A Randomized Phase II Study of the Administration of Prebiotics and Probiotics During Definitive Treatment With Chemotherapy-radiotherapy for Patients With Squamous Cell Carcinoma of the Anal Canal (BISQUIT)
Phase: PHASE2
Conditions: Anal Cancer Squamous Cell
Sponsor: AC Camargo Cancer Center

 
A randomized phase II study of the administration of 
prebiotics and probiotics during definitive treatment 
with chemotherapy-radiotherapy for patients with 
squamous cell carcinoma of the anal canal (BISQUIT) 
 
Rachel Riechelmann (Main Investigator)1, 
Marcos Camandaroba1,2, Virgilio Souza1,2, Celso Abdon Mello1, Vladimir Lima1,3, 
Maria Leticia Gobo Silva4, Marcelo Cavicchioli5, Dirce Carraro3, Emmanuel Dias Neto3, 
Diana Noronha Nunes3, Louise De Brot6, Samuel Aguiar Jr7. 
Departments: 1- Clinical Oncology; 2- Postgraduate student; 3- CIPE; Radiation 
therapy; Nuclear Medicine; 6 - Pathological Anatomy; Pelvis and Coloproctology 
 
AC Camargo Cancer Center - Brazil 
 
ID: 2625-18 
 
March, 05, 2019 
 
 
 
 
 
Introduction 
Although anal canal squamous cell carcinoma (ACSCC) is rare in developed countries, it 
has shown an annual increase of 4% in its incidence in Brazil (1), and according to data 
from the Oncocenter Foundation of São Paulo (FOSP), 2,338 cases were diagnosed in 
2000 and 2016. 
The standard treatment for localized ACSCC (without distant metastases) is definitive 
chemo-radiotherapy (Ch-RT) concomitant with administration of a fluoropyrimidine 
(5FU or capecitabine) combined with mitomycin or cisplatin, which provides cure rates 
of 60-80 % depending on the staging (2). When there is no complete remission, 
surgical rescue through anal amputation is the only potentially curative modality. 
However, this strategy is associated with great morbidity, besides negative emotional 
and social impacts, with consequent reduction of quality of life. Therefore, 
interventions that may increase the chance of cure in ACSCC should be investigated. 
The main risk factors for ACSCC are human papillomavirus (HPV) infections and 
immunosuppression, including human immunodeficiency virus (HIV) infection. Chronic 
HPV infection and HIV-induced immunosuppression point to research strategies that 

strengthen the immune system to reduce the risk of developing ACSCC. In the 
metastatic setting, the use of immune checkpoint inhibitors, such as anti-programmed 
death protein-1 (PD1) antibodies, were shown to be promising in ACSCC patients, 
promoting response rates of approximately 25% (3). However, there is no evidence of 
modulation interventions of the immune system in patients with localized ACSCC. 
Recently, studies have shown that the composition of the intestinal microbiota 
influences the onset of colorectal cancer (4), and may even disrupt the effects of 
chemotherapy in this neoplasm. A preclinical study in animal model showed that E. coli 
impaired the antitumor effect of fluoropyrimidines, drug used in colorectal cancer and 
ACSCC (5). The intestinal microbiota also participates in a large set of metabolic 
processes (such as reduction, hydrolysis, dehydroxylation, etc.) involved in drug 
metabolism. For example, some intestinal bacteria have β-glucuronidases that cleave 
glucuronide from the inactive metabolite of irinotecan (SN-38G), a drug used in 
gastrointestinal tumors, releasing active metabolite (SN38) in the intestine, causing 
diarrhea and colitis (6). Ciprofloxacin has been shown to inhibit this enzyme by 
suppressing the diarrhea associated with irinotecan in an experimental model of mice 
(6). Mycoplasma hyorhinis encodes a thymidine phosphorylase that strongly restricts 
the cytostatic activity of pyrimidine nucleoside analogues (6). 
On the other hand, the replacement of the intestinal microbiota "carcinogenic" 
(Fusobacterium spp and Bacteriodes fragilis) (7) by a protective microbiota 
(Bifidobacterium and Lactobacillus) (8) has been the reason of numerous investigations 
with prebiotics and probiotics. According to the International Scientific Association of 
Probiotics and Prebiotics, probiotics are composed of living organisms which, when 
administered, promote health benefits, such as antimicrobial action against intestinal 
pathogens, modulation of the immune system, reduction of cholesterol levels, 
reduction of colitis and prevention of colorectal cancer (7). Kefir is an example of 
probiotic. Already prebiotics are inert ingredients that promote alteration in the 
composition or activity of the gastrointestinal microflora, conferring health benefits 
(7). Example of prebiotic is polysaccharide inulin. Studies with these compounds have 
been conducted, showing promising results. A small placebo-controlled trial using B. 
breve breve (Yakut®) in children undergoing chemotherapy for a variety of neoplasms 
has shown that this group had fewer episodes of fever and less frequency of use of 
intravenous antibiotics compared to controls (6). There are also studies that suggest 
that the alteration of the intestinal flora can increase the effectiveness of 
immunotherapy as a form of modulation of the immune system in several animal 
models of colorectal cancer (9). In addition, the use of this strategy could have a 
modulatory effect on local and systemic toxicity of the treatment, possibly reducing 
the morbidity of the treatment, as already suggested by studies in cervical carcinomas 
(10). 
Despite the strong scientific rationale, there are no studies that have evaluated the use 
of probiotics or prebiotics in order to increase the effectiveness of conventional Ch-RT 
treatment in ACSCC. Therefore, based on the assumptions that (1) there is a need for 
research that increases the cure rates of the definitive treatment of Ch-RT in ACSCC; 
(2) ACSCC is a virus-associated tumor in many cases and therefore potentially 
immunogenic; (3) immunotherapy is a promising strategy in ACSCC; and (4) that pre- 

