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Identification of new urine biomarkers for the detection of prostate cancerRigau Resina, Marina 28 July 2011 (has links)
El càncer de pròstata (CP), és la segona causa de mort per malaltia oncològica en els homes del món
occidental. S’estima que un de cada sis homes desenvoluparà un càncer d’aquest tipus al llarg de la
seva vida. El CP afecta com el seu nom indica, a la pròstata. La pròstata, és un òrgan glandular de
l’aparell genital-urinari masculí. Té la mida d’una nou i es localitza sota de la bufeta, envoltant la uretra
i davant del recte. La seva funció és secretar productes que s’afegiran al líquid seminal amb la finalitat
de nodrir i protegir els espermatozous.
El actual diagnòstic del CP es basa en una triada diagnòstica que consta de; l’anàlisi dels nivells de
PSA (Prostate Specific Antigen) en sèrum, el tacte rectal (TR) i finalment la biòpsia prostàtica. Quan els
nivells de PSA en sèrum es situen per sobre de 4 ng/mL i/o el TR és sospitós, l’uròleg pot estimar quina
és la probabilitat que el pacient estigui afectat per un CP i per tant decidir la necessitat de practicar o
no una biòpsia prostàtica, que permetrà establir el diagnòstic definitiu. La introducció del test del PSA,
a finals dels anys 80, s'ha traduït en una millora del diagnòstic precoç del CP, moment en el qual les
opcions de tractament per el són eficaces. No obstant això, malgrat aquesta detecció primerenca, la
mortalitat per CP no ha disminuït significativament en els últims anys. El principal problema del PSA
com a marcador d’screening és que aquest presenta un nivell d’especificitat baix (30% aprox) i alhora,
un valor predictiu negatiu baix. Això es tradueix en que al voltant d'un terç de tots els homes sotmesos
a una biòpsia seran positius per CP. Com a resultat dels seus persistents nivells sèrics de PSA, però els
resultats negatius de la biòpsia, aquests homes es sotmeten a repetides biòpsies. Tot i el importants
avenços en la investigació de biomarcadors de CP, alguns homes encara estan sobre-diagnosticats de
CP “indolent”, mentre que d’altres moren de malaltia agressiva que s'ha diagnosticat massa tard.
Aquesta situació es coneix com "dilema diagnòstic".
És per tot això, que el càncer de pròstata, es beneficiaria de l’existència de nous marcadors
d’screening més específics i alhora d’un diagnòstic menys invasiu. Per altra banda, una millora en el
diagnòstic evitaria un gran nombre de biòpsies innecessàries i conseqüentment un important estalvi
econòmic en el cost sanitari actual. La recerca de nous marcadors en el càncer de pròstata suposa un
camp de treball important en la detecció precoç d’aquest tipus de càncer. Donada la situació de la
pròstata a l’organisme, sota la bufeta i envoltant la uretra, les secrecions i inclús les mateixes cèl·lules
prostàtiques, ja siguin normals o malignes, poden trobar-se presents en l’orina. És per això que
considerem l’orina com una font important d’informació, a través de la qual es podria arribar a
determinar quina situació s’està donant a l’òrgan en qüestió. Altres estudis evidencien l’existència de
potencials biomarcadors en l’orina que podrien ajudar en la millora del diagnòstic del CP. A nosaltres
ens ocupa l’estudi d’aquelles molècules de RNA així com de les molècules protèiques que es troben a
l’orina.
Suposem doncs, que un massatge prostàtic enriqueix la mostra d’orina de tot tipus de molècules
proteiques. Així doncs, la nostra hipòtesi de treball recolza que, l’orina després d’un massatge prostàtic
pot ser el fluid ideal per a la recerca de nous biomarcadors capaços de discriminar entre pacients amb
o sense càncer de pròstata.
L’objectiu principal de l’estudi és diagnosticar el CP assimptomàtic per us sistema minimament
invasiu, que consisteix en l’anàlisi de l'ARN o de la proteïna en l'orina obtinguda després d’un
massatge prostàtic i, superar la baixa especificitat de l’actual sistema d’screening (PSA en sèrum)
mitjançant l'ús de biomarcadors addicionals (RNA o proteina) per tal de reduir el nombre de biòpsies
innecessàries (reduir els costos socio-econòmics, així com la reducció dels efectes secundaris no
desitjats).
