TITLE: Development of a Hybrid Optical Biopsy Probe to Improve Prostate Cancer Diagnosis

Transcription

1 AD Award Number: W81XWH TITLE: Development of a Hybrd Optcal Bopsy Probe to Improve Prostate Cancer Dagnoss PRINCIPAL INVESTIGATOR: Hanl Lu CONTRACTING ORGANIZATION: Unversty of Texas at Arlngton Arlngton, TX REPORT DATE: June 2010 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medcal Research and Materel Command Fort Detrck, Maryland DISTRIBUTION STATEMENT: Approved for publc release; dstrbuton unlmted The vews, opnons and/or fndngs contaned n ths report are those of the author(s) and should not be construed as an offcal Department of the Army poston, polcy or decson unless so desgnated by other documentaton.

2 REPORT DOCUMENTATION PAGE Form Approved OMB No Publc reportng burden for ths collecton of nformaton s estmated to average 1 hour per response, ncludng the tme for revewng nstructons, searchng exstng data sources, gatherng and mantanng the data needed, and completng and revewng ths collecton of nformaton. Send comments regardng ths burden estmate or any other aspect of ths collecton of nformaton, ncludng suggestons for reducng ths burden to Department of Defense, Washngton Headquarters Servces, Drectorate for Informaton Operatons and Reports ( ), 1215 Jefferson Davs Hghway, Sute 1204, Arlngton, VA Respondents should be aware that notwthstandng any other provson of law, no person shall be subject to any penalty for falng to comply wth a collecton of nformaton f t does not dsplay a currently vald OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED 1Jun TITLE AND SUBTITLE Revsed Annual 1 Jun May a. CONTRACT NUMBER Development of a Hybrd Optcal Bopsy Probe to Improve Prostate Cancer Dagnoss 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Hanl Lu, Ph.D 5e. TASK NUMBER Emal: hanl@uta.edu 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER The Unversty of Texas at Arlngton Arlngton, TX SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medcal Research and Materel Command Fort Detrck, Maryland DISTRIBUTION / AVAILABILITY STATEMENT Approved for publc release; dstrbuton unlmted 13. SUPPLEMENTARY NOTES 11. SPONSOR/MONITOR S REPORT NUMBER(S) 14. ABSTRACT Hypothess: a mult-modal optcal spectroscopc method and an ntegrated needle probe can be developed for gudng needle bopsy for prostate cancer dagnoss. Mult-modal optcal measurements to be utlzed for the study are (1) lght scatterng spectroscopy (LSS), (2) auto-fluorescence spectroscopy (AFS), and (2) auto-fluorescence lfe-tme measurements (AFLT). Our specfc ams are: Am 1: to develop a mult-modal, optcal spectroscopc nstrument, whch allows the measurements of (1) LSS, (2) AFS, and (3) AFLT. The proposed system wll be portable, can be used for n vvo measurements, and collect and present the data n real tme. Am 2: to ntegrate the optcal fbers, whch collect lght scatterng and auto-fluorescence from the prostate tssue, nto a transrectal-ultrasound, needle-bopsy probe. In the development phase, the optcal sgnatures of prostate cancer can be collected wth the bopsy tssues and dentfed along every tract of needle bopses. Am 3: to collect optcal sgnals of control and cancer tssues ex vvo along wth the regular human needle bopsy, followed by classfcaton algorthm development to dscrmnate cancer tssues. Am 4: to perform n vvo measurement from human subjects to obtan the accuracy and senstvty of the ntegrated probe n order to provde real-tme, on-ste, mproved gudance for prostate cancer tssue bopsy. 15. SUBJECT TERMS Technology Development, optcal spectroscopy, transrectal probe, optcal bopsy, auto-fluorescence spectroscopy, fluorescence lfe-tme measurement. 16. SECURITY CLASSIFICATION OF: Unclassfed 17. LIMITATION OF ABSTRACT a. REPORT U b. ABSTRACT U c. THIS PAGE U 18. NUMBER OF PAGES 15 19a. NAME OF RESPONSIBLE PERSON Hanl Lu 19b. TELEPHONE NUMBER (817) Standard Form 298 (Rev. 8-98) Prescrbed by ANSI Std. Z39.18

3 Table of Contents SF Table of Contents Introducton Body of the Report Key Research Accomplshments and Reportable Outcomes Conclusons References