and probiotics can stimulate the immune system through modulation of the intestinal 
microbiota, and improve oncological outcomes, we propose a randomized phase II 
study of the use of pre-probiotics during definitive treatment of Ch-RT for patients 
with ACSCC located. 
The primary hypothesis of this study is that addition of pre- and probiotics increases 
the proportion of patients with complete clinical and radiological response after Ch-RT 
to ACSCC. Secondary hypotheses are that pre- and probiotics increase the metabolic 
response measured by positron emission computed tomography (PET-CT) with 18F-2-
fluoro-2-deoxy-D-glucose fluorodeoxyglucose (18-FDG) and promote greater control of 
local disease after Ch-RT; and reduce local and systemic toxicity of treatment. 
 
Hypotheses: 
Primary: The addition of pre- and probiotics increases the proportion of patients with 
complete clinical and radiological response after Ch-RT to ACSCC. 
Secondary: Pre- and probiotics increase the metabolic response measured by PET-CT 
18-FDG and promote greater control of local disease after Ch-RT; and reduce local and 
systemic toxicity of treatment. 
 
Objectives: 
Primary: Evaluate whether the addition of pre- and probiotics during Ch-RT increases 
the post-Ch-RT complete clinical and radiological response rate for ACSCC. 
Secondary: To evaluate if the addition of pre- and probiotics influences the metabolic 
response by PET-CT 18-FDG; the complete clinical and radiological response rate at 6 
months; colostomy free survival at 12 months and disease-free survival; toxicities of 
Ch-RT treatment are reduced; to assess whether the presence of HPV in the tumor 
tissue correlates with clinical outcomes; and promotes variation of systemic immune 
parameters (11). 
Outcomes: 
Primary: Complete clinical and radiological response rate at 6-8 weeks post-Ch-RT, 
defined as absence of disease visible on clinical examination and magnetic resonance 
imaging (MRI) of the pelvis (or pelvic tomography if contraindicated to MRI) and 
without disease at a distance, through tomography of the chest and abdomen. This 
moment was chosen because it is when we evaluate response to Ch-RT in the routine, 
according to national and international guidelines. 
Secondary:  
(1) Metabolic response by 18-FDG PET-CT, comparing the mean pre-and post-Ch-RT 
volume-capture measurements of each patient at 6-8 weeks post Ch-RT. As there are 
no studies evaluating ACSCC volume-uptake measures, the volume-withdrawal value 

(s) of the volume-uptake measures that is most associated with the complete clinical 
and radiological response at 6-8 weeks post-Ch-RT will be determined a posteriori. 
(2) Complete clinical and radiological response rate at 6 months, defined as absence of 
disease visible to the clinical and pelvic MRI (or pelvic tomography) and without 
disease at a distance, through tomography of the chest and abdomen; 
(3) Progression / disease free survival, defined as the time from day 1 of the Ch-RT 
treatment to local or remote relapse, or death from any cause, whichever occurs first. 
Individuals with no evidence of disease at the time of study analysis will be censored at 
that point in the analysis. 
(4) Proportion of patients without colostomy 12 months after termination of Ch-RT. 
(5) Toxicity frequency: Adverse events of grade 2 or higher by the Common Toxicity 
Criteria for Adverse Events (CTCAE) version 4.0, through study completion. 
(6) Incidence of positivity to HPV in tumor tissue through genotyping, through study 
completion. 
(7) Variation of systemic immune parameters, defined by variation in the total number 
of lymphocytes, neutrophil / lymphocyte ratio (NLR) and lymphocyte / monocyte ratio 
(RLM) evaluated by comparison of values before pre-and probiotic use and collection 
values of blood during treatment, already performed in the routine, through study 
completion. 
 
Exploratory 
(1) Collection of plasma, leukocytes, faeces and anal swab before and after the use of 
pre-and probiotic for subsequent analysis of ctDNA (mutation profile and gene 
fusions), activation profile of immune cells, dosage of cytokines and intestinal 
microbiota. These biological materials will be stored for future studies, after 
completeness of the proposed study. 
(2) Evaluation of tumor microbiota of paraffin-shaped anatomo-pathological material 
from ACSCC biopsies filed at the service. This analysis will be carried out in the future, 
as part of future studies, after completeness of the proposed study. 
 
Methods:  
Design: 
 
A randomized prospective, open-label, phase II study of oral prebiotics and probiotics 
during definitive treatment of Ch-RT for patients with localized ACSCC versus same 
treatment without the addition of pre-or probiotics. Treatment of Ch-RT (using 
fluoropyrimidine [5FU or capecitabine] associated with cisplatin or mitomycin as a 
chemotherapy protocol) will occur according to institutional protocol and no change in 
standard treatment is planned in this protocol. 

