biomarkers for treatment, relapse and curebiomarkers for ... · biomarkers for treatment, relapse...
TRANSCRIPT
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Biomarkers for Treatment, Relapse and CureBiomarkers for Treatment, Relapse and Cure
7th National Conference on Laboratory Aspects of Tuberculosis
John T. Belisle, Ph.D.
Mycobacteria Research Laboratories Department of Microbiology, Immunology, and Pathology
Colorado State University
Pathogenesis ofPathogenesis of TB and DiagnosticsTB and Diagnostics
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Pathogenesis of Pathogenesis of TB and DiagnosticsTB and DiagnosticsInhalation of M. tuberculosis
Host-Pathogen interactions
Continued growth of bacteria and granuloma, liquefaction of the granuloma
Bacteria cease to grow,lesion calcifies
Dissemination of theinfection, active disease (5%)Reactivation (5%)
Immune suppression
No disease, dormant infection (90%)
Prognostic MarkerPrognostic Marker
Differential DiagnosticDifferential Diagnostic
Pathogenesis ofPathogenesis of TB and DiagnosticsTB and Diagnostics
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Pathogenesis of Pathogenesis of TB and DiagnosticsTB and DiagnosticsInhalation of M. tuberculosis
Host-Pathogen interactions
Continued growth of bacteria and granuloma, liquefaction of the granuloma
Bacteria cease to grow,lesion calcifies
Dissemination of theinfection, active disease (5%)Reactivation (5%)
Immune suppression
No disease, dormant infection (90%) •• Adult Adult vsvs Pediatric TuberculosisPediatric Tuberculosis
•• Advanced Advanced vsvs Early Pulmonary Early Pulmonary Tuberculosis Tuberculosis
•• HIV CoHIV Co--Infection Infection
•• Marker of protection Marker of protection
•• NonNon--Pulmonary TuberculosisPulmonary Tuberculosis•• Drug Resistance/Disease Drug Resistance/Disease
Resolution Resolution
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Diagnostic Targets (Assays)Diagnostic Targets (Assays)
End Product Measurement Induced Product Measurement
Diagnostic Targets (Assays)Diagnostic Targets (Assays)
MicroscopyMicroscopyBacteriologyBacteriologyMicroscopyMicroscopyBacteriologyBacteriology
Skin Test/ DTHSkin Test/ DTHIn Vitro In Vitro T Cell AssayT Cell AssaySkin Test/ DTHSkin Test/ DTHIn Vitro In Vitro T Cell AssayT Cell Assay
Clinical SymptomsClinical SymptomsAntibody DetectionAntibody DetectionA ti D t tiA ti D t ti
Clinical SymptomsClinical SymptomsAntibody DetectionAntibody DetectionA ti D t tiA ti D t tiAntigen DetectionAntigen DetectionMolecular DetectionMolecular DetectionHost Immune ResponseHost Immune Response
Antigen DetectionAntigen DetectionMolecular DetectionMolecular DetectionHost Immune ResponseHost Immune ResponseHost Immune ResponseHost Immune ResponseHost Immune ResponseHost Immune Response
Diagnostic Targets (Products)
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Diagnostic Targets (Products)Immunological
Antibody Based Protein-Protein
-Lipid -Carbohydrate
T cell basedT cell based-Protein-Lipids
Product Based (Antigen detection)-Whole Cells -Proteins-Lipids-Carbohydrates-Nucleic Acid S ll M l l-Small Molecule
Major Challenges for TB Biomarker Discovery
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Major Challenges for TB Biomarker Discovery
1. Target multiple disease states vs single disease states
2. Target Prognostic vs Diagnostic
3 A Pl tf T t3. Assay Platform Target
4. What are the characteristics of the biomarkers?(Target Product Profile)(Target Product Profile)
5. Unbiased vs Biased (hypothesis driven) approach.
6. Variability in patient population
7. Variability in the bacterium (strain variability and variable y ( yphenotypes)
Immediate Needs for TB Biomarker Discovery
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Immediate Needs for TB Biomarker Discovery
One of the greatest needs: One of the greatest needs: Acceleration of anti-tuberculosis drug development and tools that allow for more efficient and cost effective clinical t iltrails.
