US 2006O154278A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0154278 A1 Brody et al. (43) Pub. Date: Jul. 13, 2006 (54) DETECTION METHODS FOR DISORDERS (60) Provisional application No. 60/477,218, filed on Jun. OF THE LUNG 10, 2003. (75) Inventors: Jerome S. Brody, Newton, MA (US); Publication Classification Avrum Spira, Newton, MA (US) (51) Int. C. Correspondence Address: CI2O I/68 (2006.01) Ronald I. Eisenstein, Esq. G06F 9/00 (2006.01) NXON PEABODY LLP (52) U.S. Cl. ................................................... 435/6; 702/20 100 Summer Street Boston, MA 02110 (US) (57) ABSTRACT (73) Assignee: The Trustees of Boston University, Bos The present invention is directed to prognostic and diagnos ton, MA tic methods to assess lung disease risk caused by airway Appl. No.: 11/294,834 pollutants by analyzing expression of one or more genes (21) belonging to the airway transcriptome provided herein. (22) Filed: Dec. 6, 2005 Based on the finding of a so called “field defect” affecting the airways, the invention further provides a minimally Related U.S. Application Data invasive sample procurement method in combination with the gene expression-based tools for the diagnosis and prog (63) Continuation of application No. PCT/US04/18460, nosis of diseases of the lung, particularly diagnosis and filed on Jun. 9, 2004. prognosis of lung cancer. Patent Application Publication Jul. 13, 2006 Sheet 2 of 42 US 2006/0154278 A1 10']>Jml,dll/MdTSNTSWHIMSENEJJOISIT d Patent Application Publication Jul. 13, 2006 Sheet 4 of 42 US 2006/0154278 A1 o y qe96ZZIZ Patent Application Publication Jul. 13, 2006 Sheet 6 of 42 US 2006/0154278 A1 zevecs'0|| 8/9980°0 GIZI69°0 SOTE00*0 svo600,0 689800’0 8£0000'0 OZGZOOWIN 619?.00WIN 092€.00WN 966200WIN Patent Application Publication Jul. 13, 2006 Sheet 7 of 42 US 2006/0154278 A1 V,±0IJI Patent Application Publication Jul. 13, 2006 Sheet 9 of 42 US 2006/0154278 A1 Patent Application Publication Jul. 13, 2006 Sheet 10 of 42 US 2006/0154278 A1 GT3°5)I,HI Patent Application Publication Jul. 13, 2006 Sheet 11 of 42 US 2006/0154278 A1 Patent Application Publication Jul. 13, 2006 Sheet 12 of 42 US 2006/0154278 A1 „H3'50IJI Patent Application Publication Jul. 13, 2006 Sheet 13 of 42 US 2006/0154278 A1 93“OICH Patent Application Publication Jul. 13, 2006 Sheet 14 of 42 US 2006/0154278 A1 DBS258 NON-SMOKER 600 TUMOR 400 N (LOHRATIO = 64) 200 SIZE 147.84 HEIGH 22 600 NON-MALIGNANTLUNG 400 2OO SIZE 147.85 HEIGHT HEIGHT 752 626 BOO SMOKER SOO TUMOR 400 (LOHRATIO = .99) 200 SIZE 149.77 HEIGHT HEIGHT 943 615 900 NON-MALIGNANT LUNG 600 300 SIZE SIZE 149.77 151.73 Example of LOH analysis. The panels are the electropherograms resulting from the amplification of the DNA templates from tumor and nonmalignant lung tissue (normal). Allelic loss was calculated using a normalized allele height ratio (LOH ratio) equation in which R= (A1)(N2)/(A2) (N1) where A1 and A2 are the heights