Eligible patients who consent to participate in this study will be randomized 1: 1 in two 
groups: 
 
Prebiotics and probiotics group, which will receive standard nutritional 
guidance from the institutional routine and prebiotics in combination with 
probiotics, starting one (1) week prior to initiation of Ch-RT and daily 
throughout the treatment up to 6 to 8 weeks post-Ch- moment of response 
evaluation (primary outcome). 
 
Control group that will receive standard nutritional guidance only prior to 
initiating Ch-RT. 
Randomization will be simple and conducted independently by clinical trial support 
staff who are not participating in the study. The random sequence will be generated by 
computer program. 
Both groups will receive nutritional orientation by nutritionist of the institution before 
randomization, according to institutional routine. 
Both groups will be evaluated for: (1) food inventory, which will be conducted by the 
study research team through food questionnaire (12); (2) assessment of response to 
treatment as per routine; (3) assessment of metabolic response to the study; (4) 
assessment of adverse treatment events, as per routine; (4) kinetics of systemic 
immune parameters (NLR and MLR), according to the analysis of hemograms collected 
in the routine; (5) HPV tumor screening; (6) collection of biological materials (blood, 
plasma, feces, anal / rectal swab) that will be stored for future studies. Therefore, the 
interventions of this study, besides the use of pre- and probiotics, are items (3), (5) and 
(6). 
The following clinical data will be collected prospectively and structured in an 
electronic database: sex, age at start of use of pre-and probiotics, clinical staging 
(according to institutional routine), HIV status, smoking, alcoholism, comorbidities 
such as any clinical condition requiring pharmacological therapy), concomitant 
medications of chronic use, use of antibiotics and / or anti-inflammatories within 30 
days or during the study, body mass index prior to the use of pre-probiotics, laboratory 
parameters (blood count, performance status, symptoms associated with ACSCC, QT 
regimen, type of RT technique (IMRT vs 3D), adverse events during Ch-RT, results of 
symptomatic, clinical, radiological and metabolic responses and food questionnaire 
before, during week 3 of Ch-RT and at the end of the study. 
 
Study population: eligibility criteria  
Patients will be recruited from Clinical Oncology, Coloproctology or Radiotherapy 
outpatient clinics and must meet all the criteria below Inclusion and none of Exclusion: 
Inclusion criteria 
 
Patients older than 18 years; 

 
Confirmed histological diagnosis of squamous cell carcinoma / squamous cell 
carcinoma of the anal canal (ACSCC); 
 
Patients with localized ACSCC (≥ T2N0M0, according to American Joint 
Committee on cancer 8th edition) staged by conventional imaging methods 
according to institutional routine; 
 
Indication of starting definitive treatment with Ch-RT in the institution. HIV-
positive patients may be included; 
 
Free informed consent signed by the patient or legal representative. 
Exclusion Criteria 
 
Perianal squamous cell carcinomas; 
 
Clinical condition leading to difficulty in swallowing; 
 
Patients with a contraindication to receiving Ch-RT, ie receiving only 
radiotherapy or not receiving polychemotherapy; 
 
Clinical condition that, due to the investigator's judgment, prevents adherence 
to the study 
 
Active infection requiring antibiotic therapy. 
 
Intervention 
Patients in group A (experimental) will receive Symbioflora®, a food supplement 
containing probiotics represented by high amounts (109) of bacteria of the genus 
Bifidobacterium and Lactobacillus and prebiotics composed of non-digestible fruit-
oligosaccharides (inulin). This food compound is already used in the A.C. Camargo 
Cancer Center by the Nutrition team. 
Both will be administered orally, in the form of sachets, dissolved in half a glass of 
water, once a day, preferably at a fixed time, from 7 days (+/- 2 days) before the day 1 
cycle 1 of Ch-RT, during the whole treatment of Ch-RT, until 6 to 8 weeks after 
termination of Ch-RT, when the response evaluation (primary outcome) will be 
performed. The last day of pre-and probiotic will be the day of MRI (or tomography) of 
the pelvis. 
The investigators will provide dispensing of the pre- and probiotic to start the study, 
with sufficient amount until the next medical visit, when new dispensation will occur. 
The evaluation of adherence to the use of pre-and probiotic will be made through 
questioning during the medical visits and counted in the medical record. 
Adverse events associated with the use of Symbioflora® are infrequent and include 
flatulence, abdominal discomfort and rarely, diarrhea. Patients with severe 
immunosuppression (not the case of patients who are treated by Ch-RT for ACSCC) 
may develop intestinal infection by the probiotic component. 

Patients may discontinue pre-and probiotic use for any of the following reasons: 
disease progression, death, adverse event (s) leading to permanent interruption of Ch-
RT, pre-and probiotic intolerance, withdrawal of patient consent (or legal 
representative) or need for new systemic oncologic therapy or surgery. 
Patients in both groups will receive nutritional counseling, with a low residue diet, 
according to standard routine and prior to randomization (Group A) or before Ch-RT 
(Group B). 
 
Concomitant medications 
In general, concomitant medications considered necessary for the treatment and 
safety of the patient are allowed, and their use is documented in the patient's medical 
records and in the clinical file. 
Concomitant administration of investigational medicinal products is not permitted. The 
use of growth factors is allowed at the discretion of the investigator for patients with 
neutropenia, according to institutional routine. The use of antibiotics during the study 
should be avoided as long as it does not interfere with patient safety. 
 