1. ANÀLISI TRANSCRIPTÒMICA:
1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al.,
Prostate. 2010 Dec 1;70(16):1760-7. En el primer estudi, mitjançant l’anàlisi de transcripts per RTqPCR
en 215 (34% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge
prostàtic), es va determinar la utilitat de PSGR (Prostate Specific G-coupled receptor), previament descrit com a sobre-expressat en mostres de teixit de CP, com un nou biomarcador, comparable a
PCA3 (ja conegut), per a la detecció del CP en orines (taula 1). Per l'anàlisi de la corba ROC vàrem
determinar els valors de l’Area sota la corba (AUC) de PSGR (AUC 0,68) i PCA3 (AUC 0,66). Fixant la
sensibilitat a 95%, aconseguim un valor d’especificitat de 15% per PSGR i el 17% de PCA3. Alhora
vàrem veure que al combinar els dos biomarcadors (PSGRvPCA3) mitjançant un anàlisi “multiROC”, els
resultats van millorar significativament en termes de rendiment del biomarcador (AUC 0,73) com de la
seva especificitat (34%). En conclusió, podem dir que PSGR presenta un comportament similar al
biomarcador estàndard per CP en orina (PCA3), però, és necessàri la realització d’estudis futurs per
millorar encara més el rendiment d'aquesta prova.
1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau
et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. En un segon estudi, vàrem estudiar la possibilitat
de multiplexar diferents biomarcadors, per tal de ser capaços de guanyat especifcitat sense perdre
sensibilitat en la detecció del CP. Donada l’heterogeneïtat del CP la idea d’utilitzar diferents marcadors
per a la seva detecció es presenta atractiva. Mitjançant l’anàlisi de transcripts, per RTqPCR, en 215
(37% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge
prostàtic), vàrem determinar la utilitat de PSMA (Prostate Specific Membrane Antigen), un marcador
descrit anteriorment com a sobre-expressat en el CP, com a biomarcadors per a la detecció de CP en
orina. L’anàlisi de corbes ROC revela un rendiment similar PSMA (AUC 0,62), PSGR (AUC 0,65) i PCA3
(AUC 0,60) (Taula 1). Per tal de millorar l'eficàcia diagnòstica d'aquests biomarcadors es van combinar
PSMAvPSGRvPCA3 (3M), utilitzant un anàlisi de multiROC. L’AUC del 3M (AUCm) (0,74) millora
notablement, en comparació amb els valors individuals per a cada un dels marcadors. D'altra banda, si
combinem 3M amb el valor de PSA en sang l’AUCm (0,77) va ser encara millor (taula 11). Si fixem la
sensibilitat a 96%, l'especificitat del model 3M manté una especificitat del 34%, mentre que no es va
observar augment en d’aquest valor quan ho combinem amb PSA en sang (34%) (Taula 11). El
comportament d'aquests tres marcadors en una subpoblació de l’estudi que presentava nivells de PSA
en sang entre 4-10 ng/mL i sense informació d’una biòpsia prèvia "zona gris del PSA" és de PSMA
(AUC 0,74), PSGR (AUC 0,66) i PCA3 (AUC 0,61). Per al model combinat (3M), l’AUCm és de 0,82
(Taula 11). Fixant la sensibilitat al 96%, l'especificitat del model combinat (3M) manté una especificitat
del 50%. En conclusió, aquests resultats demostren que la combiació de diferents marcadors
proporciona una major especificitat a la prova sense comprometre el valor de sensibilitat. Expresat en
el nombre de biopsies que s’haurien pogut estalviar, els valors se situen al voltant del 34% de biòpsies
estalviades.
