Patent Publication Number: US-6035258-A

Title: Method for correction of quantitative DNA measurements in a tissue section

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     This invention, in some of its embodiments, uses U.S. Pat. No. 5,918,038 to Freed for General Method for Determining the Volume and Profile Area of a Sectioned Corpuscle. 
    
    
     BACKGROUND 
     1. Field of Invention 
     This invention relates to the interpretation of data from an assay of a biological cell sample, and more particularly, to correction of DNA quantitative measurements in a tissue section. 
     2. Description of Prior Art 
     Cancer diagnosis and prognosis is largely dependent on the pathologic examination of tissue surgically removed from a patient. The specific diagnosis is made by a pathologist, who classifies the tumor by site and by cell of origin after examining stained histologic sections of the fixed, paraffin-embedded cancer tissue. The prognosis depends on many factors, including the specific diagnosis, the presence and pattern of tumor metastasis, the extent of tumor at its site of origin and its proximity to vital structures, and the tumor grade as assessed by a pathologist. In some organs, such as the prostate, the usual determinants of prognosis are inadequate to provide a patient-specific prognosis, especially when such is desired prior to definitive therapy. Consequently, other prognostic indicators have been sought. 
     One prognostic indicator which has been valuable in the cancers of certain organs is DNA ploidy, which is the ratio of the quantity of DNA in a cancer cell to that in a normal cell in the resting phase of its growth cycle. In general, tumors with normal resting-phase cellular DNA content (diploid) have a better prognosis than those with twice that amount (tetraploid), and these in turn have a better prognosis than those with abnormal DNA content which is not tetraploid (aneuploid). 
     The cellular DNA is located in the nucleus. Various methods have been developed for measuring the DNA content of whole nuclei. These methods do not make it possible for the measured cells to be correlated with their position or appearance in a standard histologic section. Thus, it is likely that normal cells will be measured together with tumor cells. Also, distinct areas of tumor cannot be measured separately. An even more important consideration is that very small samples, such as prostate thin core biopsies, are unsuitable. 
     All of these limitations have been overcome by measuring the DNA content of nuclei and partial nuclei in Feulgen-stained standard histologic sections. A new problem is created, however, by the inevitable inclusion of partial nuclei among the analyzed nuclei. In many sections, because the nuclear diameter exceeds the section thickness, all of the nuclei in the section will be partial. In U.S. Pat. No. 5,235,522 to Bacus for Method and Apparatus for Automated Analysis of Biological Specimens, an apparatus and method for measuring the DNA content of nuclei in tissue sections is described, as well as a method for correction of DNA measurements necessitated by the analysis of partial nuclei. Bacus and Bacus also have described this method of correcting DNA measurements in A Method of Correcting DNA Ploidy Measurements in Tissue Sections, published in Modern Pathology, Vol. 7, pp. 652-664, 1994. The Bacus correction makes three assumptions: 1) all nuclei are spherical, 2) all nuclei have a homogenous intranuclear DNA distribution, and 3) all nuclei with a profile area greater than πT 2  /4, where T is the section thickness, have been sectioned such that the center of the nucleus lies midway between the top and bottom of the tissue section. In an actual tissue section, the nuclei deviate from perfect spheres, the DNA distribution may not be homogeneous, and the nuclei are sectioned at essentially random positions and orientations. It is clear that the Bacus correction undercorrects most of the measurements, and would overcorrect some of the measurements, such as those made on a central section of an ellipsoidal nucleus aligned with the plane of the section, or those made on a centrally-sectioned nucleus in which most of the DNA is concentrated centrally. The Bacus method creates a histogram (FIG. 1) from the corrected DNA measurements, which shows a large number of bars 1 to the left of the bar representing the true whole-nucleus DNA content 2. These bars 1 represent partial nuclei for which the Bacus correction was insufficient, but may also be interpreted or misinterpreted as subpopulations of cells with different DNA content, or as cells at different points in the cell cycle. In many cases, such a misinterpretation might result in the classification of a prognostically favorable tumor as unfavorable. In cases of undercorrection or overcorrection of the modal DNA quantity, a tumor might be considered more or less prognostically favorable than would be inferred from its true ploidy. Also, histograms of quantitative DNA measurements in whole-nucleus preparations show very discreet peaks, reflecting discreet ploidy values of the measured nuclei; but when partial nuclei are measured, whether or not the measurements are corrected, the peaks are very blurred and may not be distinguishable as such (FIG. 1). The Bacus method allows the operator to define classes of attributes, thereby excluding many unwanted partial nuclei from analysis; such an approach is helpful but may be of limited value because the a priori classes may not accord with the natural classes in the specimen being analyzed. 
