Neoadjuvant chemotherapy in muscle-
invasive urinary bladder cancer:
Studies on treatment response, tumor draining
lymph nodes and blood transfusion
Robert Rosenblatt
Department of Surgical and Perioperative Sciences, Urology and
Andrology
Umeå 2020
Responsible publisher under Swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
Dissertation for PhD
ISBN: 978-91-7855-315-0 (print)
ISBN: 978-91-7855-316-7 (pdf)
ISSN: 0346-6612 New Series Number 2091 Cover photo credit: By http://www.scientificanimations.com/ - http://www.scientificanimations.com/wiki-images/, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=81897226
Electronic version available at: http://umu.diva-portal.org/
Printed by: Tryckservice, Umeå universitet
Umeå, Sweden 2020
To Béatrice, Sasha, Noah and our newly born son
The first principle is that you must not fool yourself - and you are
the easiest person to fool.
RICHARD P. FEYMAN
i
Contents
ABSTRACT ......................................................................................... iii
LIST OF PAPERS IN THE THESIS ............................................................ v
RELATED PAPERS NOT IN THE THESIS ................................................. vi
ABBREVIATIONS ............................................................................... vii
POPULÄRVETENSKAPLIG SAMMANFATTNING .................................... xi
BACKGROUND .................................................................................... 1
Urinary bladder cancer ............................................................................. 1
The lymphatic system ............................................................................. 11
Tumor immunology ................................................................................ 12
Sentinel node detection .......................................................................... 13
Adoptive immunotherapy ....................................................................... 15
Blood transfusions .................................................................................. 15
AIM OF THE THESIS........................................................................... 21
PATIENTS AND METHODS ................................................................. 22
Paper I .................................................................................................... 22
Paper II and III ........................................................................................ 23
Paper IV ................................................................................................. 25
RESULTS AND DISCUSSION ............................................................... 26
Tumor downstaging after neoadjuvant chemotherapy (Paper I) ............... 26
Sentinel node detection after neoadjuvant chemotherapy (Paper II) ........ 29
Fewer tumor draining sentinel nodes in patients with progressing muscle
invasive bladder cancer, after neoadjuvant chemotherapy and radical
cystectomy (paper III) ............................................................................. 32
Blood transfusion during neoadjuvant chemotherapy for muscle-invasive
urinary bladder cancer may have a negative impact on overall survival
(Paper IV) ............................................................................................... 34
CONCLUSION AND PERSPECTIVES ..................................................... 38
ACKNOWLEDGEMENTS ..................................................................... 40
ii
REFERENCES ..................................................................................... 43
iii
ABSTRACT
Muscle-invasive urinary bladder cancer is a deadly disease. Mortality rates
remained unchanged for decades despite radical surgery.
After several randomized trials, we today know that cisplatin based
chemotherapy given prior to cystectomy, improves survival for every tenth
patient. Markers that predict responsiveness to chemotherapy would spare
unnecessary treatment to the majority of patients. In the search for signs of
chemosensitivity, we performed a retrospective analysis of the Nordic cystectomy
trials 1 & 2: Chemo treated patients had an almost doubled increase in tumor
downstaging compared to the controls. More importantly, this group presented
with a reduced absolute risk of death of more than 30% compared to the rest of
the patients. These results were presented in paper I.
Many cancers spread through the lymphatic system. Usually, there is at least one
tumor draining lymph node, referred to as the sentinel node. If this node is free
of metastases, there is no lymphatic spread of the disease, and consequently, no
use of excavating all neighboring lymph nodes.
Sentinel node detection, is an established method in breast cancer, penile cancer
and malignant melanoma. Based on the same principles, members of our group
developed a similar detection technique in bladder cancer. Unfortunately,
sensitivity and specificity were too low to rely on this method as a diagnostic tool
for lymphatic spread. Instead, it turned out in recent years that sentinel nodes in
muscle invasive bladder cancer are valuable for translational research-lines -
mainly in tumor immunology. As for example, sentinel nodes contain tumor
specific T cells that are useful in adoptive immunotherapy.
In paper II, we set out to test whether sentinel node detection was feasible after
chemotherapy and/or tumor downstaging. In a prospective cohort of patients, we
saw no difference in detection rates between the groups. Thus, we concluded,
neither chemotherapy nor downstaging appeared to hamper the identification of
sentinel nodes.
The concept was expanded in paper III. After recruiting more patients to the
cohort mentioned above, the average numbers of sentinel nodes in different
categories of patients were compared. We saw a pattern of decreased number of
sentinel nodes in those with locally advanced tumors. It seemed that the number
of sentinel nodes had prognostic implications.
iv
In the last study, published in paper IV, we wanted to widen our knowledge on
the clinical effects of blood transfusion. Mounting data suggests that
perioperative blood products have a negative impact on long term survival after
cancer surgery. How much allogenic blood was given during the chemotherapy
prior to surgery? It turned out that one third of the bladder cancer patients
received blood, and these patients demonstrated a significantly worse overall
survival.
Neoadjuvant chemotherapy has added a new beneficial dimension to the
treatment of muscle invasive bladder cancer. In these four studies, we addressed
the effects of chemotherapy on pathoanatomical outcomes, on tumor lymphatics
and further; we are suggesting consequences of neoadjuvant chemotherapy in
conjunction with blood transfusion. It appears that the immune system is
involved in all aspects investigated above. Most likely, an improved scientific
understanding of the immune system will be crucial for future bladder cancer
treatment options.
v
LIST OF PAPERS IN THE THESIS
I. Rosenblatt R*, Sherif A*, Rintala E, Wahlqvist R, Ullen A, Nilsson S, Malmström PU, the Nordic Urothelial Cancer Group. Pathologic downstaging is a surrogate marker for efficacy and increased survival following neoadjuvant chemotherapy and radical cystectomy for muscle-invasive urothelial bladder cancer. European Urology. 2012;61(6):1229-38.
II. Rosenblatt R, Johansson M, Alamdari F, Sidiki A, Holmström B, Hansson J, Vasko J, Marits P, Gabrielsson S, Riklund K, Winqvist O, Sherif A. Sentinel node detection in muscle-invasive urothelial bladder cancer is feasible after neoadjuvant chemotherapy in all pT stages, a prospective multicenter report. World J Urol. 2017;35(6):921-7.
III. Alvaeus J, Rosenblatt R, Johansson M, Alamdari F, Jakubczyk T, Holmström B, Hemdan T, Huge Y, Aljabery F, Gabrielsson S, Riklund K, Winqvist O, Sherif A. Fewer tumour draining sentinel nodes in patients with progressing muscle invasive bladder cancer, after neoadjuvant chemotherapy and radical cystectomy. World J Urol. 2019.
IV. Rosenblatt R*, Lorentzi G*, Bahar M, Asad D, Forsman R, Johansson M,Shareef M, Alamdari F, Bergh A, Winqvist O, Sherif A. Blood transfusions during neoadjuvant chemotherapy for muscle-invasive urinary bladder cancer may have a negative impact on overall survival. Scand J Urol. 2020:1-6
*Shared first authorship
Paper I was reproduced with the kind permission from Elsevier
Paper II and III were reproduced with the kind permission from Springer Nature
Paper IV is an accepted article published by Taylor & Francis in Scandinavian Journal of
Urology on 2020 Feb, available online http://wwww.tandfonline.com/DOI. The paper was
reproduced with their kind permission.
vi
RELATED PAPERS NOT IN THE THESIS
Rosenblatt R, Jonmarker S, Lewensohn R, Egevad L, Sherif A, Kalkner
KM, Kälkner KM, Nilsson S, Valdman A, Ullén A. Current status of
prognostic immunohistochemical markers for urothelial bladder cancer.
Tumour Biol. 2008;29(5):311-22
Rosenblatt R, Valdman A, Cheng L, Lopez-Beltran A, Montironi R,
Ekman P, Egevad L. Endothelin-1 expression in prostate cancer and high
grade prostatic intraepithelial neoplasia. Anal Quant Cytol Histol.
2009;31(3):137-42.
Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt R, Tolf A, et
al. FOXP3 and survival in urinary bladder cancer. BJU Int.
2011;108(10):1672-8.
Shah CH, Viktorsson K, Kanter L, Sherif A, Asmundsson J, Rosenblatt
R, Lewensohn R, Ullén A. Vascular endothelial growth factor receptor 2,
but not S100A4 or S100A6, correlates with prolonged survival in
advanced urothelial carcinoma. Urol Oncol. 2014;32(8):1215-24.
vii
ABBREVIATIONS
ABC Advanced bladder cancer
ACD Anemia of chronic disease
AI Anemia of inflammation
BCG Bacillus Calmette-Guérin
CCI Charlson comorbidity index
CD40L Cluster of differentiation 40 ligand
CIS Carcinoma in situ
CMV Cytomegalovirus
CR Complete response
CT Computer tomography
CTL Cytotoxic T lymphocyte
cTNM Clinical tumor node metastasis
DC Dendritic cell
DCE MRI Dynamic contrast-enhanced magnetic resonance imaging
ddMVAC dose dense Methotrexate Vinblastine Adriamycin Cisplatin
DSS Disease specific survival
EBRT External beam radiotherapy
ECOG Eastern cooperative oncology group
E-LND Extended lymph node dissection
ER Estrogen receptor
viii
EV Extracellular vesicles
FFP Fresh frozen plasma
FNR False negative rate
FP False positive
GC Gemicitabine cisplatin
GI Gastrointestinal
GSTM1 Glutathione-S-transferase mu 1
Hb Hemoglobin
HEV High endothelial venules
HLA Human leukocyte antigen
HR Hazard ratio
IL Interleukin
INF- γ Interferon gamma
L-LND Limited lymph node dissection
LM Lymphatic mapping
LND Lymph node dissection
MIBC Muscle-invasive bladder cancer
miRNA micro ribonucleic acid
MMT Multimodality treatment
MR Magnetic resonance
MVAC Methotrexate Vinblastine Adriamycin Cisplatin
NAC Neoadjuvant chemotherapy
ix
NAT-2 N-acetyltransferase 2
NCT Nordic cystectomy trial
NMIBC Non-muscle-invasive bladder cancer
NNT Numbers needed to treat
OR Odds ratio
ORC Open radical cystectomy
OS Overall survival
PBC Packed red cell concentrate
PBT Perioperative blood transfusion
PD Progressive disease
PD-L1 Programmed death-ligand 1
PDGF Platelet-derived growth factor
PMP Platelet-derived microparticles
pTNM Pathological tumor node metastasis
RARC Robotic-assisted laparoscopic radical cystectomy
RC Radical cystectomy
RCT Randomized controlled trial
RR Relative risk
SD Stable disease
SN Sentinel node
SNd Sentinel node detection
SNdef1 Sentinel node definition 1
x
SNdef2 Sentinel node definition 2
S-LND Standard lymph node dissection
SE-LND Super-extended lymph node dissection
TGF- β Transforming growth factor beta
TNFa Tumor necrosis factor alpha
TNM Tumor node metastasis
TRALI Transfusion related acute lung injury
TRIM Transfusion related immunomodulation
TURB Transurethral resection of the bladder
VEGF Vascular endothelial growth factor
VEGFC/D Vascular endothelial growth factor C/D
UBC Urinary bladder cancer
xi
POPULÄRVETENSKAPLIG
SAMMANFATTNING
I Sverige är urinblåsecancer den fjärde vanligaste cancerformen hos män och
åttonde vanligaste hos kvinnor. Sjukdomen uppträder som regel efter 65 års
åldern. Rökning är en stark riskfaktor, vilket delvis kan förklara varför män
hittills varit mer drabbade. Det vanligaste symtomet är synligt blod i urinen. Då
är det viktigt att man utreds med kontraströntgen av urinvägarna samt
kameraundersökning av urinblåsan (cystoskopi). Den slutgiltiga diagnosen får
man efter provtagning av tumören på operation. Vävnadsbitar från tumören
skickas på mikroskopisk analys vilket avgör tumörens allvarlighetsgrad.