1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia”
Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. El tercer estudi, està centrat en la utilitat,
del ja conegut biomarcador per CP en orina (PCA3), per a l’identificació una lessió pre-maligna a la
pròstata, així com la seva utilitat en la selecció dels homes amb biòpsies negatives, que requereixen la
repetició del procediment. La troballa de les lesions de HGPIN (High grade preneoplastic intraepithelial
lesion) és una indicació freqüent de la repetició de la biòpsia. Tot i la controvèrsia al voltant de la
definició de HGPIN com a lessió pre-maligna i la necessitat de repetició de la biòpsia, encara avui en el
nostre hospital s’indica el control exhaustiu del pacient, així com la repetició de la biòpsia en un
periode de relativament curt de temps. Per a aquest estudi es van seleccionar un subgrup de pacients
amb HGPIN (n=64) i com a grups control, es van seleccionar els homes amb CP (n=83) i homes amb
patologia benigne de la pròstata (BP) (n=97). Després de l’anàlisi dels transcripts de PCA3 per RTqPCR
en mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), i de
l'anàlisi de la corba ROC, es van obtenir els següents resultats; HGPIN vs CP (AUC 0,63) i BP vs PC
(AUC 0,71). Aquests resultats demostren la capacitat dels transcripts de PCA3, en orina obtinguda
deprés d’un massatge prostàtic, per a l’identificació d’homes amb CP així com la identificació de
pacients amb HGPIN.
2. ANÀLISI PROTEÒMICA COMPARATIVA
L’objectiu principal d’aquesta segona part és determinar un perfil proteòmic a l’orina capaç de
diferenciar entre pacients amb CP i controls sans. Per dur a terme aquest objectiu ens vàrem plantejar
fer una part inicial de discovery, que vàrem complementar amb “data mining” i finalment vàrem
verificar mitjançant una tècnica proteòmica independent.
2a) Per la part de discovery es va dur a terme l’anàlisi comparativa de “Differential in Gel
Electrophoresis (DIGE). Es van fer dos experiments independents, amb mostres de sobrenedants
d’orines (on trobem la fracció soluble de les proteïnes). En el primer experiment es van comparar 6
mostres de proteïna total de pacients amb CP i 6 mostres controls. En el segon experiment es van
comparar 9 mostres, de proteïna pre-tractada amb una tècnica de depleció per les proteïnes
majoritàries, de pacients amb CP i 9 mostres de pacients control. Un total de 24 proteïnes (9 sobre- i 15
infra-expresades) vàren ser identificades per MALDI-TOF. Una anàlisi informàtic de les proteïnes va
mostrar que la majoria d'aquestes proteïnes identificades es secreten els components de diverses
xarxes ben conegudes, el càncer funcionals i la inflamació, com ara NFКB, PDGFBβ i β-catenina. D'altra
banda, a causa del fet que hem utilitzat sobrenadants d'orina, on podem trobar proteïnes solubles
secretades, la majoria de les proteïnes identificades (62%) es van localitzar en l'espai extracelular.
Mitjançant una tècnica de proteòmica dirigida (Selected Reaction Monitoring: SRM) es van quantificar
de forma relativa, sense l’adició d’estandards interns marcats, un total de 15 (dels 24 biomarcadors) en
una població independent de 50 mostres (38% amb CP). Després d'una anàlisi de regressió logística de
les dades obtingudes a través de l'assaig de SRM, es va obtenir un panell de set pèptids que
corresponen a 5 proteïnes, capaços de distingir entre les mostres de CP i mostres controls amb una
sensibilitat del 95% i una especificitat del 78 %.
2b) La segona part de l’estudi de proteómicas es basa en la verificació de les proteïnes prèviament
qualificades i alhora l’estandarització dels mètodes de preparació de mostres per al seu posterior
anàlisis mitjançant la tècnica de SRM. Les 5 proteïnes que formen part del panell identificat, juntament
amb altres proteïnes biologicament rellevants, definides a la nostre fase de discovery, i juntament amb
altres proteïnes descrites a la literatura, vàren ser escollides per una segona fase de qualificació i
verificació en una població independent de 107 pacients, mitjançant un assaig de SRM en el qual es
pretén quantificar de forma absoluta (adició de pèptids estàndards marcats), un total de 42 proteïnes.
Prèviament a l’evaluació d’aquestes proteïnes, es va dur a terme una estandarització del protocol
d’extracció de proteïna en mostres d’orina, amb l’objectiu de maximitzar la senyal obtinguda en els
experiments de SRM. La precipitació amb Acetonitril (a temperatura ambient) seguida de la digestió
de les proteïnes en solució amb condicions de ratio 1:10 (trypsine:protein), “over-night” i l’adició de “NAcetyl
cysteine”, proporcionen els millors valors de quantificació absoluta. L’anàlisi estadístic de les
dades generades d’aquesta segona fase de qualificació i verificació de les proteïnes està encara
inacabat, de manera que ara per ara no podem conclou-re res.