Evaluation of Outcomes 
Efficiency Outcomes  
Primary Outcome: 
Patients will perform imaging examinations with chest and abdomen CT scans and 
pelvic NRMs preferably within 21 days (+/- 3 days) of pre-and probiotic use (group A) 
or isolated standard nutrient orientation (Group B) and in 6-8 weeks after completion 
of Ch-RT and will be assessed clinically by complete physical examination, including 
anal inspection and rectal examination, at the same times. Regarding the evaluation of 
response, patients will be classified, in the evaluation of the primary outcome, with or 
without complete response (absence of tumor in the clinical examination and by pelvic 
MRI); however, it will be noted if there is partial response (any reduction), stable 
disease (same measures) and progression (any growth); we will not use the RECIST 
criteria because they are not reliable in the primary ACSCC assessment. Patients with 
contraindication to pelvic MRI may perform pelvic tomography for response 
evaluations. 
Assessment of symptoms that may be related to disease progression will be monitored 
at each medical visit. 
 
Secondary Outcomes: 
In addition to evaluating tumor response according to conventional imaging and 
physical examination already used in the routine, both groups will also be evaluated by 
metabolic response with 18-FDG PET-CT. PET-CT scans will be performed prior to the 

use of pre-and probiotic (within 15 days) and after 6-8 weeks after termination of Ch-
RT. The evaluation of metabolic response by PET-CT will be done by a method that 
takes into account the volume of the capturing lesions and their intensity of uptake, 
total lesion glycolysis (TLG) (13). 
The methodology will be according to a study published by the researcher: 14 TLG is a 
measure that takes into account the volume and intensity of the uptake, being 
produced by multiplying the volume of the disease by the average Standardize Uptake 
Value (SUV) within that volume. It will be calculated using AW Volume Share 5 
(General Electric, Waukesha, WI). The minimum values of SUVs used to define the 
limits of the lesion (s) will be determined by visual analysis and the total volume of 
interest (VOI) that circumscribes the primary tumor and lymph nodes will be calculated 
automatically by the switch program. The VOI will be multiplied by the average SUV 
within that volume. Regions of physiological or inflammatory uptake above the SUV 
boundaries will be excluded from the images. The TLG value will be calculated for both 
PET-CT 18-FDG tests and according to published methodology (13). The variation of 
TLG values between the two examinations will be calculated by subtracting the value 
of the second TLG from the first one and dividing the result by the value of the first 
TLG. In patients with no initial uptake of 18-FDG PET-CT (TLG = 0), we will use the 
symbolic value of TLG = 0.1 to allow calculations of TLG variation. 
Patients will also be evaluated for complete clinical and radiological response within 6 
months after the termination of Ch-RT and from that moment on, they will initiate 
routine follow-up (described below) to evaluate disease-free survival outcomes and 
colostomy at 12 months . 
 
Toxicities 
For evaluation of adverse events, patients will be seen before the start of the study, 
within 72h of the pre- and probiotic dispensing (group A) or within 7 days of the 
initiation of Ch-RT (group B), every 15 days (+/- 3 days) during Ch-RT, according to 
institutional routine, and at the end of the study (in the evaluation of primary 
outcomes, 6-8 weeks post Ch-RT) with complete clinical examination and routine 
laboratory tests (blood count, urea, creatinine and others, if clinically indicated). 
Patients who present grade 2 intolerable or grade 3 toxicity will have the treatment 
dose reduced and / or radiotherapy interrupted as routine. All adverse effects will be 
graded and evaluated whether or not they are related to treatment. Patients requiring 
hospitalization should be immediately identified by the Principal Investigator of the 
study. 
As a dietary supplement, we do not expect to see serious adverse events or to lead to 
intolerance. However, if there is intolerance, the amount of pre-and probiotic will not 
be allowed to be reduced, but we will recommend discontinuation. In this case, the 
patient is still in the study, if he wishes. 
Adverse events will be assessed according to the Common Adverse Event Toxicity 
Criteria (CTCAE) version 4.0. An adverse event for the purposes of this protocol is the 

appearance of (or worsening of the pre-existing) signal (s), symptom (s) or undesirable 
medical condition (s) occurring after signature of free and informed consent, even if 
the event is not considered to be related to the study drug (s). The occurrence of 
adverse events should be obtained through direct questions to the patient or when 
they are voluntarily reported by the patient during or between visits or through 
physical examination or laboratory tests. 
Whenever possible, each adverse event should be evaluated to determine: 
1. Severity (mild, moderate, severe and life threatening) or (CTCAE grade 4.0) 
2. Their relationship to each study drug (suspected / suspected) 
3. Its duration (starting and ending dates or continuing in the final exam) 
4. Conduct adopted (no dosage adopted, dosage of study drug adjusted / temporarily 
discontinued, study drug permanently discontinued as a result of said adverse event, 
concomitant medication given, non-medicated therapy, hospitalization / extended 
hospitalization). 
 
