Patent Application: US-52551706-A

Abstract:
the invention relates to materials and methods for evaluating the prognosis of a patient presenting with chronic myelogenous leukaemia and for cll therapy .

Description:
lymphocytes can be isolated from whole blood by density centrifugation using histopaque - 1077 ( sigma ). this is a polysucrose ( 5 . 7 g / dl ) and sodium diatrizoate ( 9 . 0 g / dl ) solution which is adjusted to a density gradient 1 . 077 g / cm 3 and facilitates the sedimentation of erythrocytes and granulocytes whilst trapping mononuclear cells ( including lymphocytes ) at the plasma - histopaque interface . 1 . remove a 3 . 0 ml aliquot of histopaque - 1077 from the fridge ( in a 15 ml conical tube ) and equilibrate to room temperature . 2 . carefully layer up to 8 . 0 ml of whole blood onto the histopaque , close the screw cap and then centrifuge at 400 × g ( 1500 rpm ) for 30 mins . 3 . remove tube from centrifuge and carefully remove the upper layer to within 0 . 5 cm of the opaque interface using a pasteur pipette and discard . 4 . transfer the opaque interface ( containing the mononuclear cells ) with a sterile pasteur pipette into a sterile 15 ml conical tube . 5 . add 10 ml of sterile phosphate buffered saline to the cell suspension and mix by gentle aspiration . 6 . centrifuge the tube at 300 × g ( 1200 rpm ) for 10 mins , aspirate the supernatant and discard . 7 . add 10 ml of 0 . 87 % w / v of ammonium chloride solution to lyse contaminating red cells . gently aspirate and leave at room temperature for 5 mins . 8 . centrifuge the tube at 300 × g ( 1200 rpm ) for 10 mins , aspirate the supernatant and discard . a number of studies have demonstrated the importance of v h gene mutation status as a prognostic marker in cll . ( 1 - 4 ) the underlying biological rationale for this is controversial but is likely to be related to the fundamental biological differences between cells that have or have not undergone somatic hypermutation in response to antigen . 1 . lymphocytes should be isolated as described above and aliquoted ( 2 × 10 6 cells ) and frozen (− 70 ° c .) until dna extraction can be performed . 2 . dna was extracted using qiaamp dna blood midi kit . in a 15 ml centrifuge tube , 2 × 10 6 peripheral blood mononuclear cells were added to 200 μl of qiagen protease . 3 . to this 2 . 4 ml of buffer al was added and the solution was mixed thoroughly by vortexing . the solution was incubated at 70 ° c . for 10 minutes . 4 . ethanol ( 2 ml ) was added to the sample and mixed by vortexing . half the solution was added to the qiaamp midi column and this was centrifuged at 1850 × g for 3 minutes . 5 . the flow - through was discarded , and the rest of the solution was added to the column which was spun at 1850 × g for 3 minutes . the filtrate was discarded . 2 ml of buffer aw1 was added to the qiaamp midi column and this was centrifuged at 4500 × g for 1 minute . 6 . 2 ml of buffer aw2 was then added to the qiaamp midi column and this was centrifuged at 4500 × g for 15 minutes . 7 . the flow - through and collection tube was discarded and the qiaamp midi column was placed in a fresh 15 ml centrifuge tube . 300 μl of buffer ae was pipetted directly onto the membrane of the qiaamp midi column , and with the cap closed , it was incubated at room temperature for 5 minutes and then centrifuged for 5 minutes at 4500 × g . 8 . to obtain maximum dna concentration , 300 μl of eluate containing the dna was reloaded onto the qiaamp midi column and was incubated at room temperature for 5 minutes . it was then centrifuged at 4500 × g for 5 minutes . the dna was kept for further down stream assays such as polymerase chain reaction . the dna previously extracted was then used in a multiplexed pcr reaction with the biomed - 2 primers . 9 . a 5 μl solution was made up of 0 . 5 μg of dna sample , 10 pmol of each primer , 2 nm of dntps ( deoxy nucleotide triphosphates ), 1 u amplitaq gold and 10 × pcr buffer ii . 10 . the dna thermo cycler ( abi ) was used as follows : denaturation at 94 ° c . for 15 minutes ; 35 cycles of 94 ° c . for 30 seconds , 58 ° c . for 30 seconds and 72 ° c . for 30 seconds ; and a final cycle of 10 minutes at 72 ° c . 11 . the pcr products were analysed on the agilent bioanalyzer . the products were then sequenced directly using 3 ′ j h consensus primer in an automated abi prism 3100 genetic analyser using big - dye terminators . 