4 Annual Progress Report Ths report presents the specfc ams and accomplshments of our prostate cancer research project durng the year of fundng sponsored by the US Department of the Army. It covers our actvtes from May 1, 2009 to August 31, Introducton It s known that effectve treatments for prostate cancer are hghly assocated wth accurate and early detecton of prostate cancer. The current tools for detecton of prostate cancer have serous shortcomngs. So, the fnal dagnoss for prostate cancer reles on needle bopsy through transrectal-ultrasound gudance. Snce ultrasound s not able to show any cancerous lesons, the current dagnostc needle bopsy for prostate cancer s rather Blnd : the bopsy samples were collected almost blndly wthout knowng whether or not the bopsy lesons are hghly specous for cancer. The current dagnostc method n needle bopsy has three major defcences: (1) t may mss cancers that are present and have large hgh-grade leson, (2) t can underestmate the severty of cancer that present, and (3) t may fnd only tny low-grade cancers that lkely do not cause problems. Because of these defcences, the current prostate cancer patents are ether under or over treated: under treatments cause patents lves; over treatments cause patents unnecessary mpotence or ncontnence. The hypothess for ths study s that a mult-modal optcal spectroscopc method and an ntegrated needle probe can be developed for gudng needle bopsy for prostate cancer dagnoss. Mult-modal optcal measurements to be utlzed for the study are (1) lght reflectance spectroscopy (LRS), (2) auto-fluorescence spectroscopy (AFS), and (2) auto-fluorescence lfe-tme measurements (AFLM). The project has four specfc ams: Am 1: to develop a mult-modal, optcal spectroscopc nstrument, whch allows the measurements of (1) LRS, (2) AFS, and (3) AFLM. The proposed system wll be portable, can be used for n vvo measurements, and collect and present the data n real tme. Am 2: to ntegrate the optcal fbers, whch collect lght scatterng and auto-fluorescence from the prostate tssue, nto a transrectal-ultrasound, needle-bopsy probe. In the development phase, the optcal sgnatures of prostate cancer can be collected wth the bopsy tssues and dentfed along every tract of needle bopses. Am 3: to collect optcal sgnals of control and cancer tssues ex vvo along wth the regular human needle bopsy, followed by classfcaton algorthm development to dscrmnate cancer tssues. Am 4: to perform n vvo measurement from human subjects to obtan the accuracy and senstvty of the ntegrated probe n order to provde real-tme, on-ste, mproved gudance for prostate cancer tssue bopsy. Ths report wll summarze the work we performed from May 1, 2009 to August 2010 and the correspondng achevements obtaned durng ths perod of tme, as gven below. 3

5 2. Body of the Report The PI and her research team have made sgnfcant efforts from to accomplsh the proposed ams, resultng n several publshed journal and conference papers. We wll gve a bref report n the followng sub-sectons. 2.1 Background Prostate cancer s the most commonly found male cancer n the Unted States and s the second leadng cause of death from cancer n men [1]. It was estmated that n 2009, the number of new prostate cancer cases reported would be 192,280, resultng n 27,360 deaths [2]. At present, dgtal rectal examnaton (DRE), prostate specfc antgen (PSA) blood test and transrectal ultrasound (TRUS) guded bopsy are the clncally avalable technques for prostate cancer screenng and dagnoss. Among these, current gold standard for prostate cancer detecton s TRUS guded needle bopsy, whch nvolves resecton of a core of prostate tssue wth the gudance of an ultrasound probe. However, though ultrasound s able to provde anatomcal mages wth hgh spatal resoluton, t lacks the senstvty n dfferentatng tumor from normal tssue, especally at early stages. Ths defcency makes TRUS guded bopsy a rather blnd procedure nvolvng quas-random samplng of the prostate tssue [3]. The mplcatons of ths drawback are false negatves, and oversamplng of tssue due to non-specfc targetng. A standard bopsy procedure nvolves resecton of cores, and t was beleved that by ncreasng the number of core bopses, the detecton rate could be ncreased [4]. However, recent studes ndcate that there s no sgnfcant mprovement n detecton rate by ncreasng number of bopsy cores (whch s referred to as saturaton bopsy [5,6]. Moreover, ncreasng the number of cores ncreases complcatons assocated wth the bopsy procedure [4]. It s therefore mperatve to develop technques that can dentfy the tumor n-vvo wth hgh specfcty and senstvty and mprove the accuracy of bopsy. It s even more crucal to develop a cancer-targeted dagnostc technque, whch can dentfy the tumor on-ste wth hgh senstvty and specfcty. Optcal spectroscopc technques can be a canddate for such needed tool to dentfy cancer from control. They may be mplemented through needle probe geometry and wll be extremely helpful n mprovng the dagnostc procedure. The frst step n ths study s to utlze an optcal fber whch can be used to obtan a pror nformaton by mappng the prostate tssue frst, followed by subsequent bopsy of selected lesons. In ths way, we can ncrease the accuracy of the bopsy procedure, substantally reduce the number of bopsy cores, and thus reduce the amount of tssue resected as well as mnmze the assocated complcatons. Specfcally, two technques have been mplemented n such geometry n the past year: (1) lght reflectance spectroscopy (LRS) and (2) auto-fluorescence lfetme measurement (AFLM). LRS has been prevously utlzed for a varety of clncal applcatons by characterzng dfferent tssue types usng ether emprcal or analytcal methods [7,8,9,10,11,12]. Recently, updated methods have been developed to quantfy the absolute values of optcal parameters from LRS wth small probe geometres [9,13]. These analytcal approaches make t more robust to be appled as a generalzed means. On the other hand, varous research groups have also mplemented laser- nduced, steady-state auto-fluorescence spectroscopy and tme-resolved lfetme measurement for cancer detecton [14]. Steady-state fluorescence measurements are easy to perform, however, they are ntensty-dependent and thus senstve to optcal propertes of tssue, ambent lght, and exctaton lght ntensty. We opted for temporally-resolved fluorescence lfetme measurements as they are ndependent of absolute ntensty of exctaton lght and fluorophore concentraton. 4