En viktig skiljelinje är om tumören växer in i urinblåsans muskelskikt. I tre
fjärdedelar av fallen gör den inte det. Då räcker det oftast med att hyvla bort all
synlig tumör samt vid behov ge sköljningar i urinblåsan med antingen cellgifter
eller immunologiskt verkande läkemedel.
I en fjärdedel av fallen växer tumören på djupet, vilket kallas för muskelinvasiv
blåscancer. Detta är en mycket allvarlig sjukdom där botande behandling innebär
att man tar bort hela urinblåsan (cystektomi). Trots denna omfattande operation
har cancercellerna i nära hälften av fallen hunnit sprida sig, oftast via
lymfbanorna. För att minska risken för spridning kan man förbehandla med tre-
fyra kurer cellgifter (neoadjuvant kemoterapi). Drygt hälften av patienterna med
muskelinvasiv blåscancer är friska nog att klara cellgiftsbehandling.
Den här avhandlingen består av fyra studier som tittar närmare på olika aspekter
av cellgiftsbehandling vid muskelinvasiv blåscancer. Upprepade studier visar att
enbart var tionde patient har nytta av cellgifterna. För att finna en tydlig flagga
för vem som gagnas, tittade vi närmare på två nordiska studier från 90-talet. Här
hade patienterna lottats mellan standardbehandling med eller utan föregående
cellgifter. I vår studie slog vi ihop patienterna och jämförde skillnaden i
tumörutbredning innan och efter operation. Det var dubbelt så vanligt att
tumören utplånats helt om cellgifter givits. Dessutom var överlevnaden mycket
bättre. Nio av tio patienter var i liv fem år efter operationen. Hos övriga, oavsett
cellgifter eller ej, överlevde bara hälften av patienterna femårsdagen.
Vid många cancerformer sker spridning via lymfbanorna. För att slippa leta
cancer i hela lymfsystemet kan det räcka att titta i den tumördränerande
lymfknutan. Hittar man ingen cancer här så är risken låg för spridning. Vid
bröstcancer gör denna teknik att många patienter besparas onödiga ingrepp. Men
vid urinblåsecancer är tekniken inte helt tillförlitligt. Däremot innehåller
xii
tumördränerande lymfknutor vita blodkroppar som exponerats för cancern. Tar
man ut dessa och låter de föröka sig i ett laboratorium kan de fungera som en
effektiv behandling om man ger dem tillbaka till patienten. Detta kallas för
adoptiv immunterapi.
För att kunna använda denna terapi i större patientgrupper ville vi undersöka om
tekniken för att spåra den tumördränerande lymfknutan fungerade för
urinblåsecancer även efter cellgiftsbehandling. Det visade sig fungera; antalet
tumördränerande knutor var oförändrat. Samtidigt hade våra medarbetare
undersökt lymfknutorna. Aktiviteten hos de cancerbekämpande vita
blodkropparna hade ökat tack vare cellgiftsbehandlingen!
I den tredje studien tittade vi på antalet tumördränerande lymfknutor i olika
patientkategorier. Vi såg ett tydligt mönster. Tumörer motståndskraftiga mot
cellgifter hade färre dränerande lymfknutor. Sedan tidigare vet vi att tumören kan
styra nybildningen av lymfbanor och stänga av kontakten med närliggande
lymfknutor. Detta för att skydda sig. Därför tror vi att tumördränerande
lymfknutor kan vara en användbar markör för prognos och val av behandling i
framtiden.
Blodbrist är vanligt hos människor med cancer. Cellgifter och kirurgi kan förvärra
situationen vilket leder till att blodtransfusioner ofta ges. Men blod är inte
ofarligt. Mottagarens immunförsvar kan överreagera och det finns risk för
spridning av infektioner. Vad som är mindre känt är att blodtransfusion i många
fall har en dämpande effekt på det tumörbekämpande immunförsvaret. Detta kan
förklara varför patienter som fått blod i samband med canceroperation uppvisar
en försämrad prognos. Ett välfungerande immunförsvar är nämligen viktigt för
att övervinna cancern.
I den sista studien undersökte vi om effekten av cellgifter försämras hos patienter
som samtidigt erhållit blodtransfusion. Det visade sig att en tredjedel av
patienterna hade fått blod. Dessa hade elakare tumören när man undersökte
urinblåsan efter operation. Ännu värre var att patienterna hade försämrad
långtidsöverlevnad. Resultatet är tankeväckande men bör tolkas med
försiktighet. Studien baseras på 120 patienter och studiens upplägg gör det svårt
att avgöra om det är blodet eller orsaken till att blod givits som förklarar
skillnaden i resultat.
Nämnda studier belyser olika delar av cellgiftsbehandling vid muskelinvasiv
blåscancer. Vi har visat hur krympning av tumören är ett starkt uttryck för svar
på behandling. Därefter visade vi att cellgifter inte är ett hinder för att hitta
tumördränerande lymfknutor. På samma tema såg vi i den tredje studien att
antalet tumördränerande lymfknutor är lägre hos tumörer som svarar på
xiii
behandling. Avslutningsvis undersökte vi förekomsten av blodtransfusion under
pågående cellgiftsbehandling. Patienter som fått blod hade elakare tumörer och
sämre prognos.
1
BACKGROUND
Urinary bladder cancer
Definitions, epidemiology and etiology
Urinary bladder cancer (UBC) is a major global health concern. In 2018
approximately 550, 000 were diagnosed and nearly 200, 000 died of the
disease (1). It is the sixth most common cancer for men, and the 17th for
women, worldwide. Europe and North America, are high incidence regions
with 17.3 men and 4.5 women per 100.000 people (1). In Sweden, 2859
patients were diagnosed and 732 died from the disease in 2018 (1). As of 31st
of December 2016, 25 848 people were living in Sweden with cancer in the
bladder or in the upper urinary tract (2). The disease is strongly related to age,
with a mean of 73 years at diagnosis. The age standardized incidence rate for
UBC in Sweden has been increasing since the 1970 for both men and women
(3). However, in similarity with most other western countries, there has been
a stabilization of incidence and decrease in mortality since the mid 90s (4).
The main reason for this is ascribed to diminished smoking habits in the
population.
Risk factors can be divided into inherited properties and external exposures.
Thus far, there are no identified genes that strongly predispose to UBC. Yet,
studies of twins have estimated that 30% of the risk is attributed to heritable
factors (5). Likewise, relatives of UBC patients have an almost doubled risk of
the disease compared to controls, independent of smoking status (6, 7).
The genetic polymorphism of N-acetyltransferase 2 (NAT-2) and Glutathione-
S-transferase mu 1 (GSTM1) appear to alter the risk of UBC. NAT-2 is a liver
enzyme that acetylates arylamine, one of 60 established carcinogens in
tobacco smoke that is strongly associated with UBC (8). The slow functioning
genotype of NAT-2 has been associated with increased risk of UBC in both
non-smokers (OR=3.41) and heavy smokers (OR = 8.57) (8). GSTM1 is a
conjugating enzyme involved in the detoxification of various carcinogens,
where the non-functional version of GSTM1 (null allele) has been associated
with increased risk of cancer (9).
Tobacco smoking is the most important external risk factor for UBC. More
than 60 carcinogenic substances have been described, including aromatic
amines, which is believed to be the most important agent in the formation of
UBC. In a meta-analysis that pooled the result of 83 original articles, there was
2
an increased risk of incidence (RR = 3.47) and mortality (RR=1.53) in current
smokers compared to non-smokers (10).
Exposure to aromatic amines and other chemicals present in dyes, rubber,
leather and paint, have resulted in increased incidence of UBC in certain
occupations. The list of exposed occupations is long, and is topped by tobacco
workers (RR= 1.72), dye workers (RR= 1.58) and chimney sweepers (R=1.53)
(11).
To this day we do not understand why men are diagnosed with UBC three to
four times more often than women. Differences in smoking habits and
exposure to environmental carcinogens have been suggested. Yet, studies that
controlled for these factors have still observed gender differences (12).
Gender-related differences in metabolizing enzymes and bladder
microbiomes might contribute to diverse ecosystems in the bladder (12). A
lowered risk for UBC has been recorded for women in the premenopause, with
late menarche, and women supplementing with estrogens and progesterone.
At the tissue level, the expression of the androgen receptor in urothelial
carcinoma decreased with increasing tumor stage (12).
Even though the incidence of UBC is higher for men, women tend to have a
worse prognosis compared to men, even after adjusting for tumor stage (13).
Data show that the lead time from hematuria to diagnosis is significantly
longer for women (14), and this might have implications for prognosis (15).
Discrepancies in treatment might account for part of these differences (16). A
meta-analysis encompassing 27,912 patients, looking specifically at outcome
after radical cystectomy (RC), saw a significantly worse cancer-specific
survival in women (HR 1.20, p=0.0001) (17). As mentioned above, estrogens
appear to have a protective role for the emergence of UBC. Yet, the type of
estrogen receptor also matters: ER is associated with favorable tumor
outcome while ER predicts a worse prognosis (18).
3
Figure 1. Schematic image of the urinary bladder with tumors of different stages.
Credit: Cancer Research UK Original email from CRUK, CC BY-SA 4.0,
https://commons.wikimedia.org/w/index.php?curid=34334316
Symptoms, staging and classification
Gross hematuria is the cardinal symptom of urinary bladder cancer and is present
in 85% of the cases. Dysuria, urgency and increased frequency are other clinical
signs. Evaluation of hematuria includes white light cystoscopy, cytology of urine
or bladder-washing specimen, computed tomography (CT) urography, and rectal
examination of the prostate. Diagnosis is based on pathoanatomical assessment
of tumor specimens after transurethral resection of the bladder (TURB) (19).
90% of UBCs are of primary urothelial origin. Other primary carcinomas of the
bladder include squamous cell carcinomas (5%), adenocarcinomas (2%),
sarcomas, signet ring cell and small cell carcinoma. Primary urothelial
carcinomas can also present with mixed histology with the most common variant
being squamous cell carcinomas and adenocarcinomas. Other variants include,
micropapillary, nested cell variant, clear cell variant carcinomas and
plasmacytoid tumors (20).
The TNM classification defines the stages of the disease (table 1). UBC extends
from papillary neoplasms (pTa) of low malignant potential to local advanced
disease with metastatic spread (pT4bN+M+). Correct pathoanatomical diagnosis
is crucial for correct management and prognosis.
4
Table 1. Classification of urinary bladder cancer. Adapted from the Guidelines on Muscle-
invasive and Metastatic Bladder Cancer, European Association of Urology (EAU) (19).