Follow-up: 
Follow-up begins after evaluation of the primary endpoint, 6 to 8 weeks post Ch-RT, 
and will occur according to institutional routine, with clinical examination, including 
anal region and rectal examination, every 8 weeks (+/- 5 days) during the first year and 
every 6 months (+/- 5 days) from the second year to the fifth year. Abdominal and 
chest tomography imaging and pelvic resonance imaging will be performed every 8 
weeks (+/- 5 days) in the first year and every 6 months (+/- 5 days) in the second and 
third years, and annual fourth to fifth year. 
Cases of persistent disease at 6 months or post Ch-RT recurrence will be discussed, as 
routine, at Tumor Board meetings. 
 
Methodology for HPV screening in tumor tissue through genotyping 
 
The Roche LINEAR ARRAY HPV test will be used, according to the routine of the service. 
This is an in vitro qualitative test for the detection of Human Papilloma Virus. Thirty-
seven HPV genotypes are detected by this method, including high and low risk types 
[6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52 (MM7), 83 (MM7), 53, 54, 55, 56, 
58, 61, 62, 64, 66, 67, 68, 69, 70, 71, 84 (MM8), IS39 and CP6108]. HPVs 33, 35, 52 and 
58 make up the region of the so-called high-risk HPV POOL. 
 
The LINEAR ARRAY HPV Genotyping Test is based on four main processes: sample 
preparation; polymerase chain reaction (PCR) amplification of target DNA using 
primers for HPV; hybridization of the amplified products with oligonucleotide probes; 
and detection of the products amplified and connected to the probe, by colorimetric 
determination. 

 
Sample Preparation 
HPV DNA is isolated by lysis of epithelial cell samples by denaturation at elevated 
temperatures. Lysis is performed in the presence of proteinase K, chaotropic agent and 
detergent. There is isolation and purification of DNA on a column and elution with 
sigma water. The human β-globin gene is isolated simultaneously and evaluates cell 
fitness, extraction and amplification of each individually processed sample. 
 
PCR amplification 
 
Target Selection 
The LINEAR ARRAY HPV Genotyping Test uses biotinylated primers to define a 
nucleotide sequence within the L1 polymorphic region of the HPV genome, which has 
approximately 450 base pairs. A group of HPV primers present in the Master Mix is 
designed to amplify HPV DNA from 37 genotypes, including 14 high-risk genotypes (16, 
18, 31, 33, 35, 39, 45, 51, 52, 56, 58 , 59, 66 and 68). The uptake probe sequences are 
located in polymorphic regions of L1 bound by these primers. An additional pair of 
primers targets the human β-globin gene to provide cell fitness control, extraction and 
amplification. 
 
 
Target Amplification 
AmpliTaq® DNA Polymerase is used for "hot start" amplification of HPV target DNA and 
ß-globin control. First, the PCR reaction mixture is heated to activate DNA Polymerase 
AmpliTaq® Gold, to denature viral DNA and human genomic DNA and to expose the 
target sequences to the primer. As the mixture cools, the primers bind to the target 
DNA. AmpliTaq Gold DNA polymerase, in the presence of Mg2 + and excess dNTPs, 
elongates the primers attached throughout the target templates to produce a target, 
double-stranded HPV DNA molecule of about 450 base pairs or a molecule of Β-globin 
DNA, 268 base pairs, termed "amplicon". This process is repeated for a number of 
cycles. The amplification occurs only in the region of the HPV genome or the β-globin 
gene that is between the primer pair. The whole genome is not amplified. 
 
In the LINEAR ARRAY HPV Genotyping Test, the selective amplification of the target 
nucleic acid of the sample is achieved by the use of an enzyme AmpErase (uracil-N-
glycosylase) and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes 
and catalyzes the destruction of DNA chains containing deoxyuridine15, but not of 
deoxythymidine-containing DNA strands. Deoxyuridine is not found in nature DNA but 
is always present in the amplicon due to the use of deoxyuridine triphosphate in 
addition to the deoxythymidine triphosphate in the Master Mix reagent; therefore, the 
amplicon is the only one containing deoxyuridine. Deoxyuridine makes the 
contaminating amplicon susceptible to destruction by the enzyme AmpErase prior to 
amplification of the target DNA. The enzyme AmpErase, which is in the Master Mix 
reagent, catalyzes cleavage of deoxyuridine-containing DNA into deoxyuridine residues 
by opening the deoxyribose chain at the C1 position. When heated, in the first step of 
thermal amplification, at the alkaline pH of the Master Mix, the amplicon DNA strand 
breaks at the deoxyuridine position, thereby rendering the DNA non-amplifiable. The 
AmpErase enzyme is inactivated at temperatures above 55øC, i.e., during the thermal 
amplification steps, there is no destruction of the target amplicon. After amplification, 

any residual enzyme is denatured by the addition of the Denaturation Solution, 
thereby preventing the degradation of any target amplicon. 
 
Hybridization Reaction 
After PCR amplification, HPV and β-globin amplicons are chemically denatured to form 
a single stranded DNA, by the addition of Denaturation Solution. The denatured 
amplicon aliquots are transferred to the appropriate tray of the typing tray, which 
contains hybridization buffer and a single LINEAR ARRAY HPV Genotyping Strip, which 
is coated with probes bands for the HPV and β-globin types. The biotin labeled 
amplicon will only hybridize to the oligonucleotide probes if the amplicon contains the 
corresponding sequence of the complementary probe. 
 