6 2.2 Report for Am 1 Am 1: develop a mult-modal, optcal spectroscopc nstrument, whch allows the measurements of (1) LRS, (2) AFS, and (3) AFLM. The proposed system wll be portable, can be used for n vvo measurements, and collect and present the data n real tme. For ths am, we have mplemented and evaluated both LRS and AFLM systems ndependently, to dfferentate between cancer and non-cancerous tssue usng laboratory tssue phantoms. LRS s used to calculate the absolute concentratons of oxy-hemoglobn, deoxy-hemoglobn and scatterng propertes of the tssue. AFLM uses a custom bult devce to measure autofluorescence lfetme of the tssue. We have tested the effcacy of usng these multple parameters as classfers to dentfy prostate cancer. The results are presented n ths report and provde us wth the footprnts for future ex-vvo and n-vvo human prostate cancer measurements Laboratory tssue phantoms and preparaton Phantom measurements were carred out to test our newly mplemented LRS and AFLM system for ths part of study. Fgure 1 shows the dagram of the combned system wth a tssue phantom. To create a homogenous phantom, a blood-ntralpd soluton was made by dlutng a stock ntralpd soluton (concentraton=20%, Baxter Healthcare Corporaton, Deerfeld, IL) wth phosphate buffered salne (PBS) and de-fbrnated horse blood (Hemostat Laboratores, Dxon, CA). We vared the concentratons of ntralpd and blood, to obtan varous combnatons of scatterng and absorpton propertes. To smulate hemodynamc changes n the phantom, 4 mg of yeast was added nto a 3-lter lqud phantom to deoxygenate the blood-contanng phantom. When the phantom was fully deoxygenated, 100% oxygen was slowly bubbled nto the soluton to re-oxygenate t. Overall, we could smulate changes n lght scatterng by alternatng ntralpd concentratons and mmc changes n lght absorpton by modfyng ether the blood volume or oxygenaton state. In the tumor-smulatng experments, we ncreased the lght absorpton by 2-3 tmes hgher than that from the baselne (healthy-tssue) phantoms to detect the dfferences n both LRS and AFL. Fg. 1 A block dagram llustratng expermental set-up for LRS system (left) and AFLM system (rght). Measurements were made sequentally, by placng the fber tps on the tssue phantom surface. A closer vew of b-furcated fber tps are shown for each fber (red = source; blue = detector). Tssue phantom 5