T - Primary Tumor
Tx Primary tumor cannot be assessed
T0 No evidence of primary tumor
Ta Non-invasive papillary carcinoma
Tis Carcinoma in situ: "flat tumor"
T1 Tumor invades subepithelial connective tissue
T2 Tumor invades muscle
T2a Tumor invades superficial muscle (inner half)
T2b Tumor invades deep muscle (outer half)
T3 Tumor invades perivesical tissue
T3a microscopically
T3b macroscopically (extravesical mass)
T4 Tumor invades any of the following: prostate stroma,
seminal vesicles, uterus, vagina, pelvic wall, abdominal wall
T4a Tumor invades prostate stroma,
seminal vesicles, uterus, or vagina
T4b Tumor invades pelvic wall or abdominal wall
N - Regional Lymh Nodes
Nx Regional lymph nodes cannot be assessed
N0 No regional lymph node metastasis
N1 Metastasis in a single lymph node in the true pelvis
(hypogastric, obturator, external iliac, or presacral)
N2 Metastasis in multiple lymph nodes in the true pelvis
(hypogastric, obturator, external iliac, or presacral)
N3 Metastasis in common iliac lymph node(s)
M - Distant Metastasis
M0 No distant metastasis
M1a Non-regional lymph nodes
M1b Other distant metastasis
5
At presentation, approximately 75% of UBCs are non-muscle-invasive bladder
cancer (NMIBC). This means that the tumors have not invaded the detrusor
muscle in the bladder wall. These lesions are further categorized into low,
intermediate and high risk of recurrence and progression and are typically
managed by TURB with or without adjuvant intravesical instillations. The high
risk for recurrence and progression demands frequent cystoscopies and repeated
TURBs (21).
The remaining 25% of UBC represent muscle-invasive urinary bladder cancer
(MIBC). For MIBC, where metastatic spread is not suspected, the mainstay of
treatment is radical cystectomy (RC) and regional lymph node dissection (LND).
An overview of the different treatment options is summarized in (table 2).
Table 2. Summary of urinary bladder cancer treatment. Adapted from the Guidelines on
Muscle-invasive and Metastatic Bladder Cancer, European Association of Urology (EAU) (19).
Non-muscle-invasive bladder cancer
Low-risk Primary, solitary, TaG1, <3cm, no CIS
TURB + immediate instillation of intravesical chemotherapy
Intermediate risk All tumors not defined as low or high-risk
TURB + repeated instillations of intravescial chemotherapy/BCG
High-risk T1/G3/CIS or multiple, recurrent and >3cm
TURB + intravescial BCG 1-3 years or RC
Muscle-invasive bladder cancer
Cisplatin-fit T2-T4aN0M0
NAC+RC High risk patient NAC naïve pT3/4 and/or pN+
RC + adjuvant (GC/MVAC)
Unfit/unwilling RC EBRT/MMT
Metastatic bladder cancer
Cisplatin-fit GC, MVAC Cisplatin unfit (PD-L1 +) Pembrolizumab/Atezolizumab Cisplatin unfit (PD-L1-) Carboplatin
Second line Pembrolizumab
6
Staging and treatment of MIBC
A correct staging is pivotal for management of MIBC. The CT urography
performed in the initial investigation, accompanied with a CT of the thorax, is
used for assessing: local extension of the tumor, suspected lymph node
metastases, spread of tumor to upper urothelial tract and other organs (22).
Magnetic resonance (MR) is better at soft tissue resolution, which is valuable for
distinguishing tumors from surrounding tissue (23). However, at this point no
imaging modality can differentiate T2 from T3a, whereas identification of T3b or
higher is feasible (24). Lymph node metastases should be suspected when a
lymph node is > 8 mm in the pelvis, and >10 mm in the abdomen (19).
Comorbidity assessment
Mortality after RC increases with chronological age: 2.3% in 70-79 year of age
compared to 4.3% in 80 years and older (25). However, biological age is a more
important prognostic factor. Sarcopenia prior to abdominal surgery, and low
preoperative serum albumin levels, are some factors that are important for
overall survival after RC (26). The Charlson Comorbidity Index (CCI) is a well-
studied tool for evaluating comorbidity in MIBC. Together with instruments
developed for assessing activity level, e.g. the Eastern Cooperative Oncology
Group (ECOG), CCI can aid in determining the appropriate treatment for a given
patient (19).
Radical cystectomy
The gold standard therapy for localized MIBC (pT2-T4aN0M0) is radical
cystectomy (RC) with bilateral pelvic lymph node dissection (LND). Some other
main indications for RC are NMIBC with very high risk, BCG resistant NMIBC,
and MIBC N1 disease (19).
The standard RC implies removal of the urinary bladder and the distal ureters,
including the excision of the entire urethra, the uterus, the cervix, the ovaries, and
the anterior vagina in women, and the removal of the prostate and the seminal
vesicles in men. In men with organ-confined disease that are motivated to
preserve their sexual ability, four organ sparing techniques are described:
Preserving the neurovascular bundle, +/-, seminal vesicles and vas deferens, +/-
parts or the whole prostate (27). In women, pelvic organ-preserving techniques
maintain the neurovascular bundle, vagina, uterus and the ovaries. In
premenopausal women, preserving the ovaries will keep the hormonal
homeostasis, whereas preservation of the uterus and vagina reduce pelvic floor
disorders. Although data on functional and oncological results are limited, the
techniques can be considered in selected cases with organ-confined disease (27).
7
Controversy remains on the importance of timing of surgery for optimal oncologic
results. Some have reported worse results after delaying definitive treatment with
>3 months (28) whereas others have shown no difference (29).
Lymph node dissection (LND)
The rationale for LND is claimed to be of both prognostic and therapeutic value.
Among patients with clinical T2-4N0M0 tumors, approximately 25% have lymph
node metastases detected in removed lymph nodes post-RC (30, 31). Patients
with positive lymph nodes (pN+) are associated with a significant risk of disease
recurrence compared to pN0 (32). Nevertheless, patients without demonstrable
lymph node metastases, still suffer from recurrences and death due to metastases.
The lymphatics of the urinary bladder flows primarily to nodes in the internal
iliac, external iliac, obturator and presacral region. In a large consecutive series
of 200 patients, Abol-Enein et al observed that lymph node metastases outside
the pelvis always preceded metastases in obturator or iliac nodes first (30). Yet,
evidence of “skip lesions” i.e. metastases occurring solely in extra-pelvic regions,
have been reported (33).
Retrospective data have pointed to an oncological benefit in favour of LND
compared to no-LND (34). More controversy concerns the extent of LND which
can be classified into four templates: Limited LND (L-LND) which is restricted to
the obturator fossa (external iliac vessels laterally, iliac bifurcation proximally,
genitofemoral nerve medially). Standard LND (S-LND), includes L-LND plus the
medial internal iliac vessel medially, and the inguinal ligament distally. In
extended LND (E-END) presacral nodes up to the aorta bifurcation are added.
Super-extended LND (SE-LND) continues proximally to the root of inferior
mesenteric artery (19).
In a systematic review including 14 studies comparing E-LND with L-LND/S-
LND there was no compelling evidence in favor of E-LND (34). More importantly,
the first randomized trial comparing 198 patients with E-LND to 203 patients
with L-LND, failed to show a significant oncological advantage in the E-LND
group. Rather, the former group presented with increased need of lymphocele
drainage within 90 days of surgery, compared to the latter (35).
Retrospective comparisons of different LND templates will encounter selection
bias (36). A patient undergoing cystectomy without LND has an unknown pN
status. The more extended LND a patient is submitted to, the more defined the
pN status will be. Therefore, retrospective comparisons of different LND
templates will compare apples with pears. For instance, if a patient with pN0 after
no or limited LND is compared to a patient with pN0 after more extended LND,
8
the first patient might have been categorized as a pN+ if a more extensive
template was applied.
Robotic vs open radical cystectomy
In 2019, a Cochrane review based on five RCTs, compared open radical
cystectomy (ORC) with robotic-assisted laparoscopic radical cystectomy (RARC)
(37). With 270 and 271 patients in each arm respectively, there was no significant
difference in time-to-recurrence or major complications (Clavien 3-5). There
were fewer blood transfusions and somewhat shorter hospital stay for the RARC
arm. However, cost-effectiveness was never addressed. Similar results were
shown in a systematic review, which also demonstrated longer operative time, but
shorter hospital stay and decreased blood loss in RARC patients (38).
Urinary diversion
After the bladder is removed, the ureters can be rerouted in three principal ways:
the abdominal wall, the urethra, or the rectosigmoid. Various parts of the
gastrointestinal tract can be used as conduit or continent pouch. The most
common diversions are the ileal orthotopic neobladder and the ileal conduit. Age,
comorbidity and cognitive functions are important factors when deciding on the
type of diversion. The orthotopic neobladder is anastomosed to the urethra.
Voiding depends on abdominal pressure, intestinal movement and sphincter
opening. This alternative is generally not recommended to patients above 80
years, and it is contraindicated to patients with psychiatric illness, tumor growth
in the urethra, restricted life probability, and renal failure. Common
complications include both day- and nighttime incontinence and
ureterointestinal stenosis. With an ileal conduit, 50% of the patients experience
early complications, e.g. urinary tract infections, ureteroileal leakage and stenosis
whereas stomal complications occur in the long-term. There are no RCTs that
compare the conduit to the neobladder, but the oncological outcome is similar
(27).
Mortality
Perioperative mortality after RC at 30 and 90 days ranges between about 1.9-3.2%
and 5.7-8% with significant increased rates for low volume hospitals (39).
Oncological outcome is strongly correlated to pT stage and pN status. In one of
the larger cohorts involving 1054 patients of urothelial carcinoma, the recurrence
free survival at 5 years was 68% for the whole cohort, 92% for pT0 bladders, 89%
for pT2N0 and 78% for pT3a, 62% for pT3b, and 50% for pT4. 24% had pN+ and
these patients had a five-year disease specific survival of 35% (40). Other
prognostic factors include margin status, lymphovascular invasion, hospital
volume and age (41). Molecular subtypes are also important: luminal papillary,
luminal unstable, luminal unspecified, stroma-rich, basal/squamous and
9
neuroendocrine-like are definitions that might be used for prognosis and choice
of treatment in the future (42).
Chemotherapy
The introduction of chemotherapy as a treatment of cancer was inspired by the
use of antibiotics to treat infections in the 1930s. However, in contrast to bacteria,
cancer cells are similar to normal cells, which makes them difficult to target
without damaging healthy tissue.
Moreover, tumor cells are genomic unstable, which give rise to subclones with
varying degrees of susceptibility to drugs. It was not until chemotherapies of
different mechanisms were combined, that cancer patients started to be cured by
chemotherapy.
In MIBC, 50% of patients progress after radical cystectomy. Thus, surgery needs
to be accompanied by other treatments for survival to improve. In the mid 70s, it
was shown that advanced UBC responded to cisplatin (43) followed by
methotrexate in the early 80s (44) and then in combinations with vinblastine (45)
and doxorubicin (46). Yet, a long going debate followed, concerning the timing of
the therapy. Should the systemic treatment be given before (neoadjuvant) or after
(adjuvant) surgery?
