The quantitative
`biochemical Abbott enzyme immunoassay
`(BIA) and the Dako immunohistochemicsl
`assay (ll-IA) incorporating a semiquan-
`tiunive H-score, have been used as stand-
`ards against which the IRA quickscore for
`the semiquantitation ofoestrogen receptor
`expression was tested. A good correlation
`was found between the quickscore and the
`EIA, which was as good as that between
`the H-score and EIA. The quickscore is a
`valid approach and there is no advantage
`in using the more rigorous H—score. A
`positive cut 011' quickscore of 2 3 has been
`suggested.
`(3 Clip: Parka! 1995;48:876—879)
`
`Immunolflstochemisuy, quickscore, oes-
`Keywords:
`trogen receptor, breast cancer.
`
`involves the addition of scores for staining in-
`tensity and the proportion of positive cells,
`which is a departure from the H—score which
`is based on multiplication of these components
`and intuitively is more appropriate if staining
`intensity reflects antigen concentration. The
`aim of this study was to asseSs whether a mo-
`dification of the quickscore to a multiplicative
`form was valid and could provide an acceptable
`alternative to the H—score. Two extra categories
`for the proportion of cells staining positively
`were also included to permit a completely neg-
`ative result, and to take into account the pres-
`ence of very small numbers of positive cells.
`
`Methods
`Ninety six primary breast cancer surgical speci-
`mens, embedded in paraflin wax, were studied.
`These tumours were taken from a cohort of
`
`119 untreated patients studied previously5 and
`the oestrogen receptor EIA, using the Abbott
`H222 antibody, and the oestrogen receptor
`IHA, using the Dako lD5 antibody, have been
`described in detail. The reduction in sample
`size to 96 tumours was necessary because 17
`mismatches had occurred between EIA and
`IHA pairs of results and in order not to con-
`found the scoring appraisal these mismatches
`were excluded from this investigation. A further
`six mmours were excluded because full results
`were not available.
`
`Oestrogen receptor determination is important
`in the study of the biology of breast cancers
`and for determining the clinical implications of
`hormone n-eaunenr.’ Recently, the immuno-
`histochemical assay (Ii-IA) has gained favour
`over the gold standard enzyme immunoassay
`(EIA) because it is cheaper, quicker and easier
`to perform and enables direct visualisation of
`malignant tumour receptor localisation in small
`tissue samples.“ One disadvantage of the im-
`munohistochemical approach is the laborious
`H—score procedure which is prone to a degree
`of discordance between sssessorv.:..I
`As the immunohlstochemical approach is
`gaining increasing support
`for
`routine as-
`sessment of oestrogen receptor expression, a
`more rapid scoring scheme would be useful
`especially when appraising large numbers of
`Sections from clinical studies. To this end a
`quickseore method such as that suggested by
`Barnes er a!‘ would expedite matters. The
`method described by Barnes er al, however,
`
`SCORING ASSESSMENT
`
`For H—score assessment, 10 fields were chosen
`at random or x 400 magnification and the stain-
`ing intensity in the malignant cell nuclei was
`scored as 0, 1, 2, or 3 corresponding to the
`presence of negative, weak, intermediate, and
`strong brown staining, respectively. The total
`number of cells in each field and the number
`of cells stained at each intensity were counted.
`The average percentage positive was calculated
`and the following formula3 was applied:
`IHA Hecore=(% of cells stained or intensity category
`1 x 1) + (% of cells
`stained at
`intensity category
`2 x 2) + (is of cells stained at intensity category 3 x 3).
`
`A H—scorc between 0 and 300 was obtained
`where 300 was equal to 100% of tumour cells
`stained strongly (3 4-).
`The immunohistochemicslly stained sec-
`tions were subsequently given a quickscore
`independently by two of the authors without
`prior consultation or recourse to clinical, bio-
`
`Genome & Co. v. Univ. of Chicago, PGR2019-OOOO2
`UNIV. CHICAGO EX. 2077
`
`academic
`of Biochemistry,
`Royal Mandela
`Hospital,
`Fulbam Road.