En conclusió, les dos linies experimentals que aquí es presneten (ARN i proteïnes) represeten resultats
prometedors. No obstant això, estudis de validació en poblacions independents (del tipus estudi
multicèntric) són necessaris per acabar amb un biomarcador o un panell de biomarcadors vàlid per al
diagnòstic del CP. Per altra banda, el seguiment dels pacients ja analitzats poden relevalar dades
interessant, com la utilitat pronòstica d’aquests biomarcadors. Els resultats obtinguts haurien de tenir
una aplicació clínica ràpida que, juntament amb els paràmetres d’screening actual (nivells sèrics de
PSA i DRE), podrien ajudar en la pressa de decisions per CP. / The prostate gland is a walnut-sized organ of the male urinary-genital tract that is located below the
bladder surrounding the urethra and in front of the rectum. The most important disease affecting
prostate is prostate cancer (PCa). PCa is the most common cause of cancer death, and it is the second
most common cause of cancer death among men in the Western world. The risk of developing this type
of cancer during a lifetime is estimated at 1 in 6 men in the US, and the risk of death due to this
disease is 1 in 36.
The current diagnosis of PCa is based on a triad consisting of diagnosis, analysis of the levels of PSA
(Prostate Specific Antigen) in serum, the digital rectal examination (DRE) and finally the prostate
biopsy (PB). When serum PSA levels are above 4 ng/mL and / or when DRE is suspected, the urologist
can estimate how likely the patient is affected by PCa and therefore decided the need to practice or not
a PB, which is the gold standard for PCa diagnosis. The introduction of PSA testing in the late 80s, has
resulted in an improvement of early diagnosis of PCa, at which time options for treatment are effective.
However, despite this early detection, mortality PCa has not decreased significantly in the last 50
years. The main limitation of serum PSA as a tumor marker is its lack of specificity (around 30%) at the
cut-off value of 4 ng/mL, and also a low negative predictive value (NPV), which results in a high rate of
negative biopsies. Elevated PSA levels can also be attributed to other factors such as benign prostatic
hyperplasia (BPH), prostatitis, etc. As a consequence of the current screening parameters, around 2/3
of the approximately 1,300,000 biopsies made yearly in the United States and 390,000 in Europe are
unnecessary. In contrast, the false positive rate of a biopsy is about zero, although the false negative
rate in the first biopsy may oscillates around 20%. As a result of their persistently elevated PSA levels,
but negative biopsy results, these men undergo repeated biopsies to rule out PCa. This situation is
called the “PSA dilemma”. For these reasons, PCa would benefit from the existence of new markers for
screening and also a more specific diagnosis less invasive. Furthermore, an improvement in diagnosis
would avoid many unnecessary biopsies and consequently a significant savings in the cost of health
today. The search for new markers in PCa is an important field of work in the early detection of this
cancer.
Urine has been defined as a liquid biopsy of the urogenital tract, and it can provide much more
information about these organs (including the prostate) than a tissue biopsy. Urine obtained after DRE
can easily serve as a mirror of what is happening within the prostate. Furthermore, urine collection can
be accomplished without disruption of clinical standard practice. It can also be repeated several times
throughout the course of the prostatic disease. For all of these reasons, urine can serve as a potential
source of prostate disease biomarkers. Nevertheless, using urine for biomarker discovery represents an
important technical challenge, both in transcriptomic and proteomic approaches. Although there are
some studies that focus on those approaches and biomarker discovery and identification, there still
exists some controversy regarding the standardization of collection procedures, sample processing,
storage and normalization. We hypothesized that the utilization of targeted genomic and proteomic
techniques on urine samples from patients suspected of having PCa can provide a pattern of
biomarkers able to efficiently distinguish between the presence or absence of a prostate carcinoma
and, further, can help to identify clinically significant prostate cancer patients. The main objective of
this study is to diagnose asymptomatic PCa by non/minimally-invasive means using RNA or Protein in
urine after prostate massage, and to overcome the low specificity of PSA by the use of additional
biomarkers to reduce the number of unnecessary biopsies (reduce financial costs for society, reduction
in unwanted secondary effects).