     OBJECTS AND ADVANTAGES 
     Several objects and advantages of the present invention are: 
     (a) to provide a method which demonstrates the relationship of all the partial nuclei in a tissue section to the corresponding intact nuclei which existed prior to sectioning; 
     (b) to provide a method to more accurately classify tumors into the correct prognostic categories; 
     (c) to provide a method which more readily distinguishes tumor cell subpopulations of different ploidy in a mixed sample; 
     (d) to provide a method in which data sets that are not amenable to correction can be distinguished from those that are amenable to correction; 
     (e) to provide a method in which randomly oriented, randomly positioned, non-spherical nuclei can be more readily analyzed; 
     (f) to provide a method which is not dependent on the limiting and incorrect assumptions upon which the prior art is based; and 
     (g) to obviate the need for assignment of the nuclei and partial nuclei in a tissue section to different a priori classes. 
    
    
     DRAWING FIGURES 
     FIG. 1 is a histogram shown to illustrate some limitations of the prior art; 
     FIG. 2 is a flow diagram of the overall process of correcting DNA quantitative measurements in a tissue section; 
     FIG. 3 is a representation of the first video screen which appears after the data has been downloaded; 
     FIG. 4 is a representation of the video screen after the data curve has been horizontally scaled to terminate on the reference line; 
     FIG. 5 is a representation of the final video screen after the reference curve has been recalculated and redrawn to coincide as closely as possible with the horizontally-scaled data curve; and 
     FIG. 6 is a representation of a reference sphere. 
    
    
     REFERENCE NUMERALS IN DRAWINGS 
     1 histogram bars corresponding to partial nuclei 
     2 histogram bar corresponding to true whole-nucleus DNA content 
     13 start program 
     14 download data 
     15 display first video screen 
     16 data curve 
     17 reference line 
     18 reference curve 
     19 reference sphere 
     20 operator prompt 
     21 operator decides to quit program 
     22 program halts 
     23 operator prompt 
     24 operator prompt 
     25 program calculates F and V i   
     26 screen display is redrawn 
     27 horizontally-scaled data curve 
     28 redrawn reference curve 
     29 statement displayed preceeding program output 
     30 print screen display 
     31 operator causes program to loop 
     32 program loops 
     33 top sectioning plane 
     34 bottom sectioning plane 
     35 first control point 
     SUMMARY 
     In accordance with the present invention, a process for correcting quantitative DNA measurements in tissues sections comprises a computer program in which the measured data are compared to a sequence of reference curves until a best-fit reference curve is found. The known attributes of this best-fit reference curve enable the calculation of the corrected DNA index. 
