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(12) Patent Application Publication (10) Pub. No.: US 2014/0127716 A1 Longo Et Al US 2014O127716A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0127716 A1 Longo et al. (43) Pub. Date: May 8, 2014 (54) BENCHMARKS FOR NORMAL CELL (60) Provisional application No. 61/240,613, filed on Sep. IDENTIFICATION 8, 2009. (71) Applicant: Nodality, Inc, South San Francisco, CA (US) Publication Classification (72) Inventors: Diane Longo, Foster City, CA (US); (51) Int. C. Alessandra Cesano, Redwood City, CA GOIN33/50 (2006.01) (US); Garry P. Nolan, South San G06F 9/72 (2006.01) Francisco, CA (US) (52) U.S. C. CPC ............ G0IN33/5041 (2013.01); G06F 19/12 (73) Assignee: Nodality, Inc, South San Francisco, CA (2013.01) (US) USPC ............. 435/7.21: 435/29: 435/7.24; 435/7.4 (21) Appl. No.: 14/072,623 (22) Filed: Nov. 5, 2013 (57) ABSTRACT Related U.S. Application Data Provided herein are methods, compositions, and kits for (63) Continuation of application No. 13/821,539, filed on determining cell signaling profiles in normal cells and com Oct. 2, 2013, Continuation-in-part of application No. paring the cell signaling profiles of normal cells to cell sig 12/877,998, filed on Sep. 8, 2010. naling profiles from a test sample. Patent Application Publication May 8, 2014 Sheet 1 of 37 US 2014/O12771.6 A1 SC: Z 2 N 2 29:NS Patent Application Publication May 8, 2014 Sheet 2 of 37 US 2014/O12771.6 A1 Patent Application Publication May 8, 2014 Sheet 3 of 37 US 2014/O12771.6 A1 se|duesque?e),TWV Patent Application Publication May 8, 2014 Sheet 5 of 37 US 2014/O12771.6 A1 FLT-WTAML that signal FLT-WTAML that signal like Healthy BMMb like FLT3-ITDAML O Healthy BMMb S FLT3 ITDAML O FLT3 WTAML -0.2 O.O 0.2 0.4 DOnOrS With OW mutational load Principal Component 1 FIG. 5 Patent Application Publication May 8, 2014 Sheet 6 of 37 US 2014/O12771.6 A1 alueW pope) SnpW Patent Application Publication May 8, 2014 Sheet 7 of 37 US 2014/O12771.6 A1 N Patent Application Publication May 8, 2014 Sheet 8 of 37 US 2014/O12771.6 A1 i CN w- C CO CN v O. to N. CN senal 3e3Sd-1a C.geSd-1z C. C LeSd-91 Patent Application Publication May 8, 2014 Sheet 9 of 37 US 2014/O12771.6 A1 O O O an O) OO N. Seo +Of % 09 N CN C 9Sd peonpu Off-O Patent Application Publication May 8, 2014 Sheet 10 of 37 US 2014/O12771.6 A1 Patent Application Publication May 8, 2014 Sheet 11 of 37 US 2014/O12771.6 A1 s||30.IIejo96e?u0013d 06080/09070Z0|| Patent Application Publication May 8, 2014 Sheet 12 of 37 US 2014/O12771.6 A1 Patent Application Publication May 8, 2014 Sheet 13 of 37 US 2014/O12771.6 A1 LXIV-dT£LTHOZZOVÝ £LVLS-d’T-ISO-9OGG069-dOGLVLS-d’T-ISO-9OGG069-d'O Patent Application Publication May 8, 2014 Sheet 14 of 37 US 2014/O12771.6 A1 s||30.IIego96e?u00d8d 06080/09070Z0|| Patent Application Publication May 8, 2014 Sheet 15 of 37 US 2014/0127716 A1 Patent Application Publication May 8, 2014 Sheet 16 of 37 US 2014/O12771.6 A1 adÅ?oueudeoeguns G/80°/| Patent Application Publication May 8, 2014 Sheet 17 of 37 US 2014/O12771.6 A1 Suoendo Ileo Jueled JOLLIX18ld)980Z39 (XEld) liv60-OOSE Patent Application Publication May 8, 2014 Sheet 18 of 37 US 2014/0127716 A1 Suoendo Ileo WOueW MujeeH cSN SSS SSSS dS&S SS28SNS NS v- Š R ŠSS W |ESSv Š 823:ŠŠSSS 383 ŠSSSSSSSS Š Šs SSS dig dow. So SS do RSS &c., cSR oS. do RSS Š2 YSSS92 v SSS v v SSR v- v- v- v- v- v- v- v v- v SEilee1 Y eleelee Odd Old C C CO d Od v- v- v- v- v- v- v- v- v- v v- v royd r-cy d v re HH H : 5. O. O. O. Ol i O O i5 i i 3.N4 3.N4 3LSO. O. 3LO. O. YNSS3 SleSSRY v. NSS YSR i v Y.Y. v \ . coc v :v NoN owy cy. SSl onCN S SNCO wr v v. v v Se N CD S33 conics colony w 22S V-V-ty: v v v. cNcNY ov : V CNCNC v- r- y- v- v- r- v- v- w * ov Yi c\c\,: CN 2N/ v v- w wr y d CO Odd Odd w v w w v v. w v v. v s s Patent Application Publication US 2014/O12771.6 A1 DDD -- Patent Application Publication May 8, 2014 Sheet 20 of 37 US 2014/O12771.6 A1 EEEE|. E. -- HHHtt-- t Patent Application Publication May 8, 2014 Sheet 21 of 37 US 2014/O12771.6 A1 Patent Application Publication May 8, 2014 Sheet 22 of 37 US 2014/O12771.6 A1 s||aoIIego36e?u3ouad 06080/09OGO?7OZ0|| Patent Application Publication May 8, 2014 Sheet 24 of 37 US 2014/O12771.6 A1 s||30.IIego36e?u0013) 06080/09070Z0|| Patent Application Publication May 8, 2014 Sheet 