7 2.2.2 Auto-florescence lfetme measurement AFLM Instrumentaton For auto-florescence lfetme measurements, a custom-made, sngle-channel, Tme Correlated Sngle Photon Countng (TCSPC) system (ISS Inc., Champagn, IL) was employed. Fg. 1 (rght) shows an overall expermental setup for the tme doman AFLM. The system conssted of a 5- slot flter wheel for exctaton wavelength selecton (whch was drven by a 12-V-powered stepper motor), an emsson flter, a contnuously varable neutral densty (ND) flter for exctaton lght ntensty control, and a hghly senstve cooled PMT (Becker & Hckl GmbH) wth wavelength senstvty between nm. The PMT gan was controlled va PC-based card (DCC-100). A pcoseconds (ps) broadband ( nm) pulsed laser (SC - 450, Fanum Inc., Eugene, Oregon) was used as an exctaton source at the repetton rate of 20 MHz. Precse synchronzaton between the ncomng laser pulse and photon event was acheved through a PC-based sngle photon countng card (SPC-130). As shown n Fg. 1, the laser was coupled to the source fber of the b-furcated optcal probe (core dameter 100 μm); resultng fluorescence emsson was collected through the detector fber (400 μm core dameter). AFLM Data Analyss In ths part of study, we were manly targetng auto-fluorescence from Flavns, Lpo-pgments and Porphyrns as a contrast parameter to dfferentate cancer from healthy tssue. In order to acheve maxmum possble auto-fluorescence from all three endogenous compounds, an exctaton wavelength of 447 nm was chosen [15]. Whle keepng the exctaton wavelength constant, the emsson wavelengths were changed between 532 nm, 562 nm, 632 nm and 684 nm. Data collecton was done through Vnc software provded by ISS. The optcal probe was placed at ~1 mm away from the surface of the tssue phantom; the fluorescence data were collected at 5 random postons from the phantom for each emsson wavelength. The detected fluorescence decay was mported nto Matlab (The Mathworks Inc., Natck, MA) and normalzed wth respect to peak ntensty. In order to quanttatvely dfferentate auto-fluorescence decay of cancer and control, each curve can be ftted (least square non-lnear fttng algorthm) usng a two-component exponental model, as gven by: t Intensty( I) e, (1) where, (=1, 2) ndcates lfetme of each component and α (=1, 2) s ntensty contrbuton of each component to the overall fluorescence decay. The confdence nterval for each of these four ftted parameters was computed usng the confnt functon n Matlab. In order to make the comparson between normal and cancerous tssue, the mean lfetme, < >, was calculated usng equaton 2: 2 (2) 6

8 2.2.3 Lght Reflectance spectroscopy LRS Instrumentaton The LRS system conssted of a tungsten-halogen lght source (HL2000HP, Ocean Optcs, Dunedn, FL, USA), a CCD array spectrometer (USB 2000+, Ocean Optcs, Dunedn, FL USA), and a laptop computer. The spectrometer provded a spectrum rangng from 460 nm-1150 nm. A custom-made, b-furcated, fber optc probe (Fbergude Industres, Strlng, NJ, USA) was desgned and mplemented wth source and detector dameters of 100 µm, center-to-center source and detector separaton of ~100 µm, and an outer probe dameter of 800 µm. The probe was fxed on a stereotactc frame n order to control the probe tp poston and mnmze the pressure on the tssue surface. Multple data ponts were obtaned by placng the probe at dfferent spatal locatons on the healthy-tssue phantoms and then the tumor-smulatng tssue phantoms. On an average, 5 data ponts were obtaned for each tssue phantom type. LRS Data Acquston and Analyss Each acqured spectrum was dvded by a reflectance spectrum obtaned from a dffuse reflectance standard (WS-1, Ocean Optcs, Dunedn, FL, USA) to elmnate the spectral effect from the lght source, optcal fbers and CCD array detector from the reflectance spectrum taken from the tssue sample. Fgure 2 shows a schematc dagram for the setup. For system calbraton, we utlzed a gold-standard, dualchannel, tssue oxmeter (ISS, Inc., IL, USA), as also shown n Fg. 2. The ISS oxmeter could provde absolute values of optcal propertes for the measured sample under lght nterrogaton, thus provdng calbraton parameters to LRS. (a) Fgure (b) 2 Then, the spectral range from 500 nm nm was selected and a reflectance model [9] as shown n equaton (3) was utlzed to obtan absolute values of the concentratons of oxyhemoglobn ([HbO]), deoxy-hemoglobn ([HbR]) and total hemoglobn ([HbT] = [HbR] + [HbO]), along wth the reduced scatterng coeffcent (μ s ). R p ( ) ( ) s k k ( ), (3) 1 ' 2 a where R p s the measured reflectance, μ s s the reduced scatterng coeffcent, μ a s the absorpton coeffcent dependng on the concentraton of hemoglobn dervatves and k 1 and k 2 are calbraton constants that depend on the probe geometry and hardware set-up. A detaled descrpton of methods to obtan the above parameters has been prevously publshed [7]. Brefly, at frst, k 1 and k 2 were calculated for the specfc fber optc probe usng tssue phantom calbraton. Then, an optmzaton routne [16] (ant colony optmzaton) was used to obtan the parameters of μ s at 750 nm, [HbO] and [HbR], by achevng the best ft for the measured reflectance. The data processng was mplemented and executed n Matlab. Specfcally, we provde detals on data analyss/process procedures as follows. (1) Quantfcaton of hemoglobn concentratons: 7