Solutions:Biomarkers or biosignatures that:•Differentiate treatment failure and treatment success• Are useful 2 to 4 weeks into the treatment phaseAre useful 2 to 4 weeks into the treatment phase• Differentiate between durable cure and relapse
Biomarkers of Drug Treatment Biomarkers of Drug Treatment
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Host Changes
Macro- Metabolic C ll T &
gg
Gene Expression
Macromolecules
Metabolic Processes
Cell Type & Function Tissue
Architecture
Active Disease Remission or Cure Chemotherapy
Gene Expression Macro- Metabolic Cell
Cell Viabilityp ess o Macro
moleculesMetabolic Processes
Cell Architecture
Pathogen ChangesPathogen Changes
Analysis of Analysis of MetabolitesMetabolites
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Analysis of Analysis of MetabolitesMetabolites
Clinical Sample (Serum or Urine)
Processing Wet Bench
Computational Analyses
Analysis by LC-MS Validation
Computational Analyses Computational
Disease/Treatment Specific M t b li P fil
The vast majority of metabolites are found at 800 Daltons and below. Therefore the The vast majority of metabolites are found at 800 Daltons and below. Therefore the
Metabolic Profile
analysis platform was chosen to focus on metabolites of this class.analysis platform was chosen to focus on metabolites of this class.
Metabolomics and Biomarker: Rationale for the approach
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Metabolomics and Biomarker: Rationale for the approach
Disease states are associated with changes in the biochemistry of• Disease states are associated with changes in the biochemistry of a system resulting in abnormal metabolite profiles
• Metabolites rapidly change in abundance with alterations of a biological system
• Metabolites can be used to monitor host or pathogen changes
M t b lit t i ll i i i l i l ti i t• Metabolites typically require minimal manipulation prior to analyses
• Metabolic flux provides a biosignature
Metabolites are relatively unexplored as biomarkers for treatment of bacterial diseases
Selecting the Appropriate Biological Specimens for TB Selecting the Appropriate Biological Specimens for TB
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
g pp p g pg pp p g p
SputumPositive Negative C l d f di i S i-Currently used for diagnosis - Sputum processing
-Most disease pulmonary - Poor for non-pulmonary-Easy to equate biomarker diseaseabundance to CFU
Blood/Serum
abundance to CFU
Positive Negative No chemical processing Invasive
Urine
-No chemical processing - Invasive -Standard platforms available - Protein rich and complex-Multiple disease states
UrinePositive Negative -Easy to obtain -Complex-Standard platforms available -May only be useful in specificp y y p-Processing relatively simple disease states
Urine Samples Obtained and Analyses Performed
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Urine Samples Obtained and Analyses Performed
Date Sample Set Number #s
Time Points LC/MSAnalysis
DataAnalysis
April 2008
Micro Pilot* Project
20 D0, 2W, 2M, 6M Yes all time points
Yes all time points
April NAA2m* 14 D0, 2W, 1M, Yes all for Yes for D0, 2009 Set 1 1.5M, 2M, 4M D0, 2W, and
1M2W, and 1M
February2010
Stellenbosch 40 D0, 2W, 1M, 1 5M 6M
Yes all time points
Yes for D0, 2W 1M2010 1.5M, 6M points 2W, 1M, 1.5M, 6M
October2010
NAA2mSet 2
36 D0, 2W,1M, 1.5M, 2M, 3M,
No No
*All the samples were collected from adults (18 to 50 years), males and females,
4M, 7M, 8M, 9M
p ( y ), ,
cavitary tuberculosis (Chest x-ray and high bacterial burden), HIV positive and
negative in Kampala, Uganda. All patients treated with INH, RIF, PZA, EMB
Condition Tree Analysis of Molecular FeaturesCondition Tree Analysis of Molecular Features
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
yyD0 normalized data values greater or less than those for W2 or W4 by a factor of 5 fold.
Starting Mass List: Masses present in at least one condition with minimum 75 0%Molecular F t
Increased Abundance
Starting Mass List: Masses present in at least one condition with minimum 75.0%.Features
Decreased Abundance
D0 R d W2 Y ll
Features present in at least 75.0% of samples for one time point
bu da ce
D0 = Red W2 = YellowW4 = Green
time point.