12 . comparison of the derived sample sequence was made with germline sequences stored on the ig blast database ( http :// www . ncbi . nlm . nih . gov / igblast /) and the percentage sequence homology to the closest germline sequence was determined . the highest percentage homology ( expressed as a number between 0 and 100 for 100 % homology ) was used for subsequent calculations . both the percentage sequence homology and the v h gene segment usage for each patient sample was noted . a number of studies have shown that the quantification of cd38 on the surface of cll cells can be a useful prognostic tool in this condition . ( 1 - 3 ) a triple - colour flow cytometry assay allows the positive identification of malignant cll cells and the simultaneous analysis of cd38 expression . 1 . lymphocytes should be isolated as described above 1 and aliquoted into 5 ml falcon tubes ( 1 × 10 6 cells / tubes ) 2 . to one aliquot of cells the following antibodies are added : 5 μl of cd5 fluorescein isothiocyante ( fitc ) conjugated antibody , 4 μl of cd38 r - phycoerythrin ( pe ) conjugated antibody and 4 μl of cd19 allophycocyanin ( apc ) conjugated antibody . 3 . add the same volumes of isotype - matched control antibodies conjugated to the same fluorochromes to a separate aliquot of cells . 4 . incubated the tubes at room temperature ( in the dark ) for 15 mins . 5 . add 3 ml of phosphate buffered saline to each tube and_centrifuge the tubes at 300 × g ( 1200 rpm ) for 10 mins , aspirate the supernatant and discard . 6 . resuspend the cell pellet in 0 . 5 ml of a 1 % w / v paraformaldehyde solution and store in the fridge ready for flow cytometric analysis 7 . the facs calibur flow cytometer has a template document ( named “ cd38 acquisition ”), this should be utilised and 10 , 000 events acquired . 8 . analysis is performed by gating the viable lymphocytes ( using forward scatter and side scatter characteristics ). this gate is then applied to a cd5 / cd19 dotplot and a second gate drawn around the cd5 / cd19 double positive lymphocytes . this gate is finally applied to a cd38 / cd19 plot and the percentage of cd38 positive cll cells is quantified using a quadrant defined by the isotype - matched control plot of cd38 / cd19 as shown in fig4 a , 4 b , 4 c and 4 d . the percentage ( expressed as a number between 0 and 100 ) is used for subsequent calculations . cells are first fixed to preserve antigens in their natural configuration and to prevent leakage of intracellular proteins across the cell membrane . cells are then permeabilised to give antibodies access to intracellular antigens whilst leaving the morphological characteristics of the cell largely intact . this method is currently used to determine a wide range of protein expression including members of the bcl - 2 family , phopsho - specific proteins and cyclins . 1 . isolate mononuclear cells from whole blood using histopaque 1077 ( see above ). 2 . adjust the concentration of the isolated mononuclear cells to 1 × 10 7 cells / ml using pbs as a diluent ( see sop 1 ). then aliquot 100 μl of this solution to the desired number of 5 ml falcon tubes . 3 . add 5 μl of cd5 - pe and 4 μl of cd19 - apc to one tube and the same volumes of isotype - matched control reagents to another . briefly vortex tubes and incubate for 10 mins at room temperature in the dark . 4 . wash cells in approximately 2 ml of pbs and then centrifuge for 5 mins at 300 × g . remove supernatant and discard . 5 . add 60 μl of reagent a ( fixative ) to all tubes and incubate for 10 mins at room temperature in the dark . wash in pbs and centrifuge as described in step 4 . 6 . add 60 μl of reagent b ( permeabilisation ) to the cell pellet followed by 4 μl of anti zap - 70 - alexa fluor 488 or the same volume of the isotype - matched control reagent . vortex tubes for 1 - 2 secs and incubate for 10 mins at room temperature in the dark . 7 . wash in pbs and centrifuge as described in step 4 . discard supernatant and resuspend pellet in 0 . 5 ml 1 % paraformaldehyde in pbs . store tubes in the fridge until analysis by flow cytometry . ( 1 , 2 ). our work clearly demonstrates that cellular protein tyrosine phosphorylation integrates v h gene mutation status , cd38 and zap - 70 expression in an additive fashion . cd38 expression correlated with basal protein tyrosine phosphorylation ( fig1 a ) ( linear regression r 2 = 0 . 68 , p & lt ; 0 . 0001 ), whereas v h gene mutation status ( fig1 b ) ( linear regression r 2 = 0 . 32 , p & lt ; 0 . 0001 ) and zap - 70 expression ( fig1 c ) ( linear regression r 2 = 0 . 41 , p & lt ; 0 . 