`London SW3 6]]
`S Detre
`M Dawson
`
`Istituto di Anatomia
`ed Istologla
`Patologica,
`University of Par-ms,
`Italy
`G Ssccsni Jodi
`[‘1me: to:
`S Den-e.
`Accepted for publication
`17 Ianuary 1995
`
`

`

`Qm‘ckscors for immanohirtochemicni smiqmnrimrion
`
`87‘?
`
`T5538 3 _ SWIM“ “I”? 50mm" WW fl?"
`mjmffl Wmmfifl" ”1' “mg“ m”
`
`scores and a good positive correlation was ob-
`served between the IHA and the EIA (table 1).
`More importantly, the correlation between both
`of the quickscores and the EIA was very similar
`”‘0'
`cmmn" ”rm dm‘
`to that for the H—score and the EIA.
`can:
`Q=AxB v H score
`We also compared the performance of the
`3:23;
`331:1;2 ELI-lam"
`two quickscore approaches with two separate
`0'83]
`Q=axB 13 Em
`ease
`Q=a+aesia
`— assessors. Total agreement of score occurred
`rho.=regression coefficient corrected for ties; Q-quieirscore
`in 73% of cases and demonstrated a very strong
`gfimmfim flags” "“9“" ”mm" ”1‘5””
`correlation between the multiplicative and ad-
`mit-0001
`ditive quickscore (rho,=0'994). In the seven
`cases where disagreement occurred, five were
`at
`the highest
`score levels—for example,
`chemical or previously recorded H—score data. A x B =18 and 15, A +B = B and 9, and two at
`The quickscore categories were based on both
`the lowest levels—for example, A x B =1 and
`the intensity and the proportion of brown stain-
`2, A+B = 2 and 3. The maximum quickscore
`ing nuclei. In our modification the proportion was attained in many cases, especially where
`of malignant cells staining positively through-
`high EIA values occurred. Interestingly, a very
`out the section was termed category A and was
`high H—score level was never achieved.
`assigned scores from 1 to 6 (l=0—4%; 2=
`549%; 3 = 20—39%; 4 =40—59%; 5 = 60—79%;
`6:80—100%). The whole section was scanned Discussion
`at low power in order to gauge the general level The concordance between the two observers
`of intensity throughout. The average intensity,
`indicates that both quickscores are repro-
`corresponding to the presence of negative,
`ducible although more studies from other
`weak, intermediate, and strong staining, was
`laboratories are necessary to confirm this. The
`given a score from 0 to 3, respectively, and
`l'HA quickscore compared favourably with the
`termed category B. Category A was added to
`gold standard BIA. That the quickscore is of
`category B (A+B) to form an additive quick— equivalent value to the H-score, when using
`score and recorded in parallel with the product
`the same irnrnunohistochernical technique, was
`(A x B) as a multiplicative quickscore. The res-
`confirmed by the good Spearman rank cor—
`ults were tabulated and EIA and IHA results
`relation between them.
`were compared with the quickscore using
`The reason why these sections did not have
`Speannan rank correlation statistics.
`very high H—scores could have been because
`tumours in this cohort expressed oestrogen
`receptor at a low level. This, however, was not
`the case when the quickscore was applied as
`Results
`The EIA values ranged from 0 to 597 fmolesr‘ maximum scores were reported in 19% (GSJ)
`mg protein and 62% of the tumours were
`and 14% {SD} of cases.
`oestrogen receptorpositive {cutoff ?10 fmoles/
`The quickscore seems to be less prone to
`mg protein). H—scores
`ranged from 0 to
`variation than the H—score because the observer
`190. Additive and multiplicative quickscores
`evaluates the whole section, estimating an over—
`covered the full range—that is, CA+ B) = 1—9
`all impression of intensity when scoring and not
`and (A x B) =0—18, respectively. Table 1 shows
`inst 10 representative, but randomly selected,
`a comparison ofthe Spearman rank correlations
`high power fields. This difference in approach
`for the three oestrogen receptor methods and might explain the underestimation of H—score.