Principal Component Analysis (PCA) Demonstrates Urine Principal Component Analysis (PCA) Demonstrates Urine Metabolites can Differentiate Stages of TB Treatment Metabolites can Differentiate Stages of TB Treatment
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Metabolites can Differentiate Stages of TB Treatment Metabolites can Differentiate Stages of TB Treatment
MP NAA2m
Red = Day 0 (Day of Diagnosis) Blue = 2 week post treatment start
Brown = 4 or 8 week post treatment start
Stringent Conditions: - Molecular feature in at least 70% of samples from one group- Molecular feature must have at least a five fold change
between groups
Molecular Features Decreased in Abundance Following Treatment
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
TreatmentObserved Accurate Mass Calculated Formula Observed Accurate Mass Calculated Formula
126.1162 C7H14N2 234.1221 C11H14N4O2
129.0434 C5H7NO3 239.0781 C11H13NO5
131.057 C5H9NO3 241.1539 C10H19N5O2
Over 55 Over 55
135.0687 C5H11O4 245.098 C10H11N7O/C9H15N3O5
136.0395 C5H4N4O 248.1002 C9H16N2O6
137.0401 C7H7NO2 257.1014 C10H15N3O5
142.127 C6H14N4 258.147 C13H14N4O2
143.1314 C7H16N2O 263.1127 C10H13N7O2
149 0116 C7H3NO3 266 0519 C6H10N4O8 Over 55 Over 55 metabolites metabolites identified thatidentified thatdecrease in decrease in
b d b d
149.0116 C7H3NO3 266.0519 C6H10N4O8
159.126 C8H17NO2 268.0942 C14H20OS2
161.0692 C6H11NO4 272.1014 C12H12N6O2
161.0825 C10H11NO 282.0267 C6H10N4O7S
165.0428 C8H7NO3 282.1576 C14H22N2O4
165.0795 C9H11NO2 283.1282 C11H17N5O4abundance or abundance or disappear with disappear with antianti--tuberculosis tuberculosis
167.0225 C8H7O4 286.1284 C11H18N4O5
167.033 C6H5N3O3 293.1048 C10H19N3O5S
168.0286 C5H4N4O3 294.0599 C11H10N4O6
169.0854 C7H11N3O2 295.1037 C14H17NO6
170.1421 C9H18N2O 301.1524 C14H23NO6
treatment treatment 173.9949 C7H11N3O2 308.091 C16H12N4O3
174.0639 C6H10N2O4 327.1069 C18H17NO5
175.0485 C6H9NO5/C7H5N5O 338.1342 C18H17NO5
181.0753 C9H11NO3 345.0475 C16H11NO8
182.1054 C9H14N2O2 350.1511 C14H26N2O6S
202 143 C8H18N4O2 370 165 C14H22N6O6202.143 C8H18N4O2 370.165 C14H22N6O6
203.1269 C8H17N3O3 384.122 C14H20N6O5S
214.0094 C8H6O7 405.1746 C21H23N7S
218.1343 C20H20N2OS2 421.2046 C14H31N9O2S2
221.0729 C8H15NO4S 422.2133 C23H34O5S
Identified Molecular Features Decreased in Abundance Following Treatment
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Following Treatment
# Identified biomarkers candidates Formula Mass Nature of change
1 Hydroxyproline C5H9NO3 131.058243 Down by W2 then increase slightly
2 N‐Acetyl‐L‐aspartic acid C6H9NO5 175.048065 Down by W2 then increase slightly
3 Dimethyl‐L‐arginine C8H18N4O2 202.142975 Down gradually
4 N‐Acetylasparagine C6H10N2O4 174.064056 Down by W2 and then steady
5 1 M th lhi tidi C H N O 169 085129 D d ll5 1‐Methylhistidine C7H11N3O2 169.085129 Down gradually
6 L‐Phenylalanine C9H11NO2 165.078979 Down by W2 then increase slightly
7 2,2,5,5‐Tetramethyl‐3‐ C9H18N2O 170.141913 Down by W2 and then steadypyrrolidinecarboxamide
8 Pyroglutamic acid C5H7NO3 129.042587 Down by W2 and then steady
9 Acetylcysteine C5H9NO3S 163.030319 Down gradually
10 Trigonelline C7H7NO2 137.047684 Down by W2 then increase slightly
11 S‐Adenosylhomocysteine C14H20N6O5S 384.121582 Down gradually
12 L‐Tyrosine C9H11NO3 181.073898 Down by W2 and then steady
13 Alpha‐aminoadipic acid C6H11NO4 161.068802 Down by W2 and then steady
14 Quinolinic acid C7H5NO4 167.021851 Down gradually