0001 ) correlated with the tyrosine phosphorylation induced following bcr stimulation . cd38 expression did not significantly affect the change in tyrosine phosphorylation induced following bcr stimulation suggesting an independent role for this molecule in cll cell signaling . our model explained 81 % of the variation in protein tyrosine phosphorylation , following igm stimulation , as a function of v h gene mutation status , and the expression of cd38 and zap - 70 ( fig1 d ) using the following equation : cd38 = cll cell surface presence of cd38 , expressed as a %; percentage in this context means the percentage of cll cells with greater fluorescence than the isotype - matched control . v h status = v h gene mutation status , expressed as a % homology to the closest germline sequence ; and zap - 70 = cll cell zap - 70 expression , expressed as a %. percentage in this context means the percentage of cll cells with equal or greater fluorescence than the t - cell population in the same sample .] in order to take account of inter - laboratory variation , the figures quoted in the above equation are ± 10 %. 1 . take the percentage values generated using the three methods described above . note that all three measurements are required . 2 . calculated phosphotyrosine post igm ( py post igm ) using the following equation : 3 . our data shows that a calculated phosphotyrosine post igm greater than 26 ± 2 . 6 identifies a patient that will have a shorter time to first treatment . an investigation of various data transformations showed that a square root transformation of cd38 expression showed a superior correlation with basal protein tyrosine phosphorylation than the linear data model . it is noteworthy that protein tyrosine phosphorylation measurements retained significance when the patient cohort was categorized by v h status , cd38 expression or zap - 70 expression . cell signaling often results in a change in gene expression mediated by altered transcription factor activity in the nucleus of the lymphocyte . the transcription factor , nf - κb , has been reported to be elevated in cll samples 15 , 16 and has been implicated in lymphocyte cell survival 17 - 19 . however , the regulation of nf - κb by bcr signaling is poorly characterized in cll . therefore , we analysed nf - κb in cll samples using an electrophoretic mobility shift assay , before and after bcr ligation with anti - igm ( n = 35 ). our analysis revealed a surprising feature of cll b - cells : bcr stimulation had the capacity to not only activate nf - κb but also to repress it in distinct patient samples . stimulation with igm only increased nf - κb dna - binding in half the patient samples while in the other half of the cll samples , stimulation through igm resulted in a decrease in nf - κb dna - binding ( fig2 a and 2d ). this negative and positive signalling was independent of v h gene mutation status ( fig2 a , no significant correlation ) that is thought to define the ability of cll cells to signal through the bcr . negative signalling through antigen receptors has been observed in mouse model systems , most notably the induction of apoptosis by bcr stimulation of wehi - 231 , a murine b - cell lymphoma line of immature phenotype 20 , 21 . however , cll cells are the first human pathology where repression of nf - κb by antigen receptor stimulation has been observed . quantification and statistical analysis demonstrated that nf - κb activation correlated with zap - 70 expression ( fig2 b ; linear regression r 2 = 0 . 24 , p 0 . 005 ) but not with cd38 expression . when we considered the link between protein tyrosine phosphorylation and nf - κb activation , the change in phosphotyrosine , stimulated by igm , showed a correlation ( linear regression r 2 = 0 . 2 , p & lt ; 0 . 05 ,) with a more significant correlation between the levels of phosphotyrosine , post igm treatment , and nf - κb activation ( fig2 c ; linear regression r 2 = 0 . 27 , p & lt ; 0 . 01 ). fig2 c shows a threshold of protein tyrosine phosphorylation that must be reached in order to trigger an induction of nf - κb activation ( corresponding to a total phosphotyrosine mfi of 25 in these experiments ). above this threshold , eleven of twelve samples gave an induction of nf - κb , while below this value igm stimulation repressed nf - κb in ten out of thirteen samples . cll is characterised by the accumulation of b - lymphocytes and so is primarily regarded as a defect in cellular apoptosis 6 . for this reason , we measured apoptosis in cll samples with and without stimulation of the bcr with anti - igm . as has been previously reported 22 , anti - igm treatment induced apoptosis in some cll cells but protected others from spontaneous cell death . interestingly , our analysis of spontaneous apoptosis revealed no significant correlation between spontaneous cell death and any variable we measured . however , levels of cellular apoptosis post igm treatment showed an inverse correlation with the expression of cd38 and zap - 70 although these associations had quite small correlation coefficients . more convincingly , our data showed that the change in phosphotyrosine signal can explain 32 % of the variation in the change in cellular apoptosis induced by anti - igm ( fig2 e ). the largest correlation coefficient was observed when graphing the change in apoptosis induced by anti - igm against the change in nf - κb activity induced by anti - igm ( r 2 = 0 . 