1. TRANSCRIPTOMIC APPROACH: In recent years, the explosion of genomic and transcriptomic
approaches have resulted in increased biomarker discovery. The recent discovery of Prostate Cancer
Gene 3 (PCA3) in urine as a biomarker for the detection of PCa and studies to determine its
applicability in routine diagnosis represent a significant success for the scientific community in this
field. First, we wanted to characterize a new urine candidate biomarker (PSGR) to be compared with
PCA3, and second, we planned to use a panel of biomarkers, in order to improve diagnostic accuracy.
Finally, we proposed to better characterize the well-known biomarker PCA3 as a tool for the early
detection of pre-neoplastic PCa lesions, such as High grade prostatic intraepithelial neoplasia (HGPIN).
1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al.,
Prostate. 2010 Dec 1;70(16):1760-7. PSGR is a member of the G-protein coupled OR family. PSGR has
previously been described to be highly prostate tissue-specific and over-expressed in PCa tissue. Our
aim was to test whether PSGR could also be detected by RTqPCR in urine sediment obtained after
prostate massage (PM). A total of 215 urine samples were collected from consecutive patients (34%
with PCa), who presented for PB due to elevated serum PSA levels (> 4 ng/mL) and/or an abnormal
DRE. These samples were analyzed by RTqPCR. By univariate analysis we found that PSGR and PCA3
were significant predictors of PCa. A Reciever Operator Characteristics (ROC) curve was used to
assess the outcome predictive values of the individual biomarkers. We obtained the following Area
Under the Curve (AUC) values: PSGR (0.68) and PCA3 (0.66). Both markers individually overcame the
AUC value for serum PSA (0.60). Finally, we combined those markers to test if a combination of both
biomarkers could improve the sensitivity of PCA3 alone. By using a multivariate extension analysis,
multivariate ROC (MultiROC), the outcome predictive values of the paired biomarkers were assessed.
We obtained an AUC value of 0.73 for the combination of PSGR and PCA3 (PSGRvPCA3). Then, we
tested whether a combination of PSGR and PCA3 could improve specificity by fixing the sensitivity at
95%. We obtained specificities of 15% (PSGR) and 17% (PCA3) for each individual marker and 34% for
PSGRvPCA3. In summary, a multiplexed model that included PSGR and PCA3 improved the specificity
for the detection of PCa, especially in the area of high sensitivity. This could be clinically useful for
determining which patients should undergo biopsy.
1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau
et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. Much evidence points to the fact that a single
marker may not necessarily reflect the multifactorial and heterogeneous nature of PCa. The principle
that underlies the combined biomarker approach is consistent with tests offered for the detection of
PCa in tissue specimens and takes into consideration the heterogeneity of cancer development based
on a diagnostic profile. The combined model that results from these combinations provides overall
increased sensitivity without decreasing the specificity. Following the same approach than in our
previous work, we combined three biomarkers to maximize individual specificities. Prostate Specific
Membrane Antigen (PSMA), another well-known PCa biomarker, was used to test whether a
combination of PSGR, PCA3 and PSMA was able to improve the specificity of the current diagnostic
technique. We analyzed post-PM urine samples from 154 consecutive patients (37% with PCa), who
presented for PB due to elevated serum PSA levels (>4 ng/mL) and/or an abnormal DRE. We tested
whether the putative PCa biomarkers PSMA, PSGR, and PCA3 could be detected by RTqPCR in the
post-PM urine sediment. By univariate analysis, we found that the PSMA, PSGR, and PCA3 scores
were significant predictors of PCa. We then combined these findings to test if a combination of these
biomarkers could improve the specificity of an actual diagnosis. Using a multiplex model
(PSGRvPCA3vPSMA), the area under the MultiROC curve (AUCm) was 0.74, 0.77 with PSA and 0.80
with PSA density (PSAD). Fixing the sensitivity at 96%, we obtained a specificity of 34%, 34% with
PSA and 40% with PSAD. Afterwards, we specifically tested our model for clinical usefulness in the
PSA diagnostic ‘‘gray zone’’ (4–10 ng/mL) on a target subset of 82 men with no prior biopsy (34% with
PCa) and a target subset of 77 men with the PSAD information (35% with PCa). Using a multiplex
model, the AUCm was 0.82, 0.89 with PSAD. Fixing the sensitivity at 96%, we obtained a specificity of
50% and 62% with PSAD in the gray zone. This model would allow 34% of the patients to avoid
unnecessary biopsies in the gray zone (42% when using PSAD).