of the alleles from lung and N1 and N2 are the heights of the alleles from the DNA from normal tissue. For informative primers, LOH was scored as a 20% decrease in the height ratio of an allele inadenocarcinoma Compared with the height ratio of the same allele in nonmalignant lung DNA. Therefore, cases in which the LOHRatio was <0.8 or >1.25 were scored as LOH. The horizontal axis is allele size in base pairs. The Vertical axis is the allele height in arbitrary fluorescent units. In this example, the tumor tissue from the nonsmoker (top panel, arrow) demonstrates LOH at the microSatellite D8S258. Patent Application Publication Jul. 13, 2006 Sheet 15 of 42 US 2006/0154278 A1 3. 5SE5 e s E3: s s N s Se s Ps S S (TV) SSO)ITTYTYNOLION Patent Application Publication Jul. 13, 2006 Sheet 17 of 42 US 2006/0154278 A1 crocesoroctocaecaeocaeocaecaeoeaedes-Beroeaecroesroer»erbo-perocrocrocroeae |}| {{9(9.I.H. Patent Application Publication Jul. 13, 2006 Sheet 18 of 42 US 2006/0154278 A1 O O O O s | |?olo || ofLI INNsIYY. | | | | | Ovu I NI V | | | | O | A OF YW. S. PIE 56p W|W | | || 1 || O d 9 WYY EIIL's N. |PPPP | | | IAIHO9) EO V Idigy| s 2 g Patent Application Publication Jul. 13, 2006 Sheet 19 of 42 US 2006/0154278 A1 2 S O Patent Application Publication Jul. 13 2006 Sheet 20 of 42 US 2006/0154278 A1 Patent Application Publication Jul. 13, 2006 Sheet 21 of 42 US 2006/0154278 A1 1OOOOO 5 10000 1000 e 2 100 2 2 10 E R=92 0.1 0.1 1 10 1OO 1000 1 OOOO 100000 GENE EXPRESSION INTENSITY (RIGHT BRONCHUS) 100000 2 10000 2 e S2 1000 E E 100 5 10 5 R=85 0.1 0.1 1 1O 1OO 1OOO 1OOOO 10OOOO GENE EXPRESSION INTENSITY (BASELINE) FIG. 8B Patent Application Publication Jul. 13, 2006 Sheet 22 of 42 US 2006/0154278 A1 - REPLICATESAMPLES - - - - UNRELATED SAMPLES ANALYSISOFREPLICATESAMPLES WERSUSUNRELATED SAMPLES 0.0008 O.OOO 0.0006 O.0004 0.0003 O.OOO2 FOLD CHANGE (log2(DIFFERENTIAL GENE EXPRESSIONRATIO) FIG. 8C Patent Application Publication Jul. 13, 2006 Sheet 24 of 42 US 2006/0154278 A1 NORMAL AIRWAY TRANSCRIPTOME (7119 GENES) 1400 1200 1000 TOP 10% MOST WARIABLE GENES 400 200 O 50 100 150 200 COEFFICIENT OF WARIATION (Sd/mean)*100) FIG. 10 Patent Application Publication Jul. 13, 2006 Sheet 25 of 42 US 2006/0154278 A1 153F/20 156F/20 126F/2 136F/1.08 159F/12 138F/12 77F/7 53F/32 63F/12 52F/27 144F/20 122F/1.17 119F/O.67 34F/20 78F/O.5 134F/1 108F/O.25 163F/008 FIG. 11 Patent Application Publication Jul. 13, 2006 Sheet 26 of 42 US 2006/0154278 A1 NQO1 EXPRESSION REAL-TIMERT-PCR MICROARRAY 12.00 10.00 8.00 6.00 R=.98 4.00 i 2.00 O.OO % 143N 149N 145N 146C 140C 162C SAMPLE # FIG, 12A ALDH3A1 EXPRESSION REAL-TIMERT-PCR TOOO MICROARRAY 60.00 50.00 40.00 30.00 20.00 i 10.00 OOO 143N 149N 145N 146C 140C 162C SAMPLE # FIG. I2B Patent Application Publication Jul. 13, 2006 Sheet 27 of 42 US 2006/0154278 A1 REAL-TIMERT-PCR CYP1B1 EXPRESSION MICROARRAY 400.00 350.00 300.00 250.00 200.00 150.00 1OO.OO i 50.00 O.OO 143N 149N 145N 146C 14OC 162C SAMPLE # FIG. 12C CEACAM5 EXPRESSION 60.00 REAL-TIMERT-PCR MICROARRAY 50.00 40.00 30.00 20.00 i 10.00 O.OO 143N 149N 145N 146C 14OC 162C SAMPLE # FIG. 12D Patent Application Publication Jul. 13, 2006 Sheet 28 of 42 US 2006/0154278 A1 REAL-TIMERT-PCR SLT1 EXPRESSION MICROARRAY 2.00 1.OO s O.OO 2 -1.00 g -2.00 S -3.00 R=. 