67 , p & lt ; 0 . 0001 ; fig2 f ). thus , this correlation explains 67 % of the variation in cell survival post anti - igm treatment . as examples , fig2 d shows two samples , one where nf - κb was increased and so was cell survival and another where nfκ - b was decreased and there was a corresponding decrease in cell survival . importantly , this shows that the repression of nf - κb by anti - igm , i . e . negative signaling through the bcr , has functional relevance in terms of regulating cellular phenotype . we also used an nfκ - b inhibitor and investigated its effects on cll cells . treating cll cells with bay 11 - 7082 23 for 24 hours caused a dose - dependent increase in cll cell apoptosis . this shows a causative relationship , between nf - κb and cll cell survival , strongly supporting the correlation we observed . having demonstrated that calculated phosphotyrosine set a threshold for nf - κb , which in turn regulates cll cell survival , we investigated whether calculated phosphotyrosine could predict patient prognosis . to test this we used a larger database of 155 patients on which we have measured all of the prognostic markers and had information about the patients clinical course . we used the equation , described above to calculate phosphotyrosine as a function of the three prognostic markers , and set a threshold at a calculated phosphotyrosine greater than 26 . this is above the threshold that is required to result in activation of nf - κb and thus would increase cell survival . dividing patients in this way identified patients with a shorter time to first treatment . the kaplan meier analysis ( fig3 a ) shows that patients with a calculated phosphotyrosine higher than 26 have a significantly shorter time to first treatment than patients with a calculated phosphotyrosine lower than 26 ( p & lt ; 0 . 0001 ). the shape of these curves and the median time to first treatment for patients with a high calculated phosphotyrosine ( 1080 days ) is very similar to patients with unmutated v h genes ( 1085 days ). however , calculated phosphotyrosine greater than 26 identifies more patients ( 55 / 155 ) with adverse prognosis than analysis of unmutated v h genes alone which only identifies 27 patients from our cohort . we analysed binet stage a patients in our database ( fig3 b ) and patients with mutated v h genes ( fig3 c ). in both of these cases , high calculated phosphotyrosine was able to significantly differentiate patients with an early time to first treatment . this study has identified three important points about the biology of chronic lymphocytic leukaemia cells . firstly , our data shows that tyrosine phosphorylation integrates two distinct pathways : cd38 expression reflecting the basal tyrosine phosphorylation , while zap - 70 and vh status reflect the ability of the cell to respond to bcr ligation . secondly , phosphotyrosine sets a threshold above which bcr ligation can activate nf - κb . interestingly , below this threshold bcr ligation is capable of repressing nf - κb , the first time this has been observed in a human pathology . thirdly , the strong correlation between igm regulation of nf - κb and igm - mediated control of cell survival shows the critical link between these two events . this link and our nf - κb inhibition studies show that nf - κb is likely to be a valuable therapeutic target . the most important aspect of this study is our integration of the three prognostic markers to improve the identification of patients that will require early intervention . in stage a patients , with mutated vh genes , high cd38 and zap - 70 expression and adverse cytogenetics , such as 17p and 11q deletions , is uncommon making it currently impossible to identify those patients with unfavourable prognosis . our equation , integrating the three prognostic markers , makes the identification of patients that have unfavourable prognosis , with stage a