1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia”
Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. An ideal biomarker for the early detection
of PCa should also differentiate between men with isolated HGPIN and those with PCa. PCA3 is a
highly specific PCa gene, and its score in post-PM urine seems to be useful in ruling out PCa,
especially after a negative PB. The biopsy finding of an HGPIN is a frequent indication that the PB
should be repeated. The aim of this study was to determine the efficacy of post-PM urine PCA3 scores for ruling out PCa in men with previous HGPIN. The PCA3 score was assessed by RTqPCR in 244 post-
PM urine samples collected from men subjected to PB (64-isolated HGPIN, 83-PCa, and 97-benign
pathology findings (BP)). The median PCA3 score was 1.56 in men with BP, 2.01 in men with isolated
HGPIN and 9.06 in men with PCa. A significant difference was observed among the three scores (p <
0.001) and also between HGPIN and PCa (p = 0.008); however, no differences were observed between
HGPIN and BP (p = 0.128). The AUC in the ROC analysis was 0.71 in the subset of men with BP and
PCa, while it decreased to 0.63 when only men with isolated HGPIN and PCa were included in the
analysis. Finally, the median of the PCA3 scores was assessed in men with previously diagnosed
unifocal HGPIN (2.63) and in men with previously diagnosed multifocal HGPIN (1.59). No differences
were observed between unifocal and multifocal HGPIN (p = 0.56). In conclusion, the efficacy of post-PM
urine PCA3 scores in ruling out PCa in men with HGPIN is less than in men with BP. For this reason,
when HGPIN is found at PB, these results should be taken into consideration, in order to establish the
clinical usefulness of the PCA3 score as a tool for avoiding unnecessary repeated biopsies.
2. PROTEOMIC APPROACH: The high-throughput proteomic analysis of urine samples has recently
become a popular approach for the identification of novel biomarkers. Proteins secreted by cancer cells,
also referred to as "cancer cell secretomes," are a promising source for biomarker discovery. A great
advantage to these cancer-secreted proteins and/or their fragments is that in most cases, they enter
body fluids, such as blood or urine, and therefore, can be measured via non-invasive assays. Since the
protein products of PCa cells can be detected in urine, their use as a proximal body fluid in the
detection of PCa is very attractive.
2a.“The Discovery and Qualification of a Panel of Urine Biomarkers for Prostate Cancer Diagnosis”. In
this study we used DIGE proteomic analysis on 30 age-matched, post-PM urine supernatant
specimens, in order to identify the differentially expressed proteins in patients with PCa. 24 potential
biomarkers were identified, the majority of which were secreted proteins associated with several wellknown,
functional cancer pathways. Qualification of 15 of the 24 identified biomarker candidates was
then undertaken by relative quantification using an SRM-based assay (target) on 50 post-PM urine
supernatant samples (38% with PCa). After statistical analysis, 7 peptides, corresponding to 5 different
proteins, were selected. A multiplex ROC curve using those 7 peptides showed an AUC value of 0.93.
Fixing the sensitivity at 95%, we achieved a specificity of 78%.
2b. “Qualification and Verification of Urine PCa Candidate Biomarkers with Selected Reaction
Monitoring”. The qualification and verification of candidate biomarkers is a critical stage in the great
biomarker discovery pipeline. Credentialed biomarkers that have successfully passed through this
stage are considered verified biomarkers, which are of high value for translation into large-scale,
clinical validation studies. The evaluation of biomarkers in body fluids necessitates the development of
robust methods to quantify proteins in body fluids, using large sets of samples. In the present study, we
performed the qualification of a set of 42 candidate biomarkers for PCa diagnosis on a set of 107 post-
PM urine supernatant samples (36% with PCa) using SRM-based absolute quantification. Before that,
urine sample preparation and analytical procedures were optimized for SRM methodology. We
standardized preparation of the urine protein samples for SRM analysis by using 9 different protocols.