95 -4.00 m 2 -5.00 -6.00 154 159 (F 156 (F 163 (F 18O (N) quitd quitd quit.< (S) 2 yrs 2 yrs 2 yrs ago) ago) ago) SAMPLE # FIG. 12E Patent Application Publication Jul. 13, 2006 Sheet 29 of 42 US 2006/0154278 A1 AFFYMETRIX ID GENE 207847 s at muc-1TRANSMEMBRANE 204895 X at muc-4 213693 s at muC-1 TRANSMEMBRANE 214303 X at muc-5 AC 214385 s at muc-5B 217 109 at muc-4 21711.0 s at muc-4 205725 at CLARA CEL 209008 X at CYTOKERATIN 8 FIG. 13 Patent Application Publication Jul. 13, 2006 Sheet 30 of 42 US 2006/0154278 A1 AFFYMETRIX ID GENE (CELLTYPE SPECIFICITY) 205049 s at cd-79 alpha (BCELL) 205297 s at cd-79 beta (BCELL) 205264 at cd-3EPSILONASSOCIATED PROTEIN(TCELL) 210031 at cd-3 Zeta (TCELL) 206804 at cd-3 gamma (TCELL) 205982 X at SURFACTANTASSOCIATED PROTEINC (DISTALALYEOLAREPTHELIUM) 211735 X at SURFACTANTASSOCIATED PROTEINC(DISTALALVEOLAREPTHELIUM) 214199 at SURFACTANTASSOCIATED PROTEIND(DISTALALVEOLAREPTHELIUM) 21.8835 at SURFACTANTASSOCIATED PROTEIN A2(DISTALALVEOLAR EPITHELIUM) 215454 x at SURFACTANTASSOCIATED PROTEINC(DISTALALVEOLAREPITHELIUM) 203948 s at MPO (MYELOID/MONOCYTE) 203949 at MPO (MYELOID/MONOCYTE) 206120 at CD33(MYELOID SPECIFIC) 210184 at CD11c(MYELOID SPECIFIC) 204625 s at CD61 (MEGAKARYOCYTE) 211579 at CD61 (MEGAKARYOCYTE) FIG. 14 Patent Application Publication Jul. 13, 2006 Sheet 31 of 42 US 2006/0154278 A1 NEVER FORMER cuRRENT PVALUE METHOD NUMBER OF SUBJECTS 23 18 W 34 AGE 54.33 (16.64) 43.59 (12.23) ANOVA GENDER 11M/7F 27M/7F CHISO RACE 7W/19B/80 CHISQ PACK YEARS OFSMOKING 22.02 (20.30) ITEST: AGE STARTED SMOKING 19.12 (12.01) LENGTH OF TIMESMOKED (YRS) 25.18 (17.56). 24.24 (13.70) TIME PASSED SINCEQUIT (YRS)NA 10.49 (10.47) NA FIG. 15 MEAN R SAMPLES SQUARED ALL REPLICATES (TECHNICAL SPATIAL TEMPORAL, N=7) N (N=23, #PAIRWISE CORRELATIONS=253) CURRENTSMOKERS (N=34, #PAIRWISE CORRELATIONS=561) CURRENT VERSUSNEVER SMOKERS (#PAIRWISE CORRELATIONS = 782) FIG. 16 Patent Application Publication Jul. 13, 2006 Sheet 33 of 42 US 2006/0154278 A1 AFFYMETRIX A GE: GENDER: D GENE VARIABLE AGE GENDER RACE GENDER RACE MODEL 204211 X AT PRKR 2.25E-04 1.64E-05 8.82E-05 217933 SAT LAP3 2.41E-04 1.71E-06 | 1.40E-05 214001 X AT RPS10 2.86E-04 2.16E-06 || 4.25E-05 201883 SAT B4GALT1 3.01E-04 3.22E-06 9.00E-05 209969 SAT STAT1 3.23E-04 2.2E-03 1.12E-06 | 1.84E-05 209696 AT FBP1 4.09E-04 6.63E-04 1.86E-03 3.99E-04 217738 AT PBEF 9.61E-04 .19E-05 || 7.42E-04 208018 S AT HCK 1.62E-03 5.62E-05 4.76E-04 201426 SAT RPLP2 1.68E-03 1.OE-03 634E-04 218.986 SAT FU20035 1.75E-03 1.57E04 2.14E-03 214315X AT CALR 2.03E-03 8.91E-06 1.62E-04 202446.