`the individual categories of the AxB quick-
`The positive cut off for the EIA is usually
`some are compared with intervals of H-scores
`10 Emolesfmg protein in breast carcinomas.”7
`and EIA in table 2. There was no difference The positive cut off using the IHA is more
`between the additive and multiplicative quick-
`contentious and varies from a H—score of 20,
`
` 0 i' 2 .i' H 8 to 1'2 :5 :3 "Rural
`
`
`
`
`
`
`
`29
`2
`I}
`D
`0
`0
`
`0
`3
`1‘
`I}
`D
`I}
`
`D
`2
`0
`O
`D
`O
`
`0
`0
`2
`D
`I)
`0
`
`0
`(I
`l
`3
`o
`[I
`
`D
`D
`3
`5
`0
`0
`
`D
`O
`D
`5
`8
`O
`
`0
`0
`0
`4
`lo
`O
`
`0
`D
`i]
`l
`3
`U
`
`D
`D
`I}
`2
`12
`D
`
`29
`T
`‘3
`20
`33
`D
`
`
`
`
`
`
`
`
`
`
`Table 2 Freon-my of multiphase qmehtomr in [Hid and EM result when
`Quichmore =2! 3: B
`
`
`H—soore
`G
`1— I 9
`20-49
`50—99
`too-199
`200-300
`Em
`36
`0
`D
`i]
`I)
`O
`I]
`O
`2
`3
`31
`59
`24
`2
`2
`a
`5
`7
`2
`I
`o
`l"
`I)
`10—49
`16
`2
`I
`4
`5
`l
`2
`I
`o
`I}
`I)
`50-99
`12
`5
`I
`5
`I
`I]
`D
`o
`0
`O
`I)
`100—199
`5
`3
`0
`l
`1
`D
`D
`O
`D
`0
`0
`200-29?
`1
`l
`O
`O
`O
`I]
`o
`o
`i]
`0
`0
`300—399
`a
`D
`0
`0
`D
`D
`O
`0
`I)
`0
`0
`400499
`2
`l
`o
`(1|
`1
`0
`0
`{I
`D
`0
`0
`500-600
`
`
`
`
`
`
`
`
`
`
`
`31 4 2 2 4 8 I3 14 4 14Total 9'6
`‘ =32; "‘ = 29 I'Inolesimg protein.
`.
`Negative H—score s 19.
`Negative EIA 59$:qu protein.
`
`

`

`878
`
`in our laboratory,5 to 50—100 in others.” Bear-
`ing in mind our laboratory cut off values, table
`2 shows a logical demarcation between the
`positive and negative EIA and IHA subsets
`at a multiplicative quickscore ?3. Thirty six
`breast
`tumours with negative EIA results
`($9finoleslmg protein) occurred in the 0—2
`quichcore subset; no oestrogen receptor neg-
`ative breast tumours on EIA were reported in
`the 3—18 quickscore subset whereas 59 of 60
`oestrogen receptor positive breast cancers were
`observed in this classification. Only one of 60
`oestrogen receptor EIA positive tumours was
`present in the 0-2 quickscore subset. The same
`results were obtained using a H-score cut off
`<20. The one tumour negative on assessment
`using the quickscore but positive by II-IA (H-
`score=32)
`and
`oestrogen
`receptor-BIA
`(29 fmolesfrng protein) came from the same
`patient.
`The results also show that there is no differ-
`ence between adding or multiplying categories
`A and B. The additive approach, with a range
`from 1 to 9, has two disadvantages. Firstly, the
`additive (A+B) quickscore does not permit a
`completely negative result, which occurred in
`many of our oestrogen receptor negative cases,
`whereas the multiplicative (Ax B) quickscore
`does. Secondly, the range is small for the ad-
`ditive quickscore. Considering the wide range
`
`Dem, Scream‘Jorn, Dossier:
`
`of values within the quantitative methods, the
`multiplicative quickscore, ranging from O to
`18, is preferable. Without prejudicing accuracy,
`the quickscore approach takes about a quarter
`of the time taken when calculating the H—score
`because it dispenses with the need to count
`individual cells.