15 Hypoxanthine C5H4N4O 136.038513 Down by W2 then increase slightly
Validation Validation Urine Urine Samples Obtained and Analyses Performed Samples Obtained and Analyses Performed
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Urine Urine Samples Obtained and Analyses Performed Samples Obtained and Analyses Performed
Date Sample Set Number #s
Time Points LC/MSAnalysis
DataAnalysis
April 2008
TBRU PilotProject
20 D0, 2W, 2M, 6M Yes all time points
Yes all time points
April NAA2m 14 D0, 2W, Yes all for Yes for D0, 2009 Set 1 1M, 4M D0, 2W, and
1M2W, and 1M
February2010
Stellenbosch 40 D0, 2W, 1M, 6M Yes all time points
Yes for D0, 2W and 6M2010 points 2W, and 6M
October2010
NAA2mSet 2
22 D0, 2W,1M, 1.5M, 2M, 3M, 4M, 7M, 8M, 9M
No No
, , ,
MPP analyses conducted with same parameters as for the Uganda samples: MPP analyses conducted with same parameters as for the Uganda samples: Present in at least70% of one condition, changes by at least 5 fold, and has a p value < 0.05
PCA Analysis of Metabolomic Data from Stellenbosch PCA Analysis of Metabolomic Data from Stellenbosch Urine Samples Urine Samples
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Urine Samples Urine Samples
Validation of Signature with Stellenbosch Sample Set: 21 of 58
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Validation of Signature with Stellenbosch Sample Set: 21 of 58
Observed Accurate Mass Calculated Formula 129.0434 C5H7NO3135.0687 C5H11O4137.0401 C7H7NO2159.126 C8H17NO2167.0225 C8H7O4169 0854 C7H11N3O2169.0854 C7H11N3O2170.1421 C9H18N2O174.0639 C6H10N2O4175.0481 C6H9NO5181.0753 C9H11NO3202.143 C8H18N4O2221.0729 C8H15NO4S234.1221 C11H14N4O2257.1014 C10H15N3O5263 1127 C10H13N7O2263.1127 C10H13N7O2268.0942 C14H20OS2272.1014 C12H12N6O2282.0267 C6H10N4O7S293.1048 C10H19N3O5S93 0 8 C 0 9 3O5S301.1524 C14H23NO6338.1342 C18H17NO5
Validated Molecular Features (9 of 16) that Decreased in Abundance Following Treatment
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Following Treatment
# Identified biomarkers candidates Formula Mass Nature of change
1 Hydroxyproline C5H9NO3 131.058243 Down by W2 then increase slightly
2 N‐Acetyl‐L‐aspartic acid C6H9NO5 175.048065 Down by W2 then increase slightly
3 Dimethyl‐L‐arginine C8H18N4O2 202.142975 Down gradually
4 N‐Acetylasparagine C6H10N2O4 174.064056 Down by W2 and then steady
h lhi idi C O 69 08 29 d ll5 1‐Methylhistidine C7H11N3O2 169.085129 Down gradually
6 L‐Phenylalanine C9H11NO2 165.078979 Down by W2 then increase slightly
7 2,2,5,5‐Tetramethyl‐3‐ C9H18N2O 170.141913 Down by W2 and then steadypyrrolidinecarboxamide
8 Pyroglutamic acid C5H7NO3 129.042587 Down by W2 and then steady
9 Acetylcysteine C5H9NO3S 163.030319 Down gradually
10 Trigonelline C7H7NO2 137.047684 Down by W2 then increase slightly
11 S‐Adenosylhomocysteine C14H20N6O5S 384.121582 Down gradually
12 L‐Tyrosine C9H11NO3 181.073898 Down by W2 and then steady
13 Alpha‐aminoadipic acid C6H11NO4 161.068802 Down by W2 and then steady
14 Quinolinic acid C7H5NO4 167.021851 Down gradually
15 Hypoxanthine C5H4N4O 136.038513 Down by W2 then increase slightly
Summary of Urine Analysis for BiomarkersSummary of Urine Analysis for Biomarkers
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
y yy y
16 metabolites with known or predicted structures were found with decreased abundance in patients after start of drug treatmentdecreased abundance in patients after start of drug treatment.