Detection Reaction 
Upon completion of the hybridization reaction, the LINEAR ARRAY HPV Genotyping 
Strip is thoroughly washed to remove any unbound material. Streptavidin-Horseradish 
Peroxidase Conjugate is added to the strip. The Streptavidin-Horseradish Peroxidase 
Conjugate binds to the biotin-labeled amplicon hybridized with the oligonucleotide 
probes, which are present in the strand. The strip is washed to remove Streptavidin-
Uncoated Strontium Peroxidase Conjugate and a substrate solution containing 
hydrogen peroxide and 3,3 ', 5,5'-tetramethylbenzidine (TMB) is added to each strip. In 
the presence of hydrogen peroxide, streptavidin-horseradish peroxidase catalyzes the 
oxidation of TMB to form a blue staining complex, which precipitates at the positions 
of the probe where hybridization occurs. The LINEAR ARRAY HPV Genotyping Strip is 
visually read by comparing the blue stripe pattern with the LINEAR ARRAY HPV 
Genotyping Test Reference Guide. 
 
Exploratory Studies 
As part of this project, we will collect the following biological materials for 
further studies, after this study complete the recruitment and evaluation of the 
primary outcome of all patients included. 
1. We will collect a sample of 10 ml of peripheral blood pre-study, before the start 
of the use of pre- and probiotics, in the evaluation of response of 6-8 weeks 
and in 6 months post-Ch-RT. This collection will configure a liquid biopsy that 
will investigate the mutation and gene fusions and mutational load (TMB) 
profile in the samples. TMB analysis will be performed on circulating tumor 
DNA and also on pre-treatment paraffin-shaped tumor material (biopsy of the 
diagnostic routine). Both the mutation profile and the gene fusions profile and 
the BMR will be correlated with the clinical outcomes. 
 
2. 
Collection of anal / rectal swabs and feces for microbiota analysis at the same 
times as blood collections, with subsequent comparison with tumor microflora of 
stored paraffin wax pretreatment. We will also compare the pre- and post-use 
microbiota of group A pre-and probiotics, as well as microbiota variations in group B. 
We will evaluate if the anal and intestinal microbiota profile correlates with clinical 
endpoints and systemic immune parameters (NLR and MLR ). This study will be 

important to evaluate if the engraftment of the microbiota of the probiotics correlates 
with the outcome of the patients. 
 
3. 
Collection of 5 ml of blood at the same times as (1) for plasma separation for 
analysis of inflammatory cytokines and leukocytes for immunophenotyping by flow 
cytometry. The findings will also be correlated with clinical outcomes. 
 
Statistical Considerations 
Statistical plan 
 Descriptive statistics will be used to report the results of categorical and 
continuous variables. The time-to-event variables will be reported in medians and 
Kaplan-Meier curves. The degrees of toxicity will be tabulated. The number of patients 
who discontinue treatment for any reason will be presented. 
Efficacy and toxicity analyzes will be performed by intention to treat. Patients 
receiving at least one pre-and probiotic dose in addition to day 1 cycle 1 of Ch-RT in 
group A and at least day 1 cycle 1 of Ch-RT in group B will be evaluated for toxicity and 
efficacy. 
Inferential analyzes will be performed according to the type of variable of 
interest and if the comparison is intra- or intergroup. For the analysis of the primary 
endpoint, complete response rate at 6-8 weeks, as well as comparisons between 
groups of 6-month response rates and 12-month colostomy-free survival and 
frequency of toxicities, we will use the Chi-square test . For comparative analyzes 
between groups regarding disease-free survival, we will use a non-parametric log-rank 
test. For intra-group comparisons, for example, the kinetics of NLR and MLR, we will 
use the ANOVA test. For the metabolic response analyzes, the TLGs of each pre- and 
post-intervention patient will be compared by paired T test and unpaired T test, when 
the TLG drop comparison is intergroup. The TLG drop values most associated with 
complete clinical and radiological response at 6-8 weeks and at 6 months post Ch-RT 
will be presented by percentage drop of TLG and will be determined by ROC curves. 
In order to minimize possible effects of imbalances between groups on 
prognostic characteristics that may influence the outcome of the primary endpoint, we 
will conduct univariate and multivariate logistic regression analyzes, adjusting the 
analysis of the primary endpoint for other prognostic factors in addition to the pre-e 
probiotics. These factors will be: clinical stage (T2N0 vs T3 / 4 or N +), sex, HIV status, 
age at diagnosis, presence of comorbidities, need for interruption of radiotherapy, and 
depending on the number of negative cases, HPV status. Values of p <0.20 in the 
univariate analysis will be considered significant for the variable to enter the 
multivariate model. 
For all inferential analyzes, bicaudate values of p <0.05 will be considered 
statistically significant. 
 
Sample Size Calculation 
The study sample will be 75 patients. For this calculation, we considered H0 of 
complete clinical and radiological response at 6-8 weeks post-Ch-RT of 70%, based on 
literature data (3), H1 as 90%, type I bicaudate error of 10 %, 80% power and loss of 
follow-up / non-adherence of 10%.  

