The document outlines a PhD research proposal on using circulating tumor cells (CTCs) as biomarkers for renal cell carcinoma (RCC). The proposal includes 5 research projects: 1) A systematic literature review on CTCs in RCC. 2) A retrospective study analyzing CTC counts in RCC patient samples and their relationship to staging and recurrence. 3) A prospective study comparing CTC liquid biopsies to renal mass biopsies for diagnosis. 4) A randomized controlled trial comparing surgical techniques for radical nephrectomy. 5) A randomized controlled trial comparing surgical techniques for partial nephrectomy. Detailed methods, timelines and practical considerations are provided for each project.

1. 1
Programa Doutoral do Centro Académico Médico
de Lisboa (CAML)
CTCS AS RCC BIOMARKERS
F O R D I A G N O S I S , S U R G I C A L T E C H N I Q U E A N D P R O G N O S I S
STARTING DATE: April 1, 2017
by Tito Miguel Palmela Leitão
SUPERVISOR: Prof. Doutor Luis Costa

2. 2
Introduction
Hello, my name is Tito Miguel Palmela Leitão. I am a ___-year student at Centro Académico Médico de Lisboa (CAML)
majoring in PhD. I’m going to talk to you today about my research on CTCS AS RCC BIOMARKERS.

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TECHNICAL DESCRIPTION
LITERATURE REVIEW

4. 4
Renal cell carcinoma (RCC) accounts for approximately 3% of
adult malignancies and 90–95% of kidney neoplasms1. Each
year, 330,000 cases are diagnosed and more than 140,000 die
due to RCC 2, corresponding to worldwide incidence rates
ranging from 0.6/100,000 to 14.7/100,000 3. Most tumors
present in the fifth to seventh decade of life, with a median
age at diagnosis of 66 years and median age at death of 70
years3. The incidence is two to three times higher in men and
is slightly more common in blacks than in whites 3. The
incidence of renal tumors at autopsy is approximately 2%3.
LITERATURE REVIEW
Fig. 1. Ten Leading Cancer Types for the Estimated New
Cancer Cases and Deaths by Sex, United States, 2015.

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CTCs are released from tumors and enter bloodstream early
before metastasis occur. CTC detection in peripheral blood
samples can be processed by immunomagnetic, microfluidic chip
or size based enrichment technique. Blood can also be directly
processed by nucleic acid based detection to identify tumor
specific transcript presence or absence24.
After enrichment CTCs may be detected by immunofluorescence
staining for positive and negative selection markers, and further
characterized by RT-PCR, fluorescence in situ hybridization (FISH)
or genomic sequencing24.
LITERATURE REVIEW
Figure 2. Circulating biomarkers in metastatic clear cell
renal cell carcinoma 23.

6. 6
Literature Review
CTC isolation techniques24
The first observation of CTCs in the peripheral blood of a patient with metastatic cancer from
unknown primary site was made by Thomas Ashworth in 186925. Technological advances have
been improving our ability to isolate as well as harness more knowledge of these quite rare cells,
which have led to improved understanding of basic cancer biology.
CTCs enter the circulation by either passive cellular shedding from the primary tumour site or
through a dynamic process that comprises stromal invasion and subsequent intravasation into the
blood stream26–28.
Within the circulation, most CTCs will not be able to form distant metastasis. CTCs must survive
sheer stress, anoikis and evade the host immune system to extravasate at a distant site26. Once at
their new location, CTCs must adapt to the local microenvironment, where they can lay dormant in a
quiescent state or undergo proliferation to develop into metastatic foci28.

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The first observation of CTCs in the peripheral blood of a patient with
metastatic cancer from unknown primary site was made by Thomas
Ashworth in 186925. Technological advances have been improving our
ability to isolate as well as harness more knowledge of these quite rare
cells, which have led to improved understanding of basic cancer biology.
CTCs enter the circulation by either passive cellular shedding from the
primary tumour site or through a dynamic process that comprises
stromal invasion and subsequent intravasation into the blood
stream26–28.
Within the circulation, most CTCs will not be able to form distant
metastasis. CTCs must survive sheer stress, anoikis and evade the host
immune system to extravasate at a distant site26. Once at their new
location, CTCs must adapt to the local microenvironment, where they
can lay dormant in a quiescent state or undergo proliferation to develop
into metastatic foci28.
LITERATURE REVIEW
Figure 4. CTC enrichment and identification26.