Neoadjuvant chemotherapy (NAC)
Studies on breast cancer indicate that both neoadjuvant and adjuvant
administration of chemotherapy are effective in eradicating micrometastases
(47). However, in MIBC, the situation is different: Patients are older, have more
comorbidities, and will undergo cystectomy; an operation associated with more
complications and deconditioning than surgical treatment of breast cancer. Thus,
the arguments for neoadjuvant treatment are multiple: 1) The patient is in better
shape to tolerate systemic therapy. 2) Gross anatomy is unobstructed, with intact
blood vessels that can deliver the cytotoxic agents to the target organ. 3) The
chemotherapy is given as early as possible, before the tumor burden is too high.
4) Chemotherapy allows debulking effects on the tumor, which facilitates surgery,
and increased levels of pT0, pN0 and negative margins. 5) Chemotherapy does
not appear to affect surgical morbidity nor the eligibility for RC (48).
One of the early initiatives to investigate the role of NAC in UBC came from
Scandinavia. In 1992, the first Nordic Cystectomy Trial (NCS1) demonstrated a
significant survival advantage in patients with cT1-T4aNxM0 that were
randomized to two cycles of cisplatin + doxorubicin prior to standard care
(radiotherapy and RC) (49). In the follow-up study the Nordic Cystectomy Trial
2 (NCS2), patients with only MIBC were included and the chemotherapy regimen
was intensified (50). However, no distinct survival benefit was recorded in the
10
experimental arm until the two studies were combined in a joint analysis. Now an
absolute risk reduction was seen in favour of the experimental arm (p=0.045)
(48).
Today there is solid evidence for the benefit of NAC. The Advanced Bladder
Cancer (ABC) Meta-analysis Collaboration, encompassing individual patient data
of 3005 patients from eleven RCTs, (including the NCS1&2), showed a significant
benefit in overall survival for platinum-based combination chemotherapy
(HR=0.86, p=0.03) (51). The most recent updated meta-analysis of the ABC,
including four additional RCTs and 427 new patients showed an absolute risk
reduction of eight percent at five years (NNT=12,5) when gemcitabine cisplatin
(GC) or methotrexate, vinblastine, adriamycin and cisplatin (MVAC)
chemotherapy were considered (52).
Adjuvant chemotherapy
The arguments for adjuvant chemotherapy is that that patients with low risk of
metastatic disease are sorted out, and that curative surgery is performed without
delay. With this approach, patients with pT3-4 and/or pN+ disease with high risk
for relapse, would be candidates for postoperative treatment.
Yet, repeated trials have failed to demonstrate any benefit from this strategy.
Patients are often in weakened condition after cystectomy, with deteriorating
renal function that makes them unfit for chemotherapy. Four randomized trials
recruited less than 50% of their planned enrolments. In the largest of them,
EORTC 30994, 284 patients were randomized between early or deferred
chemotherapy (GC, MVAC or ddMVAC). The median overall survival was 6.74
years in the early compared to 4.60 years in the deferred (p= 0.13) (53). In an
updated meta-analysis of nine trials including 945 patients, a hazard ratio of 0.77
was seen in overall survival (p=0.049). The benefit was accentuated in disease
free survival for patients with lymph node positive disease (p=0.010) (54).
In contrast to NAC, trials on adjuvant chemotherapy have only examined tumors
that are locally advanced and/or pN+. This approach deprives patients, with a
potentially high load of undetected micrometastases (≥cT3pN0), from systemic
treatment. Subgroup analysis of cT3 tumors has shown greatest benefit from NAC
in this group, both in terms of tumor downstaging as well as in significantly
improved overall survival. Arguably, this is due to treatment of undiagnosed
micrometastatic disease (48).
11
The lymphatic system
The lymphatic vessels
Fluid is constantly formed in the interstitial space between cells. To maintain
tissue homeostasis, small vessels, lymph capillaries or lymphatics, drain
vascularized tissue from extracellular fluid. The absorbed fluid, lymph, travels
through a network of merging vessels that connects to a lymph node. In turn, one
lymph node is connected to a series of other lymph nodes. Accumulating lymph
eventually empty into the thoracic duct that returns to the blood circulation
through vena cava superior (55).
The lymph contains debris secreted by cells, but also antigens from invading
microbes and cells from the immune system e.g. memory T cells, Dendritic cells
(DC) and macrophages. They use the lymphatics to reach the lymph nodes from
the peripheral tissue (55).
The lymph node
The lymphatics enter the lymph node through afferent vessels that penetrate the
surrounding fibrous capsule. Here, in the subcapsular sinus, macrophages reside
for the purpose of recognizing and removing foreign organisms. Underneath lies
the outer cortex where clusters of B lymphocytes form follicles surrounded by
paracortex, which mainly constitutes T lymphocytes. In the center of the follicle
lies follicular DCs. After antigenic stimulation, the B cells start proliferating. Now
the center of the follicle stains lighter in the microscope, and is called a germinal
center, and the structure as a whole has become a secondary follicle (55).
The DC travels to the lymph node from the peripheral tissue upon activation by
antigens. Here, the ingested antigen is presented to T and B lymphocytes. The
lymphocytes access the lymph node through cortical blood vessels named high
endothelial venules (HEV). They are directed towards specific sites in the lymph
node by different chemokines expressed by stromal cells. The antigen receptors
on the lymphocytes are unique for a particular antigen. The binding of the antigen
to the correct receptor can activate the lymphocytes in the presence of the right
co-stimulatory signal and create an antigen specific immune response. This
makes the DC and the lymph node the crucial link between the innate and the
adaptive immunity (55).
Neolymphangiogenesis
A growing tumor needs oxygen. The hypoxic environment makes the tumor cells
secrete angiogenic factors that stimulate the formation of new blood vessels. As
blood plasma filters through the aberrant walls of the newly formed vessels, fluid
12
is accumulating in the interstitial space of the tumor. The increased pressure
partly drives the formation of new lymph vessels (56). In addition, growth factors,
such as vascular endothelial growth factor C/D (VEGFC/D), secreted by tumor
cells, follow the interstitial flow and lead to the proliferation of lymphatic
endothelial cells that lead to new lymph capillaries (57). The increase in lymphatic
vessel density facilitates the transport of migrating cancer cells. In fact, lymphatic
density and expression of VEGFC/D have been associated with tumor
progression (58).
Tumor immunology
The interplay between the immune system and cancer has caught the attention of
scientists since the late 1800s. In 1957 the hypothesis of “immunosurveillance”
was formulated by Burnet and Thomas (59). It stated that neoplastic alteration of
a cell will be detected by the immune system as foreign, and thus be removed
before they grow into tumors. A key player is the cytotoxic T lymphocyte (CTL)
which eradicate cells that express foreign antigens on their major
histocompatibility complex class I. However, when the immune system is
attenuated, as for organ transplanted or HIV/AIDS patients, a higher incidence
of certain types of cancer can appear (60).
In an immunocompetent state, the microenvironment of the tumor is crowded
with immune cells. The quantity of T lymphocytes and macrophages infiltrating
the tumor correlates to prognosis in several cancers (61, 62). For the neoplastic
cells to evade the immune system, they need to modify the expression of proteins
on the cell surface or downregulate the antitumoral response by secretion of
inhibiting cytokines (63).
In the tumor draining lymph node, naïve lymphocytes and macrophages are
activated to mount a tumor specific immune response (64). These lymphocytes
have been utilized for adoptive cell immunotherapy in UBC (65) and in colon
cancer (66). However, there are also many examples of how the tumor establishes
a tumor tolerant environment in the draining node (67). Immune suppression is
achieved by tumor-derived cytokines (68) that augment the number of regulatory
T cells while making cytotoxic T cells non-functional (56). Also, increased
interstitial flow can induce TGF-β that downregulates DCs in the lymph node (56)
and tumor-secreted exosomes that induce tolerance in draining lymph nodes
(69). In malignant melanoma, it has been shown that nodes closest to the primary
tumor were more immunosuppressed, whereas those furthest away lacked
immunological reactivity compared to intermediate nodes (64). This tumor
induced immunomodulation is perhaps a prerequisite for the tumor to colonize
“hostile” lymph nodes.
13
Sentinel node detection
The first draining lymph node of a tumor is unique, and often referred to as the
sentinel node (SN). Since many cancers spread through the lymphatic system, the
identification of the SN can be valuable for both diagnostic and therapeutic
reasons.
A lymphatic mapping (LM) can be performed by one or a combination of
techniques. A radioactive tracer is injected peritumorally so that a signal can
either be detected by a preoperative lymphoscintigraphy and/or by utilizing an
intraoperative handheld Geiger meter (Figure 2)(70). If blue dye is injected
around the tumor, blue lymphatics can be followed to a blue colored SN. The SN
is then excised and sent for pathological assessment (sentinel node biopsy).
Figure 2. Schematic illustration of intraoperative sentinel node detection by a handheld
Geiger meter. Credit: Malin Winerdal.
In malignant melanoma, breast cancer and penile cancer, the sentinel nodes
biopsy is a well-established diagnostic tool for detecting lymphatic metastases
(71, 72). If the SN is free of tumor cells, there is no need to proceed with regional
LND as the SN is said to reflect the status of the whole region of nodes. Thus, the
patient is spared from avoidable surgery and its associated comorbidities.
14
The implementation of SN biopsies in the above-mentioned cancers have
inspired others to investigate its role in other types of cancers. In UBC, the
pioneering work of Sherif et al, detected SNs in 11 of 13 patients (73). Metastases
were found in the SNs of four patients and not in any non-SNs. In another cohort
of 14 patients, two patients were without detectable SN, whereas all seven
patients that were pN+ had metastases in SNs and three in non SNs (74).
To improve detection rates, attempts to fuse the lymphoscintigraphy with single-
photon emission CT and conventional CT visualized 21 SNs in five out of six
patients (70).
In 2006, Liedberg and colleagues performed peritumoral injections with
radioactive tracer and blue dye in 75 patients (75). The first 30 patients
underwent preoperative lymphoscintigraphy, but the method was later
abandoned due to low sensitivity. Only two SNs were identified by blue dye, which
is remarkably low compared to the experiences in breast cancer. 19% of the lymph
node metastases were found in non-SN. The authors proposed that
macrometastases might have obstructed the flow to the true sentinel nodes.
Instead, the lymphatic flow was redirected, and detected as a false negative SN
(75).
Aljabery et al. injected intraoperative radiotracer and blue dye on 103 patients
with MIBC. 80% of the patients had SN detected with a sensitivity and specificity
for finding metastases of 67% and 90% respectively (76).
In a meta-analysis of eight studies (n=336) including the above-mentioned ones,
the pooled rate of detection and sensitivity were 91% and 89% respectively.
Interestingly, subgroup analysis showed reduced sensitivity rates for patients
with many pT3-pT4 tumors. This phenomenon is in line with SN detection in
other forms of cancer (77).
15
Adoptive immunotherapy
The sentinel node biopsy appears incomplete as a diagnostic tool for identifying
lymph node metastases in UBC. Instead, the technique is promising for
harvesting tumor-reactive lymphocytes for adoptive immunotherapy (78). In a
pilot study on 14 patients with UBC, the lymphocytic activity increased in SNs
after stimulation with autologous tumor extract (74). In a follow-up study, 12
patients with metastatic UBC had the lymph nodes that drained their metastatic
nodes, the so-called metinel nodes, extirpated. In vitro, T-cells from the metinel
nodes were cultured and expanded by autologous tumor extract and the
mitogenic signal IL-2 (79). After reinfusion of nine patients with expanded T-
cells, two patients showed objective radiological response with an overall survival
of 35 months and 11 months respectively (65). Adding NAC has demonstrated a
dose-dependent, immunostimulatory effect of T cells in SNs (80), a plausible way
to improve the therapy further.