disease and mutated v h genes , possible for the first time . fig5 illustrates an example of a suitable computing system environment 100 on which a system for the steps of the claimed method and apparatus may be implemented . the computing system environment 100 is only one example of a suitable computing environment and is not intended to suggest any limitation as to the scope of use or functionality of the method of apparatus of the claims . neither should the computing environment 100 be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in the exemplary operating environment 100 . the steps of the claimed method and apparatus are operational with numerous other general purpose or special purpose computing system environments or configurations . examples of well known computing systems , environments , and / or configurations that may be suitable for use with the methods or apparatus of the claims include , but are not limited to , personal computers , server computers , hand - held or laptop devices , multiprocessor systems , microprocessor - based systems , set top boxes , programmable consumer electronics , network pcs , minicomputers , mainframe computers , distributed computing environments that include any of the above systems or devices , and the like . the steps of the claimed method and apparatus may be described in the general context of computer - executable instructions , such as program modules , being executed by a computer . generally , program modules include routines , programs , objects , components , data structures , etc . that perform particular tasks or implement particular abstract data types . the methods and apparatus may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network . in a distributed computing environment , program modules may be located in both local and remote computer storage media including memory storage devices . with reference to fig5 , an exemplary system for implementing the steps of the claimed method and apparatus includes a general purpose computing device in the form of a computer 110 . components of computer 110 may include , but are not limited to , a processing unit 120 , a system memory 130 , and a system bus 121 that couples various system components including the system memory to the processing unit 120 . the system bus 121 may be any of several types of bus structures including a memory bus or memory controller , a peripheral bus , and a local bus using any of a variety of bus architectures . by way of example , and not limitation , such architectures include industry standard architecture ( isa ) bus , micro channel architecture ( mca ) bus , enhanced isa ( eisa ) bus , video electronics standards association ( vesa ) local bus , and peripheral component interconnect ( pci ) bus also known as mezzanine bus . computer 110 typically includes a variety of computer readable media . computer readable media can be any available media that can be accessed by computer 110 and includes both volatile and nonvolatile media , removable and non - removable media . by way of example , and not limitation , computer readable media may comprise computer storage media and communication media . computer storage media includes both volatile and nonvolatile , removable and non - removable media implemented in any method or technology for storage of information such as computer readable instructions , data structures , program modules or other data . computer storage media includes , but is not limited to , ram , rom , eeprom , flash memory or other memory technology , cd - rom , digital versatile disks ( dvd ) or other optical disk storage , magnetic cassettes , magnetic tape , magnetic disk storage or other magnetic storage devices , or any other medium which can be used to store the desired information and which can accessed by computer 110 . communication media typically embodies computer readable instructions , data structures , program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media . the term “ modulated data signal ” means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal . by way of example , and not limitation , communication media includes wired media such as a wired network or direct - wired connection , and wireless media such as acoustic , rf , infrared and other wireless media . combinations of the any of the above should also be included within the scope of computer readable media . the system memory 130 includes computer storage media in the form of volatile and / or nonvolatile memory such as read only memory ( rom ) 131 and random access memory ( ram ) 132 . a basic input / output system 133 ( bios ), containing the basic routines that help to transfer information between elements within computer 110 , such as during start - up , is typically stored in rom 131 . ram 132 typically contains data and / or program modules that are immediately accessible to and / or presently being operated on by processing unit 120 . by way of example , and not limitation , fig5 illustrates operating system 134 , application programs 135 , other program modules 136 , and program data 137 . the computer 110 may also include other removable / non - removable , volatile / nonvolatile computer storage media . by way of example only , fig5 illustrates a hard disk drive 140 that reads from or writes to non - removable , nonvolatile magnetic media , a magnetic disk drive 151 that reads from or writes to a removable , nonvolatile magnetic disk 152 , and an optical disk drive 155 that reads from or writes to a removable , nonvolatile optical disk 156 such as a cd rom or other optical media . other removable / non - removable , volatile / nonvolatile computer storage media that can be used in the exemplary operating environment include , but are not limited to , magnetic tape cassettes , flash memory cards , digital versatile disks , digital video tape , solid state ram , solid state rom , and the like . the hard disk drive 141 is typically connected to the system bus 121 through a non - removable memory interface such as interface 140 , and magnetic disk drive 151 and optical disk drive 155 are typically connected to the system bus 121 by a removable memory interface , such as interface 150 . the drives and their associated computer storage media discussed above and illustrated in fig5 , provide storage of computer readable instructions , data structures , program modules and other data for the computer 110 . in fig5 , for example , hard disk drive 141 is illustrated as storing operating system 144 , application programs 145 , other program modules 146 , and program data 147 . note that these components can either be the same as or different from operating system 134 , application programs 135 , other program modules 136 , and program data 137 . operating system 144 , application programs 145 , other program modules 146 , and program data 147 are given different numbers here to illustrate that , at a minimum , they are different copies . a user may enter commands and information into the computer 20 through input devices such as a keyboard 162 and pointing device 161 , commonly referred to as a mouse , trackball or touch pad . other input devices ( not shown ) may include a microphone , joystick , game pad , satellite dish , scanner , or the like . these and other input devices are often connected to the processing unit 120 through a user input interface 160 that is coupled to the system bus , but may be connected by other interface and bus structures , such as a parallel port , game port or a universal serial bus ( usb ). a monitor 191 or other type of display device is also connected to the system bus 121 via an interface , such as a video interface 190 . in addition to the monitor , computers may also include other peripheral output devices such as speakers 197 and printer 196 , which may be connected through an output peripheral interface 190 . the computer 110 may operate in a networked environment using logical connections to one or more remote computers , such as a remote computer 180 . the remote computer 180 may be a personal computer , a server , a router , a network pc , a peer device or other common network node , and typically includes many or all of the elements described above relative to the computer 110 , although only a memory storage device 181 has been illustrated in fig5 . the logical connections depicted in fig5 include a local area network ( lan ) 171 and a wide area network ( wan ) 173 , but may also include other networks . such networking environments are commonplace in offices , enterprise - wide computer networks , intranets and the internet . when used in a lan networking environment , the computer 110 is connected to the lan 171 through a network interface or adapter 170 . when used in a wan networking environment , the computer 110 typically includes a modem 172 or other means for establishing communications over the wan 173 , such as the internet . the modem 172 , which may be internal or external , may be connected to the system bus 121 via the user input interface 160 , or other appropriate mechanism . in a networked environment , program modules depicted relative to the computer 110 , or portions thereof , may be stored in the remote memory storage device . by way of example , and not limitation , fig5 illustrates remote application programs 185 as residing on memory device 181 . it will be appreciated that the network connections shown are exemplary and other means of establishing a communications link between the computers may be used . fig6 is an illustration of a cll prognosis routine 200 for determining a prognosis of a patient presenting with chronic lymphocytic leukaemia ( cll ), as provided in greater detail above . all or part of the method 200 may be implemented as one or more routines , which may be provided on a computer readable medium and / or operational with a computing system where the routine is adapted to be executed by a processor . the routine 200 may be implemented using any suitable programming languages and techniques . referring to fig6 , the routine 200 begins at block 210 where input values are received for measurements of a v h gene mutation status ( v h ), cd38 expression ( cd38 ), and zap - 70 expression ( zap70 ) obtained from lymphocytes of a human with chronic lymphcytic leukaemia ( cll ). for example , information concerning the v h gene status of an individual , information concerning the expression of cd38 in cll cells taken from the patient , and information concerning the expression of the zap - 70 in cll cells taken from the patient may be inserted into a computer , such as the computer 110 of fig5 , using a variety of means , including , but not limited to , a removable memory interface 150 , a user input interface 160 , a network interface 170 or other devices for receiving information at the computer 110 . at block 220 , a cll cell protein tyrosine phosphorylation ( py postigm ) is generated from the measurements received at block 210 . the py postigm generated at block 220 may be calculated as a sum based upon the measurements according to the weighted relationship : cd38 = the cll cell surface presence of cd38 , expressed as % of cll cells with greater fluorescence than an isotype - matched control , vh status = vh gene mutation status , expressed as % homology to the closest germline sequence , and zap70 = cll cell zap - 70 expression , expressed as % of cll cells with equal or greater fluorescence than the t - cell population in the same sample . the py postigm may be calculated according to the following formula : at block 230 , a representation of the py postigm generated at block 220 may be displayed on a display unit , such as the monitor 191 of fig5 . the representation may be provided as an alphanumeric display and / or as a graphical display . in particular , the representation may be provided by a user interface routine which provides a graphical user interface ( gui ) and which may be implemented via a computing system , such as the computer 110 . it should be recognized that the gui may include one or more software routines that are implemented using any suitable programming languages and techniques . at block 240 , a prognosis for the patient with cll may be generated based upon the py postigm generated at block 220 . the routine 200 may determine whether the prognosis generated at block 240 is a good prognosis or a poor prognosis at block 250 . for example , the determination at block 250 may include determining whether the phosphorylation status is above or below a certain threshold . in the formula indicated above , the threshold may be 26 ± 2 . 6 . alternatively , the threshold is where the expression : is greater than 50 , and , more particularly , where the above expression is greater than 52 . 95 . if the phosphorylation status is above the threshold , as determined at block 250 , the routine 200 may determine that the prognosis of the patient is poor , and provide an indication that the prognosis is poor at block 260 . as explained in further detail above , an elevated py postigm correlates with a poor prognosis . if the phosphorylation status is below the threshold , as determined at block 250 , the routine 200 may determine that the prognosis of the patient is good , and provide an indication that the prognosis is good at block 270 . at block 280 , a course of treatment for the patient may be generated based upon the py postigm generated at block 220 or based upon the prognosis generated at block 240 . 1 . shanafelt , t . d ., geyer , s . m . & amp ; kay , n . e . prognosis at diagnosis : integrating molecular biologic insights into clinical practice for patients with cll . blood 103 , 1202 - 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