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Studies of CTCs in RCC cases are limited. Compared with prostate and bladder
cancer, CTCs have been much less well studied in kidney cancer. In studies
published in the 1990s Pontes et al29 and Hioki and Sugimura30 used RT-PCR
to detect circulating tumor DNA in patients with RCC. In an early study Ashida
et al identified mutations in the von Hippel-Lindau gene in circulating cells
using RT-PCR31. Ultimately these series were limited by the low detection rate
of RCC cell mRNA in peripheral blood.
LITERATURE REVIEW
The heterogeneous natural history and propensity for recurrence in
kidney cancer is well suited for improved individualization of care
using circulating tumor cells.
Figure 5. Circulating tumour
cells (CTCs) cycle28

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TECHNICAL DESCRIPTION
OBJECTIVES

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OBJECTIVES
This PhD proposal entails research to take place in the Centro de
Investigação Clínica (CIC) of the Centro Académico de Medicina de Lisboa
(CAML), a collaboration between Centro Hospitalar Lisboa Norte (CHLN)
and Faculdade de Medicina da Universidade de Lisboa (FMUL).
First
Firstly, he proposes to make a
systematic review of the role of CTCs
in renal cell carcinoma. Molecular
biomarkers have been shown to aid
the diagnosis and disease monitoring
for other cancers, but such markers
are not currently available for
RCC18. Studies of CTCs in RCC
patients are limited in number, in
study size and by the low detection
rate of RCC cells in peripheral blood.
Second Third
With the PhD proposal, the applicant intends to research the role of CTCs as
biomarkers of RCC, namely renal mass diagnosis, nephrectomy surgical
technique, and RCC follow-up and prognosis.
Secondly, the applicant intends to do a
retrospective study on CTC count in RCC
Biobanco samples collected at the time of
radical or partial nephrectomies
conducted in the CHLN from April 2014
to April 2017. The objective is to
determine the diagnostic and prognostic
acuity of CTCs in RCC patients, namely to
assess the relationship between CTC
levels and pathological staging, final
histology and recurrence rates and lead
time before imagiological detection of
the recurrence.
Thirdly, the applicant aims to
prospectively compare peripheral blood
liquid biopsy (CTCs) with renal mass
biopsy in the diagnosis of renal masses,
establishing the sensitivity and specificity
of each approach and looking in to
combining the two methods to enhance
diagnostic accuracy. This aims to reduce
the negative predictive value of renal
mass biopsies to better support
treatment decisions, particularly
watchful waiting strategies.

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Lastly, the applicant aims to address CTCs release in blood
circulation during kidney manipulation in partial or radical
nephrectomies. Earlier work by Hioki et al, demonstrated that
cancer cells are released into circulation during or after
surgery30. In this study, CTCs were identified in 15 of 20 (75%)
patients and specifically in 2 of 17 (11.2%) patients before
surgery, 6 of 16 (37.5%) patients within 24 h after surgery, 3 of 16
(18.8%) patients on postoperative day 7, and 2 of 11 (18.2%)
patients on postoperative day 3030 (figure 6). In seven of nine
(77.8%) patients, cancer cells were present in the renal vein.
These results show that intraoperative tumor manipulation
enhances cancer cell dissemination into the circulation.
OBJECTIVES
Figure 6. Rate of detection of circulating cancer cells
with VHL gene mutation 30.

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TECHNICAL DESCRIPTION
RESEARCH PLAN, METHODS AND
TASK DESCRIPTION

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ONE
P r o j e c t
The applicant proposes to make a systematic review of the role of CTCs in renal cell
carcinoma. The review is to be performed according to Preferred Reporting Items for
Systematic Reviews and Meta-analysis (PRISMA)55 and the Cochrane Handbook for
Systematic Reviews of Diagnostic Test Accuracy56. Studies will be identified by highly
sensitive searches of electronic databases (Medline, Medline In-Process, Embase,
Cochrane Controlled Trials Register, and LILACS) and relevant Web sites57.
All studies concerning the detection of CTCs in RCC will be included.
A data extraction form will be developed a priori to collect information on study
design, patient characteristics (age, gender, comorbidities), tumour features (size,
solid or cystic pattern), CTC detection methods, and other relevant variables.
RESEARCH PLAN, METHODS AND TASK DESCRIPTION
PROJECT 1 — SYSTEMATIC REVIEW OF CTC IN RCC