9 It s well known that μ a (λ) s a functon of concentratons of deoxygenated hemoglobn, [Hb], oxygenated hemoglobn, [HbO], and water, H 2 O. The spectral dependence of µ a on [HbO], [Hb], and H 2 O for blood-perfused tssues can be wrtten as a ( ) [ HbO] HbO ( ) [ Hb] Hb ( ) [% H2O] H ( ), (4) 2O where s the wavelength n nm, ε HbO (λ), ε Hb (λ) and ε H2O (λ) are the wavelength dependent extncton coeffcents of [HbO], [Hb], and water, respectvely. [%H 2 O] represents the percentage of water n the medum. Other chromophores, such as fat and melann, may be added to ths equaton, dependng on the wavelength range of nterest and tssue type under nvestgaton. The spectral dependence of µ s can be approxmated as gven by Me theory [17]: g 1.1 ( ), (5) ' (1 g), (6) s s, (7) s a b where μ s s the effectve scatterng coeffcent, μ s s the reduced scatterng coeffcent, and g s the ansotropy factor. In eq. (7), parameters of a and b are constants and depend on scatterer szes and types. For 10% ntralpd soluton, the calculated values are a = 2.54x10 9 cm -1 and b = 2.4 [17]. (2) Calculaton and calbraton of k 1 and k 2 In ths study, we determned the values of k 1 and k 2 usng the least-squares regresson approach. By rearrangng (3), we obtan ' s ( ) k1 k2 a ( ). (8) R ( ) p Ths equaton shows that k 1 and k 2 can be determned by obtanng a lnear regresson lne that best fts μ s (λ)/r p (λ) versus µ a (λ). In prncple, we can obtan the measured spectra of R p (λ) from the LRS system wthn nm and also acheve quantfcaton of µ a and μ s at 750 nm and 830 nm from ISS oxmeter f the measurements by both LRS and ISS oxmeter are taken smultaneously. Besdes μ a values, ISS oxmeter s also able to provde derved values of [Hb], [HbO], and μ s at two wavelengths (OxplexTS). Then, wavelength-dependent absorpton spectra, μ a (λ), can be quantfed usng eq. (4) f the measured [Hb] and [HbO] as well as the hemoglobn extncton coeffcents are avalable. Moreover, gven two μ s values at two measured wavelengths (750 nm and 830 nm), t s reasonable to nterpolate and extrapolate μ s (λ) over the desred wavelength range usng eqs. (5)-(7) based on Me theory. Durng the system calbraton n the study, we followed the exact prncple or procedures gven above to acqure R p (λ), µ a (λ), and μ s (λ) from a set of blood-contanng tssue phantoms. A correspondng set of k 1 and k 2 at varous total hemoglobn concentratons and reduced scatterng coeffcents were obtaned, averaged, and calculated for ther means and standard devatons. If the relatve errors for both k 1 and k 2 were less than 10% (.e., standard devatons dvded by ther means), then the correspondng set of k 1 and k 2 were fnalzed as the system calbraton parameters for the specfc LRS system (.e., the lght source, fber probe, and spectrometer) chosen for ths study. As an example, Fg. 3(a) shows a set of measured R p (λ), µ a (λ), and μ s (λ) (blue symbols) and the lnear regresson lne (red thck lne) for our LRS probe wth k 1 beng the y-ntercept and k 2 beng the slope. Whle the nverse calculatons for an unknown sample wll be descrbed n 8