Blood transfusions
Early attempts to transfuse blood originates from the 1600s. The first successful
transfusion was in 1838 by Dr James Blundel, yet the treatments were risky and
often led to death of the patient. The discovery of blood groups by Karl
Landsteiner led to safer transfusions and a Nobel Prize in the 1930s. With
anticoagulants and refrigeration, the blood could be stored for transfusion at a
later time, which laid the basis for blood banks. In the 1940s, blood fractionation
allowed separation of blood into three main compartments: plasma (55%), buffy
coat consisting of leukocytes and platelets (<1%) and erythrocytes (45%)(81).
Today a unit of donated blood is converted into different blood products. Packed
red blood cells (PBC) can be stored up to 35 days and are used for increasing the
oxygen-carrying capacity of the receiver (Figure 3). Fresh frozen plasma (FFP)
contains plasma proteins including coagulation factors, protein C and S,
fibrinogen and antithrombin. It is used for treating coagulations deficiencies and
for reversal of anticoagulant medication. Platelet transfusion is indicated for
thrombocytopenia after hemorrhage or as a prophylaxis in patients with <10
000/μL (82).
16
Figure 3 Image of bag with packed red cell concentrate (PBC). Credit: ICSident at German WikiWikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=29492562
Blood group systems
The transfusion of donated blood is termed allogeneic blood transfusion. Foreign
blood contains many elements that can be antigenic in the receiver such as
carbohydrate and protein structures on the surface of erythrocytes, thrombocytes
and leukocytes as well as plasma proteins themselves. Antibodies with affinity to
these antigens are called alloantibodies. Matching of blood is important in order
to prevent alloantibodies from attacking the allogeneic blood. Today, there are 36
blood group systems that categorize more than 350 antigens (82).
The first discovered and most important blood group system, AB0, classifies the
erythrocytes on the type of carbohydrate structure that is attached to their cell
surface. An individual with type A will carry anti-B antibodies and vice versa,
whereas an AB individual will carry neither anti-A nor anti-B antibodies. In type
0 individuals, there are neither A nor B structures on their cells, instead both anti-
A and anti B antibodies are present in their blood. In case a type A person would
receive type B blood, the anti-B antibodies in that individual would attack all
foreign erythrocytes carrying the B antigen and vice versa. Similarly, a type 0
could neither receive blood from A, B or AB since that individual carries both anti-
A and anti-B antibodies. Other blood group systems include Rh, Lewis, Kell,
Duffy, Kidd, I and MNS Ralston, Stuart and Penman (83).
The pretransfusion testing consists of determining the AB0 and Rh phenotype by
applying antibodies against A, B and D antigens. The alloantibody screen detects
antibodies against other erythrocyte antigens.
17
Adverse reactions
Despite pretransfusion testing, adverse reactions do occur after allogeneic blood
transfusion. Lethal outcome is rare and accounts for 0,3 cases per 100 000
transfusions. Mild reactions like fever and urticaria are seen in 3%.
Immune-mediated reactions are of different kinds. Acute hemolytic reactions
present with hypotension, tachypnea, tachycardia and fever. The condition
occurs when preformed antibodies destruct donor erythrocytes. A less dramatic
presentation is the delayed hemolytic reaction which arises when allogenic blood
induces the production of alloantibodies detectable 1-2 weeks post transfusion.
Febrile nonhemolytic reactions can appear when antibodies are directed against
donor leukocytes and HLA antigens. The most feared complication is Graft-
versus-host disease. Here, donor T lymphocytes recognize host HLA antigens as
alien and attack the host. Another, mostly fatal, complication is transfusion-
related acute lung injury (TRALI). Donor antibodies aggregate with host
leukocytes in the blood vessels of the lungs leading to increased capillary
permeability and pulmonary edema (82).
Infectious complications are rare. Blood donations are tested for HIV, hepatitis B
and C. Cytomegalovirus can be transmitted through infected donor leukocytes.
Receivers at risk such as immunosuppressed patients and neonates should
receive leukocyte-depleted blood products (83).
Anemia in the cancer patient
In 2005, the European Cancer Anaemia Survey (ECAS) enrolled more than 15
000 patients with solid and hematological malignancies of various stages and
treatments. The Patients were followed for up to 6 months. The prevalence of
anemia at enrollment was 39.3% (Hb < 12.0 g/dL) and 67.0% of the patients were
anemic at least once during the survey. Anemia was often seen after
chemotherapy, and increased with the number of cycles given (84).
In a study of MIBC, 53.3% of patients were anemic prior to RC, and NAC was an
independent predictor for preoperative anemia (85).
18
There can be many causes of anemia in cancer patients of which some are listed
below (86):
1. Anemia of chronic disease/inflammation ACD/AI.
2. Tumor bleeding (eg GI bleeding, hematuria).
3. Malnutrition (deficiency in iron, folate, B12)
4. Renal failure with defective erythropoietin production.
5. Bone marrow toxicity from chemo/radiotherapy
6. Bone marrow metastases
7. Autoimmune hemolytic anemia
In solid tumors, the most common etiology of anemia is that of chronic
disease/inflammation. In similarity with other chronic inflammatory conditions,
cancer can cause an increased activation of macrophages and T cells. These
immunocytes secrete cytokines like TNFα, IFN-γ and IL-1 that suppress the
erythropoiesis in the bone marrow and interfere with iron utilization. In addition,
the cytokines induce hepcidin production in the liver. This polypeptide hormone
blocks ferroportin in the enterocytes, so that iron is retained in the enterocytes
establishing a state of sequestrated anemia. Thus, it might be challenging to
differentiate ACD/AI from iron deficiency. High doses of intravenous iron have
been proposed as means to evade the hepcidin barricade (86).
Regardless of the etiology, the high prevalence of anemia in cancer patients make
them predisposed to receive allogeneic blood transfusion during the course of the
disease (87). Apart from the above described adverse reactions, there is evidence
that blood transfusions can affect long term oncological outcome; an effect known
as transfusion related immunomodulation (TRIM).
Transfusion related immunomodulation
In 1973 Opelz et al observed improved renal-allograft survival in patients that
received allogeneic blood (88). This beneficial immunomodulatory effect, later
referred to as transfusion related immunomodulation (TRIM) became utilized
by renal transplant units to increase graft tolerance. However, in 1981, concerns
were raised whether the immunosuppressive effect of blood could lead to tumor
progression in cancer patients (89). In the following years, many observational
studies saw an association between blood transfusions and increased cancer
recurrence, and also increased risk for postoperative bacterial infections (90). In
19
addition, a number of RCTs revealed increased mortality in patients receiving
leucocyte containing vs leukocyte depleted blood products in cardiac surgery (91).
Hence, donor leukocytes were suggested as an important mechanism in TRIM,
partly through the release of cytokines and inflammatory lipids (92).
In the late 1990s, blood products were leukocyte reduced as a routine in many
western countries. Febrile transfusion reactions and CMV transmissions
decreased. In vitro studies observed reduction in the proinflammatory cytokine
IL-1β, compared to non-leukocyte reduced blood (93), whereas levels of IL-6, IL-
10 and TNFα were repeatedly high (93, 94). Yet, both leukoreduced and non-
leukoreduced blood increase the level of T regulatory cells (95).
Prolonged blood storage has been linked to tumor growth in animal models.
Release of degraded lipid membrane-derived factors and raised tissue hypoxia
are plausible tumor promoting factors (96). In addition, storage of blood leads to
the deterioration and apoptosis of blood cells. Leukocytes and platelets release
the immunosuppressive TGF-β. Apoptotic cells present phosphatidylserine on
their surface, which attracts phagocytic cells like macrophages, that in turn
triggers release of TGF-β (97).
Moreover, the disruption of erythrocytes leads to the release of hemoglobin and
non-heme-bound iron. The cell-free hemoglobin binds nitric oxide, resulting in
vasoconstriction, hypertension, and inflammation. These factors have been
attributed to the increase in multi-organ failure and mortality seen after massive
transfusions (92).
Lastly, microparticles can have immunomodulatory effects. These extracellular
vesicles (EV) contain lipids, proteins and miRNA. They are constantly discarded
from all cells, a process that is exacerbated by the handling of blood ex vivo. The
effect of EVs on the immune system depends on many factors: cell origin of EV,
different methods of manipulation of blood products, storage time etc. A myriad
of effects on the immune system has been observed including neutrophil priming,
transfer of miRNA, expression of CD40L and the stimulation of T cells (98).
TRIM and clinical outcome
In 1981, Gantt et al raised the question whether blood transfusion could have a
negative impact on clinical outcome in cancer patients (89). Since then, a number
of studies, predominately of retrospective design, have addressed this specific
issue. Today, there are meta-analyses encompassing 5000-25,000 patients who
have investigated the impact of perioperative blood transfusion for patients
undergoing surgery for colorectal cancer (99), lung cancer (100), urinary bladder
cancer (101), and prostate cancer (102). They all show an increased risk of
mortality associated with allogeneic blood transfusion.
20
Whether autologous blood is safer is not completely clear. Intraoperative blood
salvage or cell salvage, is a medical procedure that allows reinfusion of blood
during surgery. It was speculated that this form of autotransfusion could
reintroduce malignant cells from the surgical removal of the primary tumor (103).
In 1993, 475 patients were randomized between autologous and allogeneic blood
transfusions in patients undergoing surgery for colorectal cancer. The risk of
recurrence increased after transfusions of both autologous and allogeneic blood
(104). In contrast, data from surgery of hepatocarcinoma concluded that
autologous blood transfusion does not increase recurrence of disease (105).
21
AIM OF THE THESIS
The aim of this thesis was to elucidate different clinical aspects of neoadjuvant
chemotherapy (NAC) on muscle-invasive bladder cancer (MIBC).
Paper I. 1. To clarify the role of tumor downstaging as a surrogate marker for
survival after NAC in MIBC.
Paper II. 2 To assess the feasibility of sentinel node detection in MIBC after NAC
and/or after completely downstaged (pT0) tumors post-NAC.
Paper III. 3. To assess the number of sentinel nodes in radical cystectomy for
MIBC as a prognostic marker in NAC-treated patients.
Paper IV. 4. To evaluate the extent of and the effects of blood transfusions
during NAC on pathoanatomical outcome and overall survival in patients with
MIBC undergoing radical cystectomy (RC) with curative intention (cT2-
T4aN0M0).
22
PATIENTS AND METHODS
Paper I
Cohort NCS1
In 1985-1989 the Nordic Cooperative Bladder Cancer Study Group included 325
patients from Sweden, Norway and Finland, who were randomized between +/-
NAC and standard care (low dose irradiation and RC) or standard care alone.