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TWO
P r o j e c t
Study population: patients who underwent partial or radical nephrectomy at the CHLN
Sample Repository: Biobanco CAML
Study Period: April 2014 to April 2017
Objective: To determine the diagnostic and prognostic acuity of CTCs in RCC patients,
namely to assess the relationship between CTC levels and pathological staging, final
histology and recurrence rates and lead time before imagiological detection of the
recurrence.
Samples: Peripheral blood drawn at the time of specimen removal; surgical specimen
RESEARCH PLAN, METHODS AND TASK DESCRIPTION
PROJECT 2 — RETROSPECTIVE STUDY CAN CTCS REPLACE RENAL BIOPSIES AND
PREDICT STAGING IN RCC DIAGNOSIS? — LIQUID BIOPSY OF RENAL MASSES

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THREE
P r o j e c t
The applicant aims to prospectively compare peripheral blood liquid biopsy (CTCs) with
renal mass biopsy in the diagnosis of renal masses, establishing the sensitivity and
specificity of each approach and looking in to combining the two methods to enhance
diagnostic accuracy. This aims to reduce the negative predictive value of renal mass
biopsies to better support treatment decisions, particularly watchful waiting strategies.
RESEARCH PLAN, METHODS AND TASK DESCRIPTION
PROJECT 3 — ACURACY OF PERIPHERAL BLOOD LIQUID BIOPSY (CTCS) AND RENAL
TUMOUR BIOPSY IN THE DIAGNOSIS OF RENAL MASSES — A PROSPECTIVE STUDY

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FOUR
P r o j e c t
The applicant proposes a two-arm
prospective single-center single-
surgeon randomized controlled trial
(RCT), comparing direct vs. late pedicle
ligation in radical nephrectomy.
The population of the study will be
every patient presenting to the renal
cancer consultation of CHLN which is
diagnosed with a solid or complex
cystic renal mass. A flowchart with the
patient pathway and study protocol is
presented in figures 7 and 8.
RESEARCH PLAN, METHODS AND TASK DESCRIPTION
PROJECT 4 — SINGLE-SURGEON RCT COMPARING DIRECT VS. LATE PEDICLE LIGATION
IN RADICAL NEPHRECTOMY
7
8

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FIVE
P r o j e c t
The applicant proposes a second two-arm prospective single-center single-surgeon RCT
comparing en bloc renal pedicle clamping vs. renal artery only clamping in partial
nephrectomy.
The hypothesis is that renal vein clamping may reduce CTC dissemination during partial
nephrectomy. In both groups the surgeon will directly dissect the pedicle at its origin in
the great vessels, with no kidney manipulation. Renal vein blood samples will be
collected at pedicle isolation and patients assigned to group 1 will have both renal artery
and vein clamping, whereas in group 2 only the artery will be clamped. The surgeon
completes the partial nephrectomy and a second renal blood sample will be collected
after hilum unclamping and parenchymal hemostasis is achieved.
RESEARCH PLAN, METHODS AND TASK DESCRIPTION
PROJECT 5 — SINGLE SURGEON RCT COMPARING EN BLOC RENAL PEDICLE CLAMPING
VS. RENAL ARTERY ONLY CLAMPING IN PARTIAL NEPHRECTOMY

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TIMELINE AND MILESTONES
PROJECT 1 — SYSTEMATIC REVIEW ON CTCs IN RCC
April-May
2017
Systematic literature review
and data collection
June-September
2017
Article drafting and
submission for publication in
indexed periodic peer-
reviewed journal

19. 19
TIMELINE AND MILESTONES
PROJECT 2 — RETROSPECTIVE STUDY CAN CTCS REPLACE RENAL
BIOPSIES AND PREDICT STAGING IN RCC DIAGNOSIS? — LIQUID
BIOPSY OF RENAL MASSES
April-May
2017
Literature review and data
collection
June-September
2017
Oct-December
2017
Statistical analysis
Article drafting and submission
for publication in indexed
periodic peer-reviewed journal

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TIMELINE AND MILESTONES
PROJECT 3 — ACURACY OF PERIPHERAL BLOOD LIQUID
BIOPSY (CTCS) AND RENAL TUMOUR BIOPSY IN THE
DIAGNOSIS OF RENAL MASSES — A PROSPECTIVE STUDY
April-May
2017
Literature review,
protocol drafting
and registration
April-May
2017 June
2017
• Trial registration
• Submission of
protocol for
publication
• Beginning of patient
recruitment
(expected frequency:
3 patients per week)
May
2018
Interim analysis of
results (patient
safety check)
Protocol submission
for approval by
Ethics’ Committee
May
2019
End of patient
recruitment
May-September
2019
Article drafting and
submission for publication in
indexed periodic journal