10 the next sub-secton, an llustraton of the measured and ftted reflectance s shown n Fg. 3(b). The accuracy of parameters k 1 and k 2 crtcally affects the goodness of ft n the nverse calculatons, and thus nfluences the accuracy of measured optcal propertes of tssues under nterrogaton. It s noteworthy to pont out that k 1 and k 2 depend hghly on the whte sample measurement for elmnatng nstrumentaton effects on the measured spectrum of the sample under examnaton. Incorrect measurement of the whte sample wll lead to serous errors for the ftted parameters,.e., [Hb], [HbO], a and b n eq. (7). Us'/R μs / Rp (cm-1) (cm -1 ) (a) Ua μ a (cm-1) ) Whte sample measurement can vary wth the followng parameters: dstance between the fber tp and sample surface, ntegraton tme, lght source ntensty. Measurng dfferent types of tssue requres a dfferent ntegraton tme for the detector. Fg 4 shows the measurement geometry from the whte sample. Snce the reflectance curve needs to be dvded by the reference curve from the whte sample, care should be taken to standardze the procedure. Ths can be done ether by fxng the dstance and collectng whte sample at varous ntegraton tmes to create a lookup table, or by scalng the whte sample reflectance curve based on one sngle measurement. The latter method s based on our observaton that the relatonshp between reflectance spectra and ntegraton tmes s approxmately lnear. Fgure 4 (3) Inverse calculaton The nverse calculaton of eq. (3) deals wth determnaton of tssue chromophore concentratons and effectve scatterng coeffcents from the measurements of LRS taken on the tssue surface. The mathematcal problem of nverson can be practcally solved by an approach of functon mnmzaton usng an optmzaton algorthm. The algorthm searches for an optmal set of values, as represented by x=( [Hb], [HbO], a, b), from the gven mult-parameter space that best match the measured R p ( ) usng the least- squares analyss. It can be mathematcally expressed as follows: M f ( x) 1 2 ( measured) ( predcted) R p ( ) R ( ), (9) p Normalzed Reflectance (A.U.) Fgure Wavelength Lambda (nm) (nm) where f(x) represents an objectve functon and needs to be mnmzed by fndng an optmal set of the parameters, x opt, M s the number of wavelengths, R p ( ) (measured) and R p ( ) (predcted) are the reflectance values (at wavelength λ ) measured by the optcal probe and predcted by eqs. (3) to (7), respectvely, wth the parameter set, x, to be optmzed. Evolutonary algorthms are wdely utlzed n the feld of optmzaton. The algorthms work on a populaton of values rather an ntal guess, makng the soluton ndependent of an ntal guess. Ther nherent characterstcs make them sutable to search for a global mnmum, rather than beng stuck n local mnma. The ant colony optmzaton (ACO) algorthm, ntroduced by M. Dorgo [18], s a probablstc evolutonary (b) Measured Data Ftted Data 9

11 technque for solvng computatonal problems. Ths basc technque was modfed n ths study to sut functon mnmzaton [16]. Equaton (3) s very non-lnear and spanned from 500 nm to 850 nm. In order to obtan optmal ft between the measured and calculated reflectance curve, we had to ncorporate multple sequences, wthn each of whch the objectve functon for mnmzaton, eq. (9), was selected somewhat dfferently. Frst, the entre spectrum (500 nm 850 nm) was utlzed for fttng and the ftted values of [Hb], [HbO], a and b n eq. (7) were obtaned. Next, the μ s ( ) values (or a and b) were fxed wth 15% bounds used n sequence one. A smaller spectral regon of nm that has a strong hemoglobn absorpton band was selected for re-fttng to mprove goodness of fttng for [Hb] and [HbO]. Fnally, after refnng the ftted parameters of [Hb] and [HbO], they were fxed wthn 20% bounds used n sequence two. The entre spectrum ( nm) was re-ftted, leadng to the fnally optmzed values of [Hb], [HbO], a and b. Overall, three fttng sequences wth changng parameter bounds and spectral regons were utlzed n order to obtan an optmal ft for scatterng and absorpton parameters. An example to show comparson between the measured and ftted reflectance s already gven n Fg. 3(b) Results: Algorthm valdaton for absolute quantfcaton by LRS In order to valdate the algorthm for absolute quantfcaton, we created tssue phantoms and performed the measurements wth both our sngle-channel LRS system and a tssue oxmeter (ISS Oxmeter), whch was used as a gold standard devce for comparson. Fgure 5(a) shows comparsons between the values obtaned from ISS tssue oxmeter and those quantfed wth our method when a change n oxygen saturaton(o 2 Sat) was acheved by addng yeast to the lqud phantom. The measurements were made at 12 dfferent tme ponts n the O 2 Sat range of ~20%-80% durng the oxygenaton and deoxygenaton process. It s clear from Fg. 5(a) that oxygen saturaton(o 2 Sat) values measured by both modaltes are very consstant n the O 2 Sat range of 20%-80%. The absolute errors were also calculated at each data pont (n=12) and are plotted n Fg. 5(b): average absolute errors for O 2 Sat, [Hb] and [HbO], along wth standard error of mean (SEM) were 3.0±0.7 %, 0.9±0.2 M, and 0.9±0.2 M, respectvely. Smlar experments were repeated several tmes, and the results were very smlar to those seen n Fgs. 5(a) and 5(b). The relablty of the algorthm was also evaluated by comparng the changes n total hemoglobn concentraton, [HbT], and μ s for each set of the above measurement. Snce yeast dd not nduce much change n ether [HbT] or μ s, these two parameters should deally reman constant when the oxygenaton state was altered. Fgure 5(c) shows the measured values of [HbT] and μ s ( at 750 nm and 830 nm) wth changng O 2 Sat. As expected, those values are found to be constant across the saturaton range of 20-80% wth small SEMs as [HbT] = 28.0 ±0.4 M, μ s (750 nm) = 10.46±0.05 cm -1, and μ s (830 nm) = 9.34±0.04 cm -1. Another set of experments were conducted to test the consstancy of μ s when Intralpd volume was vared from 75 ml to 170 ml wthn the gven lqud phantom volume. Fgure 5(d) shows a comparson of values obtaned by the ISS oxmeter and LRS system at 830 nm. The mean dfference (wth standard devaton) between the scatterng values obtaned by two methods was calculated to be 0.02±0.13 cm -1, demonstratng almost perfect match between the two methods. After comprehensve algorthm valdaton for LRS, we were able to quantfy optcal propertes and hemodynamc parameters, namely μ a, μ s, [HbO], and [HbT], of nterrogated tssues by the optcal probe. The LRS system was ready for studes usng ex vvo human prostate specmens. 10