Patients with urothelial, squamous cell, and undifferentiated carcinoma of the
bladder, T1G3NxM0 and T2-T4aNxM0 were included. Staging included
cystoscopy, bimanual palpation, iv pyelography, and chest radiography. CT was
not obligatory. All patients received 4 Gy daily for five consecutive days targeting
the bladder, and lymph nodes in the iliac and obturator region. The
chemotherapy consisted of 2 cycles of 70mg/m2 cisplatin and 30mg/m2
doxorubicin. 311 patients were eligible of which 151 in the experimental arm and
160 in the control. 21 and 26 patients respectively were not cystectomized. In the
experimental arm, ten patients received no chemotherapy, whereas eight patients
received one course. 58 patients had T1 disease. At cystectomy, only enlarged
lymph nodes in the iliac and obturator regions were removed
Cohort NCS2
317 patients with T2-T4NXM0 were recruited between 1991-1997 from Sweden,
Norway and Finland. Squamous and adenocarcinoma of the bladder were
excluded. Staging encompassed TURB, bimanual palpation, and iv pyelography.
No preoperative radiation was used. The chemotherapy regimen comprised three
cycles of cisplatin 100mg/m2 and methotrexate 250mg/m2 given with a three-
week interval. 309 patients were eligible, 155 in the experimental arm and 154 in
the control. 14 patients received no chemotherapy, nine received one course cycle,
and 14 received two cycles. 23 patients were not cystectomized compared to 15 in
the control. Cystectomy included dissection of obturator and iliac nodes.
Method and statistics
The aim of the analysis was to compare clinical data and pathologic outcome in
patients with urinary bladder cancer cT2-T4aN0M0 from NCS1&2. Data was
retrieved from the Regional Oncologic Centre in Uppsala/Örebo (Regionalt
Onkologiskt Centrum Uppsala/Örebro region).
23
From the above described 311 eligible patients in NCS1, all 53 patients with T1
tumors, 5 patients with missing cT-stage, and 50 patients with missing pNx and
pTx were excluded, resulting in 203 patients for further analysis. From the 309
eligible patients in the NCS 2, one patient with cTx, and 62 patients with pNx/pTx
were excluded, resulting in 246 patients. A total of 449 patients remained for a
joint analysis. The experimental arms of NCS1&2 were grouped together (n=225)
and so were the control arms (n= 224).
The primary endpoint was the rate of pathologic downstaging, i.e. a pathologic
TNM lower than the clinical TNM. The secondary endpoint was overall survival,
measured from date of randomization to death or last follow-up. SPSS 19 was
used for statistical analyses. Chi-squared tests compared categorical data.
Differences in survival were plotted in Kaplan-Meier curves, and tested by log-
rank. A Cox-regression model was used to assess factors of importance for overall
survival. P≤ 0.05 was deemed significant.
Paper II and III
Patients
In 2013, a prospective non-randomized, multicenter trial was initiated at the
Urological Department, University Hospital of Umeå (Norrlands
universitetssjukhus) and the Department of Surgical and Perioperative
Sciences, Division of Urology and Andrology, Umeå University in cooperation
with the Department of Medicine, Unit of Translational Immunology,
Karolinska Institutet, Stockholm. Patients with suspected muscle-invasive
urinary bladder cancer were recruited from up to 10 different urological
centers in Sweden. Blood, urine, and specimens from macroscopically healthy
bladder and bladder tumors were collected at the time of the first TURB.
Patients were excluded if histopathology showed non-muscle invasive
urothelial bladder cancer, primary bladder cancer of non-urothelial origin,
benign tissue, patients unfit for radical cystectomy, logistical problems,
incurable cancer, robot-assisted cystectomy or prior BCG treatment. Based on
clinical routines at the recruiting center, patients were either selected for
neoadjuvant chemotherapy plus radical cystectomy, or radical cystectomy
alone.
Method and statistics
Sentinel node detection was performed on the day of cystectomy prior to
surgery. 1 ml of 80 MBq Technetium was deposited intravesically by
cystoscopy and a Williams injection needle (Cook Medical®), at four positions
around the primary tumor or around the resection scar in case of visual pT0.
24
The patient then proceeded for cystectomy in the same surgical session. After
the bladder specimen was removed, the lymph node dissection was
performed. All dissected lymph nodes were registered and probed by a Geiger
meter. Radioactivity was quantified in counts per minute (CPM).
Two definitions of SN were used: SN definition 1, (SNdef1): a lymph node of
10 CPM. SN definition 2 (SNdef2), the lymph node with the highest CPM (the
hottest) and with CPM 10% of the hottest node in each patient.
Paper II
For the analysis presented in paper II, a total of 99 patients were recruited
between May 2013 and December 2015 from six different centers. 34 were
excluded due any of the above-mentioned reasons of which 18 was due to
NIMBC. 65 patients remained for cystectomy and SNd of which 47 patients
were fit for NAC.
Paper III
The cohort of paper II was expanded. 230 patients with suspected MIBC were
recruited between May 2013 and December 2018 from 10 different centers.
114 were excluded of which the majority (n=56) was due to NIMBC. 116
patients were enrolled for RC and SND of which 83 patients were fit for NAC.
Only SNdef1 was used in the analysis.
Statistics
SPSS 23 was used for statistics in paper II and SPSS 25/26 for paper III. The
mean and median rates of true SN and false positive SN were compared using one
way ANOVA. Categorical variables were tested with chi-squared tests. A general
linear model (ANCOVA) was used for testing clinical factors (independent
variables) of value for SN outcome (dependent variable). In paper II, independent
variables were first tested univariately. Variables with a p-value <0.05 were then
tested together in a multivariate general linear mode. In paper III four clinical
variables were tested directly in a multivariate analysis (ANCOVA).
25
Paper IV
Patients, methods and statistics
All patients undergoing radical cystectomy for UBC in 2008-2014 were retrieved
from four urological centers in Sweden: the University Hospital of Umeå,
Sundsvall-Härnösand County Hospital, Gävle County Hospital and Västmanland
County Hospital (n= 372). Patients with muscle-invasive urothelial urinary
bladder cancer (cT2-T4aN0M0) that underwent NAC and RC (n= 120) were
included for further analysis. Medical records, including charts of red cell
concentrates administered from time of diagnosis until discharge from the
hospital after the cystectomy were collected. The patients were divided into two
groups: blood yes that received blood transfusions during NAC and blood no that
were blood naïve during NAC. Clinicopathological factors were tested using t-test
and Chi-square, whereas survival rates between the groups were compared using
Kaplan-Meier and Log Rank test. IBM SPSS statistics version 24 and 26 were
used.
26
RESULTS AND DISCUSSION
Tumor downstaging after neoadjuvant chemotherapy (Paper
I)
From the original 620 patients in NCS 1 & 2, 449 patients were eligible for the
retrospective analysis. The number of patients remained balanced between the
groups with 225 patients in the experimental arm, and 224 patients in the control.
The aim of the analysis was to compare the rate of tumor downstaging between
the arms, and to see if these findings were associated with survival benefits.
The notion that preoperative chemotherapy could impact pathologic outcome in
MIBC is not new. In 1988, a phase I trial on neoadjuvant chemotherapy on T2-4
tumors, showed that 33% of the cystectomized patients downstaged to pT0 (106).
Since then, numerous trials have presented pathological response rates between
14-38% after cisplatin based neoadjuvant chemotherapy (107). One of the more
recent and largest of these trials, the Intergroup 0080, randomized 307 eligible
patients between neoadjuvant MVAC+ RC versus RC alone. 38% of the patients
downstaged to pT0 in the NAC arm and 15% in the control (108). In a multicenter,
retrospective, real-world assessment of pathologic response rates, 22.7% were
pT0N0 after at least three cycles of NAC (109). In the same study, there were no
difference in downstaging rates between MVAC and GC.
Figure 4. Percentage of complete downstaging tumors (pT0N0) stratified by treatment arm and
clinical stage
27
In the present analysis, the rate of patients with completely downstaged tumors
(pT0N0) was almost doubled (22.7%) in the arm that received preoperative
chemotherapy compared to the controls (12.5%) (p=0.006). In patients with cT3
tumors, the difference was almost three-fold (Figure 4). In patients that
downstaged to a non-invasive state or less (pT0/pTis/pTaN0), the same pattern
emerged (not shown).
In survival analysis, patients were stratified by treatment arms and downstaging
group: 88.2% of the patients with complete downstaging tumors after NAC were
alive after 5 years. This translated into an absolute risk reduction of 31.1%
compared to downstaging tumors in the control arm (p=0.001). Furthermore, the
latter group retained no survival benefits compared to patients with non-
downstaging tumors in the controls (p= 0.550) nor in the experimental arm
(p=0.612) (Figure 5).
Figure 5. Kaplan-Meier curve plotting overall survival stratified by treatment arms and
pathoanatomical outcome. Downstaging tumor after NAC (blue). Downstaging tumor without NAC
(green). Non-downstaging tumor after NAC (yellow). Non-downstaging tumor without NAC (purple
28
In the multivariate analysis, the combination of downstaging and NAC was an
independent positive prognostic factor when downstaging was defined as pT0N0
(HR= 0.26), <pT1 (HR= 0.43) and organ confined disease (HR = 0.42). In the
original paper of 2012 we postulated that the increased downstaging of the
primary tumor after chemotherapy was translated into a survival benefit through
its efficacy on micrometastatic deposits. In line with this argument, we saw how
the discrepancy in downstaging and survival between the treatment arms was
most prominent for clinical T3 tumors (ARR=40.5%, p=0.023). In an autopsy
study of UBC it was clearly displayed how the frequency of metastases increased
with the increment in pT stage (110). Thus, it is very likely that locally, more
advanced tumors (i.e. cT3) would yield a higher rate of dissemination, and
thereby benefit more from systemic treatment compared to clinical T2 tumors.
Interestingly, our results deviate from Intergroup 0080 where pT0 patients in
both arms experienced an 85% survival rate at five years (108). In 2012, when our
results were published, we argued that one of the reasons for these discrepancies
could be that NCS1&2 represented a more homogenous group of patients derived
from a smaller recruitment area of 41 hospitals in three Nordic countries. In
contrast, patients in the Intergroup 0080 were enrolled over an 11-year period
and came from 126 institutions.
Moreover, there were different chemotherapeutic regimens. The Intergroup
0080 used 3 cycles of MVAC compared to two cycles of cisplatin/doxorubicin in
NCS1 and 3 cycles of cisplatin/methotrexate in NCS2. Yet, this cannot explain the
low survival rate of pT0 in the control arm of our analysis.
Plausibly, differences in preoperative staging could have a role here. In the
NCS1&2, patients were staged T1-T4aNxM0 preoperatively. CT of the bladder was
optional. In NCS1 only enlarged lymph nodes in the iliac and obturator regions
were removed, whereas dissection of obturator and iliac nodes were performed
in NCS2. In contrast, patients in the Intergroup 0080 were staged T2-4aN0M0.
Yet, how the preoperative staging was accomplished, was not accounted for in the
article, and the LND included bilateral pelvic lymph nodes (108).
This leaves us with the possibility that patients from the NCS cohort might
comprise unidentified lymph node metastases to a higher degree than in the
Intergroup 0080. In that case, patients with pT0 in the control group of NCS1
and to a certain degree in the NCS2 might encounter a worse survival due to
undiagnosed and untreated lymph node metastases. Conversely, undiagnosed
lymph metastases in the pT0 of the experimental arm would have benefited from
systemic treatment.