38 metabolites with predicted chemical formulas, but unknown structure were found with decreased abundance in patients after start of drug treatment.
Drug metabolites serve as markers to confirm treatment adherence.
Continued definition of structures of products will be facilitated by increased availability of metabolite databases, in particular one for M. tuberculosis.
Current data and identified compounds may add to understanding of disease state. Several of the metabolites are associated with inflammation
Can Urine Metabolites ServesCan Urine Metabolites Serves as Diagnostic Markers?as Diagnostic Markers?
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
Region of Sample
C ll tiInfection
SitPersistent C h M l i F
Night S t C E i ti Ill
Final Culture R lt
gg
Collection Site Cough Malaise Fever Sweats Co‐Existing Illness ResultSalvador pulmonary yes yes yes yes none MTB complexSalvador pulmonary yes yes yes yes none MTB complexSalvador pulmonary yes yes yes yes diabetes MTB complexBARCELONA pulmonary yes yes yes no none MTB complexBARCELONA pulmonary yes yes yes no none MTB complexBARCELONA pulmonary yes no yes no hta MTB complexBARCELONA pulmonary yes yes yes yes none MTB complexVietnam Pulmonary yes yes yes yes n/a MTBcomplexVietnam Pulmonary yes yes yes yes n/a MTBcomplexVietnam Pulmonary yes yes yes no n/a MTBcomplexVietnam Pulmonary yes yes yes yes n/a MTBcomplexVietnam Pulmonary yes yes yes no n/a NegativeVietnam Pulmonary yes yes yes yes n/a NegativeSalvador pulmonary yes no no no none NegativeSalvador pulmonary yes no no no none NegativeWinnipeg pulmonary yes yes no no copd NegativeWinnipeg pulmonary yes yes no no influenza NegativeWinnipeg pulmonary yes yes no yes copd NegativeBARCELONA pulmonary yes no no no none Negativep y y gBARCELONA pulmonary yes no no no copd NegativeBARCELONA pulmonary yes yes no no copd/chronic pancreatitis NegativeBARCELONA pulmonary yes yes no no copd Negative
Can Urine Metabolites ServesCan Urine Metabolites Serves as Diagnostic Markers?as Diagnostic Markers?
MycobacteriaMycobacteria Research LaboratoriesResearch LaboratoriesColorado State UniversityColorado State University
gg
Bl e = Confirmed TBBlue = Confirmed TBRed = TB Suspect
Acknowledgments
Mycobacteria Research LaboratoriesMycobacteria Research LaboratoriesColorado State UniversityColorado State University
Case Western Reserve UniversityDr. W. Henry Boom Dr John L Johnson
Colorado State UniversityDr. Seba MahapatraDr Mary Ann DeGroote
c o edg e ts
Dr. John L. Johnson
University of Arkansas Medical CenterDr. Kathy Eisenach
Dr. Mary Ann DeGroote Dr. Mark Sartain Dr. Suresh BhamidiDr. Paulina ZuritaB i C
Public Health Research InstituteDr. Gilla Kaplan
Brian Cranmer
University of ColoradoThomas Campbell
Joint Clinical Research Center, UgandaDr. Moses Joloba Phineas GittaGrace M an e
p
Denver Health Bill Burman
Grace Muzanye
Stellenbosch University Gerhard Walzl
UC-San Francisco Payam Nahid
Funding TBRU : NO1-AI70022FDA : U18-FD004038