`
`G Saocani Jerri was supported in part by the following grants:
`CNR ACRE} Proiect. CNR Bilateral 94.021.32.61'04 and
`MURST 40% (Italy). 513 and MD were suppormd by the
`Cancer Research Campaign.
`
`1 Osborne CK. Receptors. In: HatrisIR, Helmet: 5. Henderson
`IC. Kinne DW. eds. Breast domes. Philadelphia: IE Lip-
`pincort, 199l:301-25.
`2 Andersen]. Denomination of oestrogen receptors in parami—
`emhedded tissue. Am Chiral 1992;31:611-27.
`3 McClelllnd RA, Finlay P, Walker KL Nicholson D, Rob-
`ertson IFR, Blarney RW, er of Automated quantitation
`of unmanocytuehemieally localised oestrogen receptors in
`human breast cancer. Cow Rs: ”9050:3545-50
`4 Barnes DM. Dublin EA. Fisher C], Levison DA, Millis
`RR Intrnuaohisroeheaneal detection of p53 protein in
`mammary carcinoma. Hm Parke! 1993;“:46946.
`5 Saccani Jerri G, Johnston 5111), Salter J, Detre S, Dowsett
`M. Comparison of a new immunohistocbemieal assay for
`oestrogen receptoeinparaflinwaaemheddedbreast ear-
`cinonu tissue with quantitative enzyme immunoassay 3‘
`Gift! Herbal 199-1547:900-5
`6 Andersen], Bennett SM, Poulsea HS. Relationship between
`radioligand bin '
`assay, humunoenzyme assay and im-
`muaohistochemi may for oestrogen receptors in human
`breast cancer and association with rumour difi’erentiarion.
`Ear } Cm 19381437744.
`7 Kmsei B. Saabo E. Greene 61., Konrad: J. Leight GS,
`McCarty KS. Immuooqtoehemieal analysis of oestrogen
`receptors as a predictor of prognosis in breast cancer
`patients:
`comparison with
`quantitative
`blodiemical
`methods. Cancer Res 1989348305245.
`
`3 are: Puma: 1995;423:3713—3530
`
`Granulomatous bone marrow inflammation
`
`during treatment of chronic myeloid leukaemia
`with interferon alpha-2b
`
`A Siboni, T Mounts-Andersen, J Moesner
`
`Abstract
`study, partial cytogenetic response and tem-
`A patient with chronic myeloid leukaemia
`porary morphological remission was seen in
`developed bone marrow granuloroaa dur-
`46!?1 patients.‘ Interferon alpha is used in the
`treatment of both infectious and malignant
`ing treatment with interferon alpha-2b.
`Some gsanulomas had necrotic centres
`diseases.”I One of its mechanisms of action may
`and giant cells and there was marked eoa— be inhibition of formation of new blood vessels
`inophilia surrounding them. Tile gran-
`(antiangiog'enesis) in tumour areas.2
`trans-
`alumna disappeared when the interferon
`Bone marrow necrosis at blast
`treatment was
`discontinued. Myco-
`formation of chronic gt‘anulocytic leukaemia
`bacterioais was ruled out. The most likely
`treated with interferon has been described by
`explanation for the granuloma formation Kendra et al,’ but the formation ofbone marrow
`was drug hypersensitivity.
`granulomas during the treattnent of chronic
`{5’ can Pedro! 1995;48:370-380)
`myeloid leukaemia with IFN-a-Zb has not been
`described before.
`
`Keywords: interferon, bone marrow granulomas.
`
`The treatment of chronic myeloid leukaemia Case report
`with interferon alpha-2b (IFN-or—Zb) prolongs
`A 43 year old man was referred in August 1992
`survival if cytogenic response occurs: in one
`because ofa leucocyte count of89 x lD'i'l. Bone
`
`$3,“:(Emmy
`Bit-6100 Elbifl'zs
`mm“ll let
`and M010"
`A Siboni
`T Mc'mts‘hndgfiun
`Wm of
`Podium
`I Mm"
`Cowman“ In:
`Dr Anders steam,
`Sholebakken smash.
`Bfiflgfi'b’m
`'
`a
`ted in
`oil
`1 December 1994
`r p“ mm
`ma
`
`