     DESCRIPTION AND OPERATION--PREFERRED EMBODIMENT 
     In the preferred embodiment, a computer program written in TurboPascal v. 2.0 (Borland International, Inc., Scotts Valley, Calif.) runs on an IBM-compatible personal computer equipped with central processing unit, random access memory, a floppy disk drive, a hard drive, a standard keyboard, a printer, and a video monitor. FIG. 2 shows a flow chart of the program logic. When the program is started 13, the video screen prompts the operator for the name of the data file. The user enters the file name using the keyboard. The program then finds the data file and downloads 14 the data from the file, which resides on either a floppy disk or on the hard drive, into random access memory. The data file contains the following data: The file size, the nuclear profile area (A s ) and integrated optical density data (D s ) for a plurality of nuclei and partial nuclei in a Feulgen-stained histologic tissue section, the thickness of the tissue section, and the integrated optical density of an intact diploid nucleus. When data acquisition is complete, the program switches the video display to graphics mode and displays 15 the first screen (shown in detail in FIG. 3), in which the data curve 16 [(D s1 , A s1  /A t ), (D s2 ,A s2  /A t ) . . . (D sn ,A sn  /A t )] (where A t  =πT 2  /4) is plotted, suitably scaled so that all points fit on the screen of the video monitor. A reference line 17, and a reference curve 18 derived from a reference sphere 19 the diameter of which equals the section thickness, are displayed on the same screen (FIG. 3). The statement 20, &#34;Enter `q` to quit or press Enter to continue&#34;, is displayed at the top of the screen. This is the first control point 35. If the operator presses the q key and then the Enter key 21, the program returns the video display to text mode and halts 22. If the operator presses only the enter key, the statement 23, &#34;Enter nth horizontal scaling factor:&#34;, is displayed on the next line. The operator enters a real number value using the keyboard. The entered value is assigned to the data array [F 1  . . . F n  ] according to the round (n). Then the statement 24, &#34;Enter nth reference sphere volume factor:&#34;, is displayed on the next line. The operator enters a real number value using the keyboard. The entered value is assigned to the data array [V 1  . . . V n  ] according to the round (n). 
     The horizontal scaling factor, F and the reference sphere volume, V i  are then calculated 25. The video screen display (FIG. 3) is then erased and redrawn 26 (FIG. 4) showing the now horizontally scaled data curve 27 terminating o n the reference line 17, and the reference curve 18. Also displayed at the top of the screen is the statement 29, &#34;DNA Index=&#34;, followed by the calculated DNA index. (This number will not become meaningful until the end of this process.) The operator may now print the screen display 30 (by pressing the Shift and Print Screen keys together; initialization is required using the graphics command in DOS before the program is started). The operator now presses 31 the Enter key and the program loops 32 to the first control point 35 and another round begins. 
     After each round (see FIG. 5), the operator visually assesses the proximity of the terminus of the horizontally-scaled data curve 27 to the reference line 17 (always striving for a closer approximation), and the congruence of the redrawn reference curve 28 to the horizontally-scaled data curve 27 (always striving for the closest approximation to congruence). When the operator is satisfied, she may print the screen display 30. The value shown for the DNA index is now correct. 
     The reference line 17 is defined as y=(2x+1)/3 for x&gt;1 and y=x 2/3   for 0&lt;x≦1; all possible reference curves 18, 28 terminate on the reference line 17. 
     A reference curve 18, 28 is a plot of volume (V s ) versus profile area (A s ) for a representative series of sections of a reference sphere 19 of radius R (refer to FIG. 6), where the volume of the intact reference sphere (V i ) and the section thickness (T) are given. Let z=H be the equation of the top sectioning plane 33, z=L be the equation of the bottom sectioning plane 34, and Q be H or L, whichever has the smaller absolute value. (In this example, Q=L.) L=H-T. H is allowed to vary from -R to R+T in 400 equal increments; for each step, V s  is calculated according to the equation V t  =π((R 2  H-H 3  /3)-(R 2  L|-L 3  /3)), and A, is calculated according to the equation A s  =π(R 2  -Q 2 ). A set of 400 points [(V s1  /V t ,A s1  /A t ), (V s2  /V t ,A s2  /A t ) . . . (V sn  /V t ,A sn  /A t )] (where V t  =πT 3  /6 and A t  =πT 2  /4) is plotted and appears as the reference curve 18, 28 on the screen. 
     The horizontal scaling factor (F) is the product of the last-entered value Fn and all values entered on previous rounds (F 1  . . . F n ). Thus, F=Π Fn . In each round, after the last horizontal scaling factor (F n ) is entered, F is recalculated and the horizontally scaled data curve 27 [(D s1  F,A s1  /A t ), (D s2F ,A s2  /A t ) . . . (D sn  F,A sn  /A t )] is displayed. Similarly, the volume of the intact reference sphere 19, V i , is the product of the last-entered value (V in ) and all values entered on previous rounds; thus, V i  =Π vil   vin . In each round, the reference curve 28 is redrawn based on the last calculated V i . The DNA index (DI) is calculated as DI=V i  /FD, where D is the integrated optical density of an intact diploid nucleus, the value of which was downloaded from the data file. 