21. 21
TIMELINE AND MILESTONES
PROJECT 4 — SINGLE-SURGEON RCT COMPARING DIRECT
VS. LATE PEDICLE LIGATION IN RADICAL NEPHRECTOMY
April-May
2017
Literature review,
protocol drafting
and registration
April-May
2017 June
2017
• Trial registration
• Submission of
protocol for
publication
• Beginning of patient
recruitment
(expected frequency:
1 patients per week)
May
2018
Interim analysis of
results (patient
safety check)
Protocol submission
for approval by
Ethics’ Committee
May
2019
End of patient
recruitment
May-September
2019
Article drafting and
submission for publication in
indexed periodic journal

22. 22
TIMELINE AND MILESTONES
PROJECT 5 — SINGLE SURGEON RCT COMPARING EN BLOC RENAL
PEDICLE CLAMPING VS. RENAL ARTERY ONLY CLAMPING IN
PARTIAL NEPHRECTOMY
April-May
2017
Literature review,
protocol drafting
and registration
April-May
2017 June
2017
• Trial registration
• Submission of
protocol for
publication
• Beginning of patient
recruitment
(expected frequency:
1 patients per week)
May
2018
Interim analysis of
results (patient
safety check)
Protocol submission
for approval by
Ethics’ Committee
May
2019
End of patient
recruitment
May-September
2019
Article drafting and
submission for publication in
indexed periodic journal
September – March
2020
Doctoral thesis conclusion and
submission to jury for defense

23. 23
PRACTICAL ISSUES

24. 24
Sample size? Power study
I S S U E O N E
RCC Consultation in CIC?
I S S U E T W O
What is the pathway for
samples?
I S S U E T H R E E

25. 25
Who processes the samples
— INL, Biobanco,
Pathological Department?
I S S U E F O U R
eCRF?
I S S U E F I V E
Costs / Financing?
I S S U S I X

26. 26
When can we start
recruiting?
I S S U E S E V E N
Which steps are necessary?
I S S U E E I G H T
Timeframe
I S S U N I N E

27. 27
PhD tutors?
I S S U E T E N
PhD submission and
curricular component?
I S S U E E L E V E N

28. 28
Gupta, K., Miller, J. D., Li, J. Z., Russell, M. W. & Charbonneau, C.
Epidemiologic and socioeconomic burden of metastatic renal
cell carcinoma (mRCC): A literature review. Cancer Treatment
Reviews 34, 193–205 (2008).
BIBLIOGRAPHY
Choueiri, T. et al. Cabozantinib versus Everolimus in Advanced
Renal-Cell Carcinoma. N. Engl. J. Med. 373, 1814–23 (2015).
Ng, C. S. et al. Renal cell carcinoma: Diagnosis, staging, and
surveillance. Am. J. Roentgenol. 191, 1220–1232 (2008).
Capitanio, U. & Montorsi, F. Renal cancer. Lancet 387, 894–906
(2016).
Naito, S. et al. Prognosis of Japanese Metastatic Renal Cell
Carcinoma Patients in the Cytokine Era: A Cooperative Group
Report of 1463 Patients. Eur. Urol. 57, 317–326 (2010).
Bukowski, R. M. Natural history and therapy of metastatic renal
cell carcinoma: the role of interleukin-2. Cancer 80, 1198–220
(1997).

29. 29
Haaland, B., Chopra, A., Acharyya, S., Fay, A. P. & Lopes, G. de L.
Comparative effectiveness of approved first-line anti-angiogenic
and molecularly targeted therapeutic agents in the treatment of
good and intermediate risk metastatic clear cell renal cell
carcinoma. BMC Cancer 14, 592 (2014).
BIBLIOGRAPHY
Rini, B. I., Rathmell, W. K. & Godley, P. Renal cell carcinoma. Curr.
Opin. Oncol. 20, 300–6 (2008).
Lilleby, W. & Fosså, S. D. Chemotherapy in metastatic renal cell
cancer. World J. Urol. 23, 175–179 (2005).
Bullock, A., McDermott, D. F. & Atkins, M. B. Management of
metastatic renal cell carcinoma in patients with poor prognosis.
Cancer Manag. Res. 2, 123–132 (2010).

30. 30
Questions
I W O U L D B E H A P P Y T O TA K E YO U R Q U E S T I O N S .