27 of 37 US 2014/O12771.6 A1 LVLS-d(OdL qnopeeu edÅ?oue?cieoeguns o|| \ IT1 r -u == >>< IT1 Patent Application Publication May 8, 2014 Sheet 28 of 37 US 2014/O12771.6 A1 lea?Aunspueu?MouÐII30uoS103JJE,Quepuedaduo!oe-u)^AO.10 Patent Application Publication May 8, 2014 Sheet 29 of 37 US 2014/O12771.6 A1 ?OL-TWO |eA?AInS9% +96eudeGVNG Patent Application Publication May 8, 2014 Sheet 30 of 37 US 2014/O12771.6 A1 JOLLU ||"Odol 9UUOS0040.Jej Patent Application Publication US 2014/O12771.6 A1 JOLUU |#760-OC19) Patent Application Publication May 8, 2014 Sheet 32 of 37 US 2014/O12771.6 A1 SunOH v CN Co. oO CO W. CN C v- v- w - efueuo po- ayuo-d efueus) pio XWCH-d Patent Application Publication May 8, 2014 Sheet 33 of 37 US 2014/O12771.6 A1 o LO O Lo o CN w O O efueuo pio legd-d O OO CO r CN O efueuo po Zyuo-d efueuo pio XVZH-d Patent Application Publication May 8, 2014 Sheet 35 of 37 US 2014/O12771.6 A1 0 OO O OOOOOOOOOOOOO O ene/A 89 ayuo pozfo Patent Application Publication May 8, 2014 Sheet 36 of 37 US 2014/O12771.6 A1 zo||vs |.||9269 Patent Application Publication May 8, 2014 Sheet 37 of 37 US 2014/O12771.6 A1 US 2014/O12771.6 A1 May 8, 2014 BENCHMARKS FOR NORMAL CELL able elements in single cells derived from the test sample; e) IDENTIFICATION identifying one or more cell-type markers in single cells derived from the test sample; and f) gating discrete popula CROSS-REFERENCE tions of single cells based on the one or more cell-type mark ers associated with the single cells. In another embodiment, 0001. This application is a continuation of U.S. patent the method further comprises generating the statistical application Ser. No. 13/821,539, filed Oct. 2, 2013, which is model, wherein generating the statistical model comprises: d) a national stage of PCT Patent Application No. US2011/ identifying the activation level of the one or more activatable 01565 filed Sep. 8, 2011 which claims the benefit of U.S. elements in single cells derived from the plurality of normal Patent Application Nos. 61/381,067 filed Sep. 8, 2010; samples; e) identifying one or more cell-type markers in 61/440,523 filed Feb. 8, 2011: 61/469,812 filed Mar. 31, single cells derived from the plurality of normal samples: f) 2011; and 61/499,127 filed Jun. 20, 2011, all of which are gating cells in the plurality of normal samples based on the incorporated herein by reference in their entireties. This one or more cell-type markers associated with the single cells; application is a continuation-in-part of U.S. patent applica and g) generating the statistical model that specifies the range tion Ser. No. 12/877,998, filed Sep. 8, 2010, which claims the of activation levels associated with cells in the normal benefit of U.S. Patent Application No. 61/240,613, filed Sep. samples. 8, 2009, all of which are incorporated by reference in their entireties. 0005. In another embodiment, the statistical model further specifies the variance of activation levels of the one or more BACKGROUND OF THE INVENTION activatable elements associated cells in the plurality of nor mal samples. In another embodiment, the one or more acti 0002 Personalized medicine seeks to provide prognoses, Vatable elements are selected from the group consisting of diagnoses and other actionable medical information for an pStat1, pStat3, pStata, pStats, pStat6 and p-p38. In another individual based on their profile of one or more biomarkers. embodiment, the method further comprises contacting the Many of these diagnostics use classifiers which are binary test sample and the plurality of normal samples with one or statistical models trained to identify biomarkers which differ more modulators. In another embodiment, the one or more entiate diseased cells from non-diseased cells (i.e., normal modulators is selected from the group consisting of: G-CSM, cells). While these classifiers are beneficial, a major draw EPO, GM-CSF, IL-27, IFNa and IL-6. back of these methods is that they only aim to determine 0006. In another embodiment, the test sample and the similarity between two states: disease and normal. Often, plurality of normal samples are derived from individuals with disease states are heterogeneous, which complicates the iden the same race, ethnicity, gender, or are in the same age-range.
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