 
Recruitment and Study Duration 
In the A. C. Camargo Cancer Center, 30 to 35 patients with localized ACSCC are 
treated with Ch-RT annually. Therefore, the estimated recruitment time is 3 years, 
with an average of 2 patients per month, plus an 8-week follow-up for each patient to 
evaluate the outcome. Thus, it is expected to complete the study in 3.5 years from the 
first included patient. The patients included will be followed, according to institutional 
routine, for up to 5 years, with collection of clinical data for disease-free or colostomy-
free survival outcomes. 
 
Ethical Considerations 
The study will be conducted in accordance with the protocol, Good Clinical Practice 
guidelines of the International Conference on Harmonization (ICH GCP) and applicable 
local laws and regulatory requirements. The Free and Informed Consent Term (ICF) 
must be in accordance with ICH GCP guidelines, local regulatory regulations, and legal 
requirements.  
The investigator should ensure that each study patient, or his / her legal 
representative, is fully informed about the nature and objectives of the study and 
possible risks associated with participation. The investigator will obtain the written 
consent of each patient before any specific study activity is performed. The 
investigator will retain one path of each consent form signed by the patient. It will be 
emphasized that participation is voluntary and that the patient has the right to refuse 
participation or to leave in the middle of the study whenever he wishes. This will not 
impair subsequent care to the patient. Female patients will be instructed not to 
become pregnant during the treatment of Ch-RT, being advised to use contraceptive 
methods if of childbearing age, as already done in routine. There is a risk of loss of 
confidentiality if patient data are identified, however, all appropriate measures will be 
taken to prevent this from happening. 
The protocol was approved by the Local Ethics Committee. The study will be enrolled 
in the ClinicalTrial.gov international clinical trial bank. 
 
 
Funding and Sponsorship of the Study 
The patient will not incur any costs in relation to the use of pre-and probiotics or the 
18-FDG PET-CT, HPV research in biopsies or collections of biological samples. These will 
be funded by development. All of the rest (Ch-RT, laboratory tests, conventional 
imaging, medical appointments and nutritional counseling) is not part of this protocol 
because it is already part of the ACSCC patient care routine at the AC Camargo Cancer 
Center, paid by the paying source, that is, the patient's medical covenant or the 
Unified Health System. 
References: 
 

1.  
De Souza DLB, Curado MP, Bernal MM, Jerez-Roig J, Boffetta P. Mortality trends and prediction 
of HPV-related cancers in Brazil. Eur J Cancer Prev. 2013. doi:10.1097/CEJ.0b013e32835b6a43 
2.  
James RD, Glynne-Jones R, Meadows HM, et al. Mitomycin or cisplatin chemoradiation with or 
without maintenance chemotherapy for treatment of squamous-cell carcinoma of the anus 
(ACT II): A randomised, phase 3, open-label, 2×2 factorial trial. Lancet Oncol. 2013;14(6):516-
524. doi:10.1016/S1470-2045(13)70086-X 
3.  
Morris VK, Salem ME, Nimeiri H, et al. Nivolumab for previously treated unresectable 
metastatic anal cancer (NCI9673): a multicentre, single-arm, phase 2 study. Lancet Oncol. 
2017;18(4):446-453. doi:10.1016/S1470-2045(17)30104-3 
4.  
Niederreiter L1, Adolph TE1 TH. Food, microbiome and colorectal cancer. Dig Liver Dis. 
2018;50(7):647-652. 
5.  
Scott TA, Quintaneiro LM, Norvaisas P, et al. Host-Microbe Co-metabolism Dictates Cancer 
Drug Efficacy in C. elegans. Cell. 2017. doi:10.1016/j.cell.2017.03.040 
6.  
Alexander JL, Wilson ID, Teare J, Marchesi JR, Nicholson JK, Kinross JM. Gut microbiota 
modulation of chemotherapy efficacy and toxicity. Nat Rev Gastroenterol Hepatol. 2017. 
doi:10.1038/nrgastro.2017.20 
7.  
Ambalam P, Raman M, Purama RK, Doble M. Probiotics, prebiotics and colorectal cancer 
prevention. Best Pract Res Clin Gastroenterol. 2016. doi:10.1016/j.bpg.2016.02.009 
8.  
Zou S, Fang L, Lee MH. Dysbiosis of gut microbiota in promoting the development of colorectal 
cancer. Gastroenterol Rep. 2018. doi:10.1093/gastro/gox031 
9.  
Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA. The Influence of the Gut 
Microbiome on Cancer, Immunity, and Cancer Immunotherapy. Cancer Cell. 2018. 
doi:10.1016/j.ccell.2018.03.015 
10.  
Ferreira MR, Muls A, Dearnaley DP, Andreyev HJN. Microbiota and radiation-induced bowel 
toxicity: Lessons from inflammatory bowel disease for the radiation oncologist. Lancet Oncol. 
2014. doi:10.1016/S1470-2045(13)70504-7 
11.  
Oliveira SCR, Moniz CMV. Diretrizes Cancer do Canal Anal. 2017. 
https://www.sboc.org.br/images/diretrizes/diretrizes_pdfs/Cancer_do_canal_anal_vf_2017.p
df. 
12.  
Slater B, Philippi ST, Marchioni DML, Fisberg RM. Validação de Questionários de Freqüência 
Alimentar - QFA: considerações metodológicas. Rev Bras Epidemiol. 2003. doi:10.1590/S1415-
790X2003000300003 
13.  
Braghiroli MI, Mota JM, Duarte PS, et al. Evaluation of18F-FDG PET-CT as a prognostic marker in 
advanced biliary tract cancer. Nucl Med Commun. 2018. 
doi:10.1097/MNM.0000000000000810 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