Our final goal was to obtain a panel of biomarkers that alone, or in combination with the existing PCa
biomarker, would help us to better define patients with PCa. In addition, due to the large number of
samples and their pathological conditions, we would also be able to define candidate prognostic
markers. However, this study has yet to be completed.
In conclusion, the data presented in this dissertation represent a significant advance in the standard
care for PCa diagnosis. Our two approaches (RNA- and Protein-based) have begun to yield promising
results, as both have levels of specificity that exceed those of PSA. However, validation studies on
larger, multi-centric cohorts of urine samples are needed to end up with a valid PCa biomarker. The
obtained results should have a rapid application in the clinics and potentially influence, together with
actual screening parameters (serum PSA and DRE), decisions that could improve the health system, as
well as clinical, managerial and/or public practices for health outcomes in PCa.
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Detection of Occult Blood and Trace Albumin in Urine by Matrix-Assisted Laser Desorption Ionization/Time-of-Flight Mass SpectrometryChang, Ching-Wen 26 June 2006 (has links)
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noneLin, Yen-Hsiu 03 July 2007 (has links)
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Effects of cow urine and its constituents on soil microbial populations and nitrous oxide emissions : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University /Bertram, Janet. January 2009 (has links)
Thesis (Ph. D.) -- Lincoln University, 2009. / Also available via the World Wide Web.
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A study of calcium oxalate dihydrate crystallization in synthetic and human urinesThorson, Steven Thomas January 1979 (has links)
No description available.
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EFFECT OF URINARY MACROMOLECULES ON CRYSTALLIZATION OF CALCIUM-OXALATE IN SYNTHETIC URINE SOLUTIONSKraljevich, Zlatica Idalia, 1949- January 1981 (has links)
The effect that organic urinary macromolecules have on the crystallization of calcium oxalate from a synthetic urine-like solution was studied in a mixed suspension-mixed product removal (MSMPR) continuous crystallizer. Precipitation of calcium oxalate crystals occurs during the continuous passage of urine through the renal system (kidney, bladder and tubules). While in normal circumstances these crystals remain small in size and exit the system unimpeded, in the pathologic condition calcium oxalate crystals are observed to aggregate and grow beyond a critical size where there is a significant probability of being trapped inside the renal system, e.g., on the kidney wall or in the tubules. Once trapped, the crystals become a nidus for further solute deposition and aggregation, giving origin to a renal calculus or stone. It is shown that this process is significantly affected by the presence or absence of organic macromolecules that act as modifiers of crystal growth, nucleation, and aggregation. An ultrafiltration technique was used to fractionate urine specimens from normal (N) and stone-forming (SF) persons into organic compounds of different molecular weight. These compounds were then added to the MSMPR system to test their effect on calcium oxalate crystallization. Significant differences were found to exist between N and SF urines in the composition, molecular weight distribution, and total quantity of these organic macromolecular compounds. The fraction of macromolecules responsible for the major effects on calcium oxalate crystallization was isolated, and its effect on crystal growth and nucleation rates was quantified. The steady state driving force (supersaturation) in the MSMPR system was measured. Striking differences in supersaturation versus residence time behavior between N and SF macromolecules were observed. The experimental conditions under which calcium oxalate crystals agglomerate were identified. Evidence which supports agglomeration as a key mechanism in urinary stone formation is presented.
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Reference interval for urinary catecholamines and methylated catecholamines analysed using HPLCJonsson, Anna January 2012 (has links)
Catecholamines are stress hormones that are produced and released by a rare tumor called pheochromocytoma. This tumor can cause hypertension which if undiagnosed and untreated leads to death. Since good therapy is available, it is important to find the tumor in time. The most common way to diagnose the tumor is measurement of the biochemical markers; catecholamines and their metabolites, methylated catecholamines. After observation that almost all normetanephrine results for women were higher than the upper reference limit and therefore pathological, the accuracy of the present reference intervals was questioned. Therefore new reference intervals for both urinary catecholamines and methylated catecholamines were developed by analysis of 46 samples using HPLC. Creatinine was analysed in acidified urine in order to see if the results became the same as when analysed in non-acidified urine. Urinary catecholamines and methylated catecholamines were analysed using HPLC. Comparison between measurement of creatinine in acidified urine and non-acidified urine with an enzymatic method was performed using Architect ci 8200, Abbott. As suspected, there was a difference between the present and new intervals. Therefore the new intervals will be used for future diagnosis. There was no difference between the two treatments of creatinine samples wherefore it can be measured in both.In conclusion reference intervals determind in this study will be used and it was shown that creatinine can be measured in acidified urine.