12 (a) (b) (c) (d) Fgure 5 For AFLM results, we dd not obtan too much meanngful results, possbly because our tssue phantoms are hghly hemoglobn-based and are not a good lght fluorescence tssue model. As mentoned earler, the auto-fluorescence contrast comes manly from Flavns, Lpopgments and Porphyrns. Our current tssue phantoms dd not nclude much of those components. The system would be better tested and valdated when we are ready for ex vvo human prostate specmen measurements. 2.3 Report for Am 2 Am 2: to ntegrate the optcal fbers, whch collect lght scatterng and auto-fluorescence from the prostate tssue, nto a transrectal-ultrasound, needle-bopsy probe. In the development phase, the optcal sgnatures of prostate cancer can be collected wth the bopsy tssues and dentfed along every tract of needle bopses. For Am 2, we have completed and mplemented the 1 st verson of an ntegrated optcal-fber assembly that s under testng to collect both LRS and AFLM. The detaled desgn s gven below: 11

13 3. Key Research Accomplshments and Reportable Outcomes (1) Successfully developed a mult-modal, optcal spectroscopc nstrument, whch allows the measurements of (1) LRS, (2) AFS, and (3) AFLM. The ndvdual systems have been tested usng laboratory tssue phantoms. (2) Two related research works are publshed: (a) Anndta Mukerjee, Rafal Luchowsk, Amalendu P. Ranjan, Sangram Raut, Jamboor K. Vshwanatha, Zygmunt Gryczynsk, and Ignacy Gryczynsk, Enhanced Fluorescence of Curcumn on Plasmonc Platforms, Current Pharmaceutcal Botechnology, 11, (2010). (b) Anndta Mukerjee, Thomas J. Sørensen, Amalendu P. Ranjan, Sangram Raut, Ignacy Gryczynsk, Jamboor K. Vshwanatha, and Zygmunt Gryczynsk, Spectroscopc Propertes of Curcumn: Orentaton of Transton Moments, J. Phys. Chem. B, 114, (2010). 4. Conclusons and plan for next year In summary, for the perod from May 2009 to August 2010, we have mplemented two separate unts for AFLM and LRS and demonstrated that both AFLM and LRS are feasble methods to collect good optcal sgnals from tumor-smulatng tssue phantoms. However, the AFLM results from tssue phantoms may not truly reflect the actual sgnals collected from the human prostates. Further measurements from human prostate specmens wll be explored n the followng year. On the other hand, the LRS results are much relable and reflect the dfferences between healthy and tumor tssues more realstcally than those n AFLM. The senstvty and specfcty of ths technque s also reasonably hgh; the technque may allow deeper tssue measurements, as compared to AFLM, dependng on [HbR] levels n the tumor. In the comng year, we wll carry on the development for Am 2 and Am 3: (1) to ntegrate the optcal fbers, whch collect lght scatterng and auto-fluorescence from the prostate tssue, nto a 12