29
In the background section of this thesis, I reasoned that the clinical gain from
LND observed in retrospective studies is foremost an effect of improved
diagnostic staging in chemonaïve patients. It is plausible that many pT0 in NCS1
consisted of undiagnosed pN+. In that case, it is conceivable that NAC
compensates for potentially undiagnosed pN+ in NCS1, resulting in a larger
survival difference between downstaging patients in the experimental arm versus
the control.
Sentinel node detection after neoadjuvant chemotherapy
(Paper II)
65 patients were eligible for the intended analysis. 47 patients received NAC prior
to sentinel node detection (SNd) and RC and 18 patients underwent SNd and RC
alone. In accordance with the aim of the study, to assess the feasibility of SNd
after NAC and/or pT0, the cohort was divided into three groups: Group 1,
downstaging patients after NAC (n=24). Group 2, NAC patients without
downstaging (n=23). Group 3, No NAC patients (n=18).
The rate of downstaging tumors among the NAC treated was 51.1% compared to
the noNAC group 11.1%. This is congruence with reports using modern regimen
of chemotherapy (108). Patients in group 3 were older, and consisted of 50%
women compared to 20.8% and 17.4% in group 1 and 2 respectively. The mean
number of harvested lymph nodes were similar between the groups with an
average of 16.4. The total number of detected SNs were 222 according to SNdef1
and 227 according to SNdef2. The average number of SNs were 3.42 and 3.49
respectively, and when the average number was compared between the groups,
there was no statistical difference between groups for neither definition (p= 0.544
and p= 0.762). Radioactive specimen above the cut-off value, where the pathology
report showed no lymphatic tissue was labeled false positive (FP). The average FP
nodes were 0.51 and 0.46 for the two definitions, and no statistical difference was
recorded between the groups (p= 0.975, p=0.976).
30
Figure 6. Schematic illustration of detected sentinel nodes according to definition 1. The nodes were
scattered in the pelvic region in a similar fashion in the patient groups 1,2 and 3. Red arrows pointing
at red nodes indicate lymph node metastases.
In the whole cohort, only 8 patients (12.3%) had metastatic lymph nodes (n=22).
The majority of lymph node metastases (n=18) were found in non-SNs,
equivalent to a false negative rate (FNR) of 82%. Three patients had metastases
in SNs (one from group 2 and two from group 3) (Figure 6). One patient (group
3), had metastases in both SN and non-SN. In the remaining five patients (group
2), all metastases were found in non-SN.
For comparison, the studies on SNd in chemonaïve UBC patients previously
accounted for, consisted of a higher rate of metastatic lymph nodes (30-43%) but
with a lower rate of false negative SNs (0-20%) (75, 76).
When sentinel node biopsy was introduced in breast cancer the false negative
rates were around 5% or below (111). The implementation of the technique in the
broader surgical community has resulted in rates between 8-11% (111). The effect
of NAC on SN biopsies in breast cancer was systematically investigated in the
SENTINA trial. Here, more than 2000 patients were included in a prospective,
multicenter study (112). In the arm of chemonaïve patients, the detection rate was
99.1% whereas patients that first underwent NAC had a detection rate of 80.1%
and a false negative rate of 14.2% (112). In one arm, patients underwent SN
biopsies twice, before and after NAC. Less than two –thirds of the SN were
detected after NAC, and the false-negative rate was 51.6%. The number of the
lymph node metastases did not impact the detection rates, which made the
authors suggest that factors like fibrosis, and blockage of lymphatic flow were the
main factors to impair SNd after NAC (112).
The SENTINA trial is contrasted by the ACOSOG trial. Here 649 patients with
lymph node metastatic breast cancer received chemotherapy prior to SN biopsies
31
and axillary LND. The primary endpoint was FNR, which was 12.6% for the whole
cohort (113).
Despite solid data pointing at impaired SNd after NAC in breast cancer, the
present study showed no statistical difference in the number of detected SN
between the groups with an average of 3.42 per patient. In multivariate analysis,
the most important factor for SN yield was the number of harvested nodes. In
contrast, the Liedberg cohort of mainly chemonaïve patients, had a mean number
of 2.4 SNs, whereas the mean number of dissected nodes were 40 per patient (75).
The combination of low number of harvested lymph nodes and usage of NAC
might explain the low rate of detected lymph node metastasis in the present
cohort compared to Liedberg and others (75, 76). Likewise, the high rate of false
negative nodes could be attributed to NAC. It is tempting to speculate that
patients with lymph node metastases after NAC represent a distilled subgroup of
patients with more advanced disease resistant to chemotherapy. Presumably,
these patients retain chemoresistant tumor cells that obstruct and redirect the
lymphatic flow.
Nevertheless, the high false negative rate of SNs after NAC in breast cancer is
more concerning than in UBC. In breast cancer, SNB is a diagnostic tool. A missed
lymph node metastasis would lead to understaging and undertreatment due to
lack of axillary node dissection in that particular patient. In UBC, however, SNB
is not sufficient to detect all lymph node metastasis even in chemo naïve patients.
Rather, we believe the technique is valuable for identifying tumor reactive
immunocytes suitable for adoptive immunotherapy.
32
Fewer tumor draining sentinel nodes in patients with
progressing muscle invasive bladder cancer, after neoadjuvant
chemotherapy and radical cystectomy (paper III)
In paper II we investigated the feasibility of performing SNd in NAC treated
patients with MIBC. In paper III, we expanded the cohort to see if the number of
SNs were associated with pathoanatomical outcome. 230 patients were recruited
retrospectively, of which 116 were eligible for analysis. 83 patients received NAC
and were stratified by pathoanatomical outcome into Complete Responders
(pT0N0M0), Stable Disease (pTisT4aN0M0) and Progressive Disease
(pTanyN+M+). 33 NAC-naive patients were designated as no-NAC, and kept for
comparison (Figure 7).
Figure 7. Flow chart of patient inclusions in paper III. Patients were stratified into different groups
based on treatment (NAC vs noNAC) and response to NAC (CR, SD and PD).
There was no statistical difference in the distribution of age, gender or amount of
NAC between the subgroups. However, there were less advanced clinically staged
tumors in the subgroup of complete responders compared to the other subgroups
(p= 0.04) preoperatively.
The proportion of complete responders (CR) was 43.4% which is in line with
others (107, 108).
The mean number of harvested lymph nodes were 17.1, 16.3 and 13.5 in CR, stable
disease (SD) and progressive disease (PD) respectively (p=0.5). Interestingly, the
33
mean number of SNs was less than half in PD (1.4) compared to the two other
groups (p=0.034). Furthermore, the detection rate of SN, i.e. a true radioactive
lymph node, was found in 91.7% of the patients in CR compared to 58.3% in PD.
The mean number of false positive nodes were similar between the groups
(p=0.36). However, in multivariate analysis the most important factor for SN
yield was the number of harvested nodes (p=0.0004).
Could the reduced number of SN in the more advanced and plausibly
chemoresistant tumors reflect a less functioning lymphatic system? On the one
hand, tumors secrete factors that stimulate lymphangiogenesis (58). In fact,
lymph vessel density has been coupled to more advanced disease (56). Yet, as
tumors progress, there is a more extensive shedding of tumor cells into the
lymphatics, ultimately leading to the establishment of macrometastatic colonies
in the regional lymph nodes (110). The sensitivity of finding metastases in SNs is
reduced in more advanced bladder tumors (77). Conceivably, this is due to
obstructed lymphatic flow as was proposed by others (75) and most probably
observed in paper II, where the majority of metastatic lymph nodes were found
in non-SNs (114).
In addition, tumor lymphatics are oftentimes dysfunctional (115). Aberrant
lymph vessels seem to shield the tumor from attacking immune cells so that it can
maintain a tumor protective microenvironment (116). Hereby, we hypothesize
that the number of SNs detected could be an indicator of tumor lymphatic
function, and in a broader sense an indirect marker of an individual anatomical
lymphatic and tumor immunological constitution of each patient.
The association between the SN number and tumor stage is intriguing but should
be interpreted with caution for several reasons:
1) The analysis was performed retrospectively, based on post-hoc defined patient
groups. 2) The cohort of 116 patients was recruited from 10 urological centers,
where the LND was defined yet never controlled for. 3) The time between
peritumoral injections of the radioactive tracer and the SN detection depend on
many factors. In theory, logistics, surgical experience, anesthetic problems,
patient body compositions and local tumor growth might have influenced
operation time and thus delayed SNd, permitting the tracer to recede. 4) A larger
primary tumor would imply a technically more difficult injection of the tracer. 5)
The registration of the radioactivity with the handheld Geiger meter comes with
a certain degree of unpredictability. The reading can be influenced by the position
of the Geiger meter in relation to the tissue etc.
Despite the abovementioned caveats, it is a noteworthy observation that
aggressive tumors are associated with fewer sentinel nodes in our material.
34
Blood transfusion during neoadjuvant chemotherapy for
muscle-invasive urinary bladder cancer may have a negative
impact on overall survival (Paper IV)
The aim of paper IV was to map the extent of blood transfusion during
neoadjuvant chemotherapy, and to see if patients that received blood were
associated with a worse pathological and clinical outcome.
The definitive cohort consisted of 120 patients with curable muscle-invasive
urothelial bladder cancer (T2-T4aN0M0) that underwent 1-4 cycles of
neoadjuvant chemotherapy and radical cystectomy. One-third of the cohort
(n=40) were given red cell concentrate (RBC) during the course of chemotherapy
and were referred to as blood yes. The remaining 80 patients were blood naïve
from diagnosis until surgery, and were referred to as blood no. Interestingly, the
majority of patients in the whole cohort (87.5%) received perioperative blood
(PBT) and there was no statistical difference in perioperative transfusion between
the groups (p=0.079).
The two groups were similar in age, comorbidity, no of NAC-cycles and cTNM,
albeit the proportion of cT4a tumors were more than doubled in Blood yes
(p=0.314). Other differences included the percentage of women that were more
than doubled in Blood yes (p=0.033) and the hemoglobin level at time of
diagnosis that was significantly lower in this group (p=0.0001).
There was a pronounced difference in pathological outcome. Patients in the Blood
yes group had proportionally half as many pT0 tumors but more than ten times
the proportion of pT4b tumors (p=0.044). Similarly, the percentage of patients
with lymph node metastases or unknown lymph node status (pNx) were
significantly higher in the Blood yes group (p=0.007).
Survival curves revealed a 19,2% absolute risk increase at five years of
observation, for patients in the Blood yes (Figure 8). In multivariate analysis,
advanced pT stage (p<0.0001) and pNx (p=0.002) were independent markers for
unfavorable prognosis.
35
Figure 8. Kaplan Meier curve of overall survival stratified by blood transfusion status during NAC.
No blood transfusions (blue) and blood transfused (red).
This is the first time that the extent and the plausible implications of blood
transfusions during neoadjuvant chemotherapy in MIBC are investigated. As
reviewed in the introduction of this thesis, the immunomodulatory effect of
allogeneic blood has been recognized since the 1970s. This phenomenon, also
known as transfusion related immunomodulation (TRIM), has been assessed
both on a molecular as well as a clinical level.
In colorectal cancer, a Cochrane review of 36 studies including seven RCTs,
pooled data revealed an OR of 1.42 for cancer recurrence after PBT. In 14 of the
studies, PBT was an independent risk factor for recurrence (117).