     The source code is included at the end of the specification. 
     SUMMARY, RAMIFICATIONS, AND SCOPE 
     Accordingly, the reader will see that the method of this invention can be used to obtain a corrected DNA index from the measurable attributes of cell nuclei in a tissue section. The display of the data as a curve is visually appealing and has the explanatory power, lacking in histogram-based methods, of relating all the partial nuclei, whose data points lie on the data curve, to the corresponding nuclei which existed prior to sectioning. Thus, a data curve illustrates all the nuclear sections obtained from a population of nuclei sharing the same ploidy. If two or more data curves appear on the same screen display, this indicates the existence of the corresponding number of distinct nuclear subpopulations of different ploidy, which can be analyzed, each in its turn, by the method of the present invention. Discrimination of subpopulations is much more difficult in histogram-based methods, because the histogram does not relate the partial nuclei to the corresponding nuclei which existed prior to sectioning, as the method of the present invention does. A related advantage of the present invention is that it avoids the error of treating every sectioned nucleus as a centrally-sectioned sphere. The present invention can be used in conjunction with synthetic data generated by U.S. Pat. No. 5,918,038 to Freed for General Method for Determining the Volume and Profile Area of a Sectioned Corpuscle, giving the operator the opportunity to become acquainted with the problems introduced by deviations from nuclear sphericity. In this fashion, the operator will learn how to judge the appropriateness or inappropriateness, in individual cases, of attempting to correct quantitative DNA measurements in a particular tissue section. Certain limiting assumptions of prior methods were avoided in the present invention, which would be expected to result in more accurate classification of tumors into prognostic categories in some cases. 
     Although the description above contains many specificities, these should not be construed as limiting the scope of the invention but as merely providing an illustration of the presently preferred embodiment of this invention. Thus, data could be entered from a keyboard rather than downloaded from a file. Also, many specific details of the video screen displays and the program logic could be rendered differently without altering the result. For example, the choice of plotting D s  on the abscissa and A s  /A t  on the ordinate is arbitrary, these assignments could be reversed. Also, the reference line is very convenient but is not required. 
     Thus the scope of the invention should be determined by the appended claims and their legal equivalents, rather than by the example given. 
     
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PROGRAM RCM;                                                              
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const                                                                     
xscreen: integer=320;                                                     
yscreen: integer=200;                                                     
pi: real=3.141593;                                                        
type                                                                      
template=array[1..100]of real;                                            
var                                                                       
arya,aryb,naryb: template;                                                
bnfile: file of real;                                                     
fname,fname2: string[15];                                                 
datamt,i,ic,color1,colr,sggn: integer;                                    
dat,vri,y,dipdna,newthk,thkness,factorx,factory,maxarea,maxdna: real;     
increment,q1,q0,a,b,ploidy,modcrc,right,top,bottom,left,modif: real;      
ch,ch1,ch2: char;                                                         
Procedure setpt(xu,yu,rt,lt,tp,bm: real);                                 
var                                                                       
xs,ys: real;                                                              
begin                                                                     
xs:=(xu-lt)*xscreen/(rt-lt);                                              
ys:=200-(yu-bm)*yscreen/(tp-bm);                                          
plot(trunc(xs),trunc(ys),colr);                                           
end;                                                                      
Procedure readdata;                                                       
begin                                                                     
maxdna:=0; maxarea:=0;                                                    
assign(bnfile,fname);                                                     
reset(bnfile);                                                            
read(bnfile,dat); read(bnfile,dipdna); read (bnfile,thkness);             
datamt:=int(dat);                                                         
for i:=1 to datamt do                                                     
begin                                                                     
read(bnfile,arya[i]);                                                     
arya[i] :=arya[i]/dipdna;                                                 
if maxdna&lt;arya[i]  then maxdna:=arya[i];                                  
read(bnfile,aryb[i]);                                                     
aryb[i] :=aryb[i]/(3.141593*sqr(thkness)/4);                              
naryb[i]:=aryb[i];                                                        
if maxarea&lt;naryb[i]  then maxarea:=naryb[i];                              
end;                                                                      
close(bnfile);                                                            