14 combned, needle-bopsy probe; (2) to collect optcal sgnals of control and cancer tssues ex vvo along wth the regular human needle bopsy, followed by classfcaton algorthm development to dscrmnate cancer tssues. 5. References 1. A. Jemal, R. Segel, E. Ward, Y. Hao, J. Xu and M. J. Thun, "Cancer statstcs, 2009," CA Cancer J Cln 59(4), (2009). 2. A. C. Socety, "Cancer Facts and Fgures 2009," Atlanta: Amercan Cancer Socety (2009). 3. N. B. Delongchamps and G. P. Haas, "Saturaton bopses for prostate cancer: current uses and future prospects," Nat Rev Urol 6(12), (2009). 4. U. Patel, "TRUS and prostate bopsy: current status," Prostate Cancer Prostatc Ds 7(3), (2004). 5. A. Descazeaud, M. Rubn, S. Chemama, S. Larre, L. Salomon, Y. Allory, D. Vordos, A. Hoznek, R. You, D. Chopn, C. Abbou and A. de la Talle, "Saturaton bopsy protocol enhances predcton of pt3 and surgcal margn status on prostatectomy specmen," World J Urol 24(6), (2006). 6. J. S. Jones, A. Patel, L. Schoenfeld, J. C. Rabets, C. D. Zppe and C. Mag-Galluzz, "Saturaton technque does not mprove cancer detecton as an ntal prostate bopsy strategy," J Urol 175(2), (2006). 7. V. Sharma, D. Kashyap, A. Mathker, S. Narvenkar, K. Bensalah, W. Kabban, A. Tuncel, J. A. Cadeddu and H. Lu, "Optcal reflectance spectroscopy for detecton of human prostate cancer," Conf Proc IEEE Eng Med Bol Soc 2009, (2009). 8. H. Lu, H. Radhakrshnan, A. K. Senapat, C. E. Hagans, D. Peswan, A. Mathker and Y. B. Peng, "Near nfrared and vsble spectroscopc measurements to detect changes n lght scatterng and hemoglobn oxygen saturaton from rat spnal cord durng perpheral stmulaton," Neuromage 40(1), (2008). 9. G. Zonos and A. Dmou, "Modelng dffuse reflectance from sem-nfnte turbd meda: applcaton to the study of skn optcal propertes," Opt Express 14(19), (2006). 10. C. A. Gller, H. Lu, D. C. German, D. Kashyap and R. B. Dewey, "A stereotactc nearnfrared probe for localzaton durng functonal neurosurgcal procedures: further experence," J Neurosurg 110(2), (2009). 11. M. Johns, C. Gller, D. German and H. Lu, "Determnaton of reduced scatterng coeffcent of bologcal tssue from a needle-lke probe," Opt Express 13(13), (2005). 12. K. Bensalah, D. Peswan, A. Tuncel, J. D. Raman, I. Zeltser, H. Lu and J. Cadeddu, "Optcal reflectance spectroscopy to dfferentate bengn from malgnant renal tumors at surgery," Urology 73(1), (2009). 13. S. C. Kanck, C. van der Leest, J. G. Aerts, H. C. Hoogsteden, S. Kascakova, H. J. Sterenborg and A. Amelnk, "Integraton of sngle-fber reflectance spectroscopy nto ultrasound-guded endoscopc lung cancer stagng of medastnal lymph nodes," J Bomed Opt 15(1), P. K. M. Katka, L. Plon, K. Dpple, S. Levn, J. Blackwell and H. Berberoglu, In vvo tmeresolved autofluorescence measurements on human skn, Proc. SPIE 6078, 60780L (2006). 15. G.A. Wagnères, W.M. Star and B.C. Wlson, "In vvo fluorescence spectroscopy and magng for oncologcal applcatons," Photochem Photobol 68(5), (1998). 16. D. Kashyap, "Development of a Broadband Mult-Channel NIRS System for Quantfyng Absolute Concentratons of Hemoglobn Dervatves and reduced scatterng Coeffcents," Thess dssertaton (2007). 13

15 17. van Staveren, H.J., Moes, C.J.M., van Mare, J., Prahl, S.A., van Gemert, M.J.C., Lght scatterng n Intralpd-10% n the wavelength range of nm. Appled Optcs 30, Dorgo, M., Manezzo, V., Colorn, A., Ant system: optmzaton by a colony of cooperatng agents. IEEE Trans Syst Man Cybern B Cybern 26,