In UBC, the most recent meta-analysis by Cata et al in 2016, analyzed eight
retrospective studies with more than 15 000 patients. Patients transfused within
one month before or after radical cystectomy, were associated with a 27%
reduction in overall survival (101). Similarly, large meta-analyses from colon
cancer (99), lung cancer (100), and prostate cancer (102) have shown negative
oncological outcomes associated with allogeneic blood transfusions.
36
In the current analysis, there were no statistical differences in age, clinical tumor
stage, comorbidity or amount of chemotherapy prior to surgery between the
groups. However, the fact that one third of the cohort was transfused
preoperatively may signal a more advanced tumor stage underlying these
patients, than was demonstrated clinically and could be distinguished
statistically.
The similarity in age distribution is perhaps not surprising considering the fact
that the age-limit is 75 years to receive neoadjuvant chemotherapy. More
importantly, the percentage of women was significantly higher in the Blood yes.
As discussed in the introduction of this thesis, women with UBC tend to have
worse prognosis than men. Although the exact mechanism is not understood, this
might have contributed to the difference in clinical outcome.
In the multivariate analysis of the present study, advanced pTstage (>pT2) and
pNx were independent risk factors for overall survival. The fact that pNx and not
pN+ turned out as a significant risk factor warrants alertness. In the 12 patients
where lymph node dissections were lacking, eight belonged to the blood yes
group, i.e. 20% of the participants compared to 5% in the blood no group. The
reasons were local spread (n=2), extensive fibrosis (n=5) and unknown (n=5). We
interpreted the pNx as a surrogate marker for regional spread of disease.
The core question remains, is the distinct difference in pathological outcome a
consequence of the administered blood and its proposed TRIM effects or rather
an example of confounding by skewed inclusion?
Plausbibly, the immunosuppressive effect of the allogeneic blood might have
prevented full treatment effect of NAC. Apart from the direct toxic effect of the
chemotherapy on the tumor, cell death triggers release of intracellular danger
signals that alert the immune system and elicits an antitumoral response. In this
way, an immunologically “cold” tumor, i.e. without infiltrating lymofocytes, can
become “hot” after chemotherapy, surgery or radiation (118).
Beside immunosuppression, there are other factors that could promote
tumorigenesis after blood transfusion. As accounted for previously,
microparticles (MP) are constantly shed into the bloodstream (119), and the
formation is accentuated when blood is processed ex-vivo. For instance, platelet
derived microparticles (PMP), can contain growth factors like VEGF and PDGF
that fuel blood vessel and lymph vessel formation. In experimental models the
exposure to PMP resulted in tumor growth in lung, prostate and breast cancer
(92).
37
Despite interesting mechanistic concepts on the tumor enhancing effect of
allogeneic blood, the causality, i.e. the order in which blood affects tumor
outcome, and not the reverse is only speculative. Retrospective analyses can be
confounded by many factors including nutritional and functional status of the
patient, tumor stage, tumor manipulation and resectability, method of
anesthesia, use of opioids, hyperglycemia and hyperthermia (96).
The results of this small, retrospective study with a limited follow up time should
be interpreted with caution. The detrimental effect of blood transfusion shown by
others is partly blurred in the current study by the large number of patients that
received PBT in both groups: 83% in the blood no and 95% in the NAC blood yes.
These transfusion rates are extraordinarily high compared to the average
transfusions rates (38%) in other studies (101). Still, in this small cohort we study
a relatively fit and selected group of patients who all qualified for chemotherapy
prior to surgery. The results are merely an observation, yet a rather interesting
one that we believe deserves attention.
38
CONCLUSION AND PERSPECTIVES
Localized muscle-invasive bladder cancer is a lethal condition. After decades of
randomized trials, the uro-oncologic community has appreciated the use of
neoadjuvant cisplatin based chemotherapy to improve survival after radical
cystectomy. The aim of this thesis has been to elaborate on a number of aspects
related to the use of chemotherapy in the neoadjuvant setting.
First, we observed distinct pathoanatomical differences after preoperative
chemotherapy. Tumor downstaging was far more prevalent, and appeared to be
a marker for responsiveness to chemotherapy and long-term survival. Yet, the
question lingers: how do we make use of this finding in our daily clinical practice?
Early attempts to perform post chemotherapy assessment with TURB or imaging
techniques, understaged patients in 38% of the cases (106). Post-treatment
inflammation and fibrosis made prediction of complete pathological response
difficult to discern. Thus, cystectomy has remained gold standard despite
preoperative indications of response to chemotherapy. However, imaging
techniques are becoming more sophisticated. A dynamic contrast enhanced MRI
(DCE MRI) appears promising in differentiating the post-treatment effect from
residual tumor mass (120). Moreover, complete downstaging has been proposed
as a valuable marker for accelerated drug development (121) and useful in real-
world analysis to compare GC and MVAC efficacy (109).
Next, we explored the concept of tumor draining lymph nodes. Urinary bladder
cancer metastasizes to the lymphatic system at an early point. Yet, attempts to
use sentinel node detection for identifying lymph node metastases, have revealed
inconsistencies. Instead, sentinel nodes in UBC contain reactive immunocytes
useful for adoptive immunotherapy. The immunostimulatory effect of
chemotherapy makes the combination of NAC and adoptive immunotherapy
particularly appealing. In paper II we demonstrated that the number of detected
SNs was not affected by NAC nor tumor downstage, albeit an increase in false
negative rates were observed.
Hereafter, the cohort from paper II was enlarged. Interestingly, a reduced
number of SNs was observed in patients with disease progression. The finding
left us speculating: perhaps the number of SNs could be a feasible way to
determine lymphatic function? Could an intact lymphatic system, i.e. open
communication between the primary tumor and draining lymph nodes, be a good
therapeutic marker for immunomodulating drugs, such as checkpoint inhibitors?
39
Lastly, we wanted to estimate the impact of blood transfusions during
neoadjuvant chemotherapy. Anemia is common in the cancer patient and can
have many etiologies. Despite several treatment options like iron substitution and
erythropoiesis stimulating agents, clinicians often choose allogeneic blood
transfusions to treat anemia. It seems like many medical doctors are not aware of
the accumulating data that have linked blood transfusions with unfavorable long-
term outcomes after cancer surgery.
In the fourth study of this thesis, we observed more advanced tumors and worse
overall survival in patients that underwent blood transfusions during the
neoadjuvant chemotherapy. Although one must be careful with the conclusions
from this small study, the accumulating data on this topic proclaims a more
thoughtful approach to anemia and its management in the cancer patient.
40
ACKNOWLEDGEMENTS
This thesis is the result of many hard-working people including clinicians,
biologists, statisticians, teachers and staff members from laboratories, hospitals
and academic centres. I would like to extend my gratitude to a few persons in
particular:
Amir Sherif, my principal supervisor. After we met for the first time, 12 years ago,
my life took a new direction. You introduced me to bladder cancer as a research
field and urology as a clinical profession. Your working stamina is immense. Your
energy can charge my mental batteries again and again. If not for you, this or
something alike would never have happened.
Ola Winqvist, my co-supervisor. I am jealous of your intellect. When you talk on
tumor immunology, nothing feels more important. Thank you for all the valuable
feedback I received throughout the years.
Per Marits, my co-supervisor. It has been a privilege to have your support.
Börje Ljungberg for his assistance with my registration as PhD-student at Umeå
university.
Christina Ljungberg and the staff of the Department of Surgical and Perioperative
sciences, Division of Urology and Andrology.
Per-Uno Malmström. The support from someone senior and still active in bladder
cancer research means a lot.
The Nordic Cooperative Bladder Cancer Study Group, for granting me access to
invaluable data.
Julia Alvaes, co-author on paper III. Although we never met in person, it was a
pleasure to work with you. I am grateful for your many thoughtful insights.
Gabriella Lorentzi, co-author on paper IV. Only a few times have we met in
person, yet we had many intense and fruitful moments in the ether. Thank you!
David Krantz, one of my dearest friends with whom I share so many memories.
We have pushed and pulled each other, in and out from childhood, high school,
travels, medical school and research. Thank you for seeing the world differently,
it is both nurturing and challenging.
41
Malin Winerdal, former co-worker in the KI lab with deep knowledge on
immunology. Thank you for sharing your fine illustrations.
Co-workers in the KI lab. You delivered breakthrough research and kept a good
spirit despite long working hours and noisy machines: Thank you Ali Zirakzadeh,
Ciptura Adijaya Hartana, Michael Mints and Emma Ahlén Bergman.
Marcus Thuresson, statistician at Statisticon AB who assisted us in all our papers.
Anna-Maria Eriksson, Umeå university, for your good will, help and support.
The organizers of NatiOn IV, Nationell forskarskola i klinisk och translationell
cancerforskning: Ingemar Ernberg, Dan Grandér, Svetlana Bajalica Lagercrantz,
Daria Glaessgen, Ulrika Segersten, Karin Lindberg. The research school you
organized, blended interesting courses with tickling discussions that truly
developed me as a researcher.
Collaborators in all the hospitals and academic centers who provided us with
research material, logistics and thoughtful reflections: Erkki Rintala, Rolf
Wahlqvist, Anders Ullén, Sten Nilsson, Markus Johansson, Alexander Sidiki,
Benny Holmström, Farhood Alamdari, Marwan Shareef, Johan Hansson, Janos
Vasko, Tomasz Jakubczyk, Tammer Hemdan, Firas Aljabery, Ylva Huge, Katrine
Riklund, Maryam Bahar, Danna Asad, Ramona Forsman, Anders Bergh. Thank
you for invaluable resources.
Colleagues and staff at the Department of Urology, Södersjukhuset. You endorsed
my research and taught me urology as a profession. Thank you Magnus
Wijkström, Tim Fagerström, Jesper Rosvall, Ulf Norming, Ulrika Westlund,
Elisabeth Farrelly, Karin Falkman, Hans Thorsson, Ann-Helén Sherman Plogell,
Thomas Hopfgarten, Firas Tarish, Susanne Hagedorn, Malin Nyberg Isacson,
Mats Hedlund, Stefan Anania, Emma Andersson, Camilla Malm, Per Nordlund,
Abdulghafour Halawani, Yashar Khoshkar, Ziga-Maria Johansson, Pia Simonsen
and many others.
A warm thanks to all my fantastic friends. What a luxury to have you close. You
calibrate me, ensuring me a multi-dimensional life.
My dear family. Alicja och Freddy, you have indulged me with infinite quantities
of love and support. You have laid the foundation for what is me. Paula, Gustaf,
Stella, Ruben and Elle: Thank you for your enormous hospitality. With you I
always feel at ease.
42
To my extended family: My, Per, Louise, Casper, Elisabeth, John, Gustaf, Lotta,
Maxine, Fabian, Casimir: Thank you for creating a vibrant atmosphere where I
always feel welcome, in particular at Östanå, a place where I have written large
portions of this thesis.
My children Sasha, Noah and their little brother, who came to this world as I was
writing the last finishing words of this thesis. Everyday I long for our meals, our
talks, and our time together. You make sure my priorities in life are right.
At last, Béatrice, Bisse, Bitsky: my precious love. You add so much meaning and
flavour to my life. You see things I easily miss. Thank you for the enormous
support you have given me from the very start of this thesis to the intense and
thrilling end.
43
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