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Urine Proteomics for the Purpose of Biomarker DiscoveryBotelho, Diane M 20 July 2010 (has links)
Body fluids have gained widespread importance for proteomics based biomarker discovery as they have proved to reveal many candidate biomarkers for a variety of physiological diseases. Urine is a particularly favourable body fluid when profiling diseases associated with proximal tissues (kidney, urinary tract, etc.). The collection of urine is also non-invasive compared to other fluids such as blood, plasma and amniotic or cerebral spinal fluids.
The main objective of this work was to determine an optimal protocol for profiling of the urine proteome via peptide mass sequencing. Subsequently, the urine proteome was characterized as a potential source for protein biomarker(s) related to kidney obstruction. Of particular relevance to a quantitative investigation, it was found that the conventional urinary proteome workflow inadvertently introduces a sampling bias. Demonstrated here is the fact that the sediment proteins of urine, which are typically discarded prior to analysis, contain important protein constituents which were previously reported in the literature as candidate biomarkers.
A solution-based intact protein separation workflow was demonstrated for the analysis of urinary proteins. The mass-based separation platform, namely gel-eluted liquid fractionation entrapment electrophoresis (GELFrEE), incorporates the use of sodium dodecyl sulphate (SDS). As a known signal suppressant in mass spectrometry (MS), the MS tolerance of SDS in a proteome workflow was determined. Simple and effective protocols for the isolation of proteins from SDS-containing solutions are presented. Also presented is a comparison between GELFrEE and the conventional „GeLC? protocol for SDS-based protein separation, with similar numbers of proteins identified by the two platforms. Lastly, a novel strategy for isotope labelling of proteins at the intact level is presented. This intact labelling of proteins is therefore compatible with intact proteome prefractionation, as seen with GELFrEE-MS2 separation. This quantitative workflow was applied to biomarker profiling from urine samples obtained for a model (rodent) kidney obstruction. / A PhD thesis discussing optimal approaches for urine proteomics, a new strategy for separation and labelling of intact proteins for the purpose of protein quantitation and some possible biomarkers for kidney obstructions in a rat model.
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Urinary Metabolomic Signature of Pancreatic Ductal AdenocarcinomaDavis, Vanessa W Unknown Date
No description available.
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Detecting parasites loads in urine diversion toilets.Hawksworth, David James. January 2009 (has links)
In an attempt to supply sanitation to the growing communities in rural and peri-urban areas around Durban, the eThekweni Municipality has installed urine diversion (UD) toilets which have been modified to suit local conditions . These toilets are based on the ecological sanitation (EcoSan) system. The future aims are to reuse waste as a composting medium and minimize the use of water but the presence of microorganisms in the faecal waste poses a potential health risk to people in contact with it. Currently the Municipality has not deemed the waste safe for re-use but has suggested that after a one year standing period it should be free of all potential pathogens including Ascaris lumbricoiodes (human roundworm) ova. This study reports on the development of the AMBIC protocol for the recovery of Ascaris ova from the standing vaults of UD toilets. The protocol has been shown to consistently recover over 70% of Ascaris ova and has the added advantage of recovering the ova of other helminth species (Trichuris trichiura and Taenia sp.) present in a UD standing vault sample. Recoveries of Ascaris ova and ova of other parasite species, namely Trichuris and Taenia sp., are reported from waste which has been standing for one year. This is cause for concern as it shows one year is not a sufficient standing period to render the waste free of all microorganisms. Sampling from 124 UD toilet vaults that were in use, showed a high prevalence of both helminth (Ascaris lumbricoiodes, Trichuris trichiura and Taenia sp.) and protozoan (Giardia and Cryptosporidium) parasites. / Thesis (M.Sc.)-University of KwaZulu-Natal, 2009.
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