end;                                                                      
procedure redraw;                                                         
var                                                                       
ratio: real;                                                              
begin                                                                     
if ic&lt;1000 then begin                                                     
gotoxy(1,1); write(`Enter nth horizontal scaling factor:`);               
read(modif);                                                              
gotoxy(1,3); write(`Enter nth reference sphere volume factor:`);          
read(modcrc);                                                             
end;                                                                      
vri:=vri*modcrc;                                                          
graphcolormode;                                                           
colr:=1;                                                                  
ratio:=0.02;                                                              
while ratio&lt;1.02 do                                                       
begin ratio:=ratio+0.02;                                                  
setpt(ratio*ratio*ratio,sqr(ratio),right,left,top,bottom);                
end;                                                                      
ratio:=1;                                                                 
while ratio&lt;5 do                                                          
begin ratio:=ratio+0.02;                                                  
setpt((3*sqr(ratio)-1)/2,sqr(ratio),right,left,top,bottom);               
end;                                                                      
draw(0,0,0,199,1); draw(0,199,320,199,1);                                 
end;                                                                      
Procedure plotadj;                                                        
var                                                                       
d,vcalc,acalc,at,vt,t,cutpt,cz,ct,v,area,r: real;                         
begin                                                                     
t:=thkness; d:=exp(ln(t*t*t*vri)/3); r:=d/2; at:=pi*t*t/4;                
vt:=pi*t*t*t/6;                                                           
for i:=0 to 400 do                                                        
begin                                                                     
cutpt:=i*(2*r+t)/400-r; cz:=cutpt-t; if cz&lt;-r then cz:=-r;                
ct:=cutpt; if ct&gt;r then ct:=r;                                            
v:=pi*((r*r*ct-ct*ct*ct/3)-(r*r*cz-cz*cz*cz/3));                          
vcalc:=v/vt;                                                              
if abs(cz)&lt;abs(ct) then area:=pi*(r*r-cz*cz) else area:=pi*(r*r-ct*ct);   
if (ct&gt;0) and (cz&lt;0) then area:=pi*r*r;                                   
acalc:=area/at; setpt(vcalc,acalc,top,bottom,right,left);                 
end;                                                                      
colr:=2; factorx:=factorx*modif;                                          
for i:=1 to datamt do                                                     
begin                                                                     
setpt(arya[i] *factorx,naryb[i],right,left,top,bottom);                   
end;                                                                      
gotoxy(1,5); write(`DNA Index =`,(vri/factorx):8:2);                      
gotoxy(1,1); write(`     `);                                              
gotoxy(1,1); write(`Hit ENTER Key, or &#34;q&#34; to Quit`);                      
read(ch);                                                                 
end;                                                                      
begin                                                                     
GOTOXY(34,1); writeln(`PROGRAM RCM`);                                     
GOTOXY(20,4);                                                             
WRITELN(`Copyright (C) Jeffrey A. Freed, M.D. 1997`);                     
gotoxy(30,6); writeln(`All Rights Reserved`);                             
gotoxy(10,8); writeln(`This program embodies the                          
Reference Curve Method`);                                                 
gotoxy(10,9); writeln(`for correction of ploidy                           
measurements in tissue sections`);                                        
writeln(`Enter data file name:`); read(fname);                            
increment:=0.1; factory:=1; factorx:=1; modif:=1;                         
readdata;                                                                 
right:=maxdata*1.2;                                                       
left:=0;                                                                  
top:=maxarea*1.2;                                                         
bottom:=0;                                                                
newthk:=thkness;                                                          
i:=1; modcrc:=1;                                                          
if maxarea&gt;1 then factorx:=(3*maxarea-1)/(2*maxdna);                      
if maxarea&lt;=1 then factorx:=exp(3*ln(maxarea)/2)/maxdna;                  
gotoxy(1,1); writeln(`Press enter key to start`);                         
graphcolormode;                                                           
ic:=1000; while ic&gt;0  do                                                  
begin                                                                     
redraw; plotadj; ic:=ic-1; if (ch=`Q`) or (ch=`q`) then                   
begin textmode; ic:=0; end; end;                                          
END.                                                                      
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