A randomized phase II study of the administration of prebiotics and probiotics during 
definitive treatment with chemotherapy-radiotherapy for patients with squamous 
cell carcinoma of the anal canal (BISQUIT). ID: 2625-18 
 
INFORMED CONSENT FORM 
IDENTIFICATION DATA FOR THE SUBJECT OF THE RESEARCH OR LEGAL ENTITY 
1.NAME.:............................................................................. .............................................. 
IDENTITY DOCUMENT Nº: ........................................ SEX:    M □   F □ 
BIRTH DATE: ......../......../......  
ADRESS ................................................................................. Nº ...................  
APT: .................. 
DISTRICT:........................................................................     CITY............................................................. 
ZIP:........................................ PHONE: (............) ......................................... 
2.LEGAL RESPONSIBLE .............................................................................................................................. 
TYPE (degree of kinship, tutor, healer, etc.)  .................................................................................. 
IDENTITY DOCUMENT Nº:....................................SEX:  M □   F □   
BIRTH DATE.: ....../......./...... 
ADRESS: ............................................................................................. Nº ...................  
APT: ............................. 
DISTRICT: ................................................................................  
CITY: ...................................................................... 
ZIP: ..............................................  
PHONE: (............).................................................................................. 
______________________________________________________________________________________ 
 
March, 05, 2019 
RESEARCH DATA 
1. TITLE OF THE RESEARCH PROTOCOL "Randomized phase II study of prebiotic 
and probiotic administration during definitive treatment with chemoradiotherapy of 
patients with squamous cell carcinoma of the anal canal."  
2. RESEARCHER: RACHEL SIMÕES PIMENTA RIECHELMANN 
POSITION / FUNCTION: Director of the Clinical Oncology Department of the AC Camargo 
Cancer Center REGIONAL COUNCIL INSCRIPTION Nº 98131 
 
 

3. ARESEARCH RISK ASSESSMENT: 
MINIMUM RISK □ 
 
AVERAGE RISK 
□ 
LOW RISK 
X 
 
HIGH RISK 
□ 
4. RESEARCH DURATION: 3,5 YEARS 
 
1 – Study design and objective (s) 
You are being invited to participate in this clinical study to be performed at AC 
Camargo Cancer Center, São Paulo. 
You have the diagnosis of localized anal cancer (without involvement of other organs); 
this tumor is located in the region of the anus and can cause pain, difficulty to evacuate 
as well as bleeding. 
The treatment best considered for localized anal cancer is chemotherapy along with 
radiation therapy. Chemotherapy is performed with the application of drugs into the 
vein (cisplatin or mitomycin on the first day and 5-fluorouracil from the first to the fourth 
day), in the first and last week of radiotherapy. It may be used in place of 5-fluorouracil 
capecitabine, chemotherapy tablets taken every day that radiotherapy is done. 
Radiation therapy applies a dose of radiation daily into the anus and basin region by on 
average for 6/7 weeks. This treatment is standard in this and in several institutions in 
the country and the world for being the one that offers greater chance of cure. 
However, approximately 20 to 25% of patients are not cured with chemo-radiotherapy 
and therefore, research is important to understand about anal cancer. 
The reason for this study is to evaluate whether some microorganisms, given as dietary 
supplements, can improve the response in the treatment of anal cancer and thus its 
cure. These supplements are called prebiotics and probiotics. 
Probiotics are beneficial bacteria that live in the gut and improve the overall health of 
the body, bringing benefits, such as facilitating digestion and absorption of nutrients, 
and strengthen the immune system. When the intestinal flora is in imbalance, the 
intestine ends up being populated by pathogenic or "bad" bacteria, which compromise 
the immune system and leave the organism susceptible to diseases. Prebiotics are 
nondigestible foods and fibers that have the function of stimulating growth and / or 
activating the metabolism of some beneficial gut bacteria. In this way, prebiotics 
constitute the "food" of probiotic bacteria. 
In this study for which you are being invited to participate voluntarily, we aim to 
evaluate the benefit (fewer side effects, better treatment response and cure), the use of 
prebiotics and probiotics given during chemo- radiotherapy for anal cancer. We 
idealized this study because, despite the scientific logic, we do not know if probiotics 
prebiotics benefit anal cancer patients. 
 
2 – Procedures to be carried out and their purposes 
All patients who agree to participate in the study will receive, in addition to standard 
chemo-radiotherapy treatment, nutritional counseling and will be randomly selected to 
receive prebiotics and probiotics. 
You will be in one of the groups below: 
Prebiotics and probiotics group: They will receive standard nutritional guidance from 
the institutional routine and prebiotics in combination with probiotics. Both will be 
administered orally as a sachet dissolved in water once a day, preferably on a fixed 
schedule, starting 7 days before the first day of chemo-radiotherapy, throughout the 

chemo-radiotherapy treatment, and up to 6 at 8 weeks after completion of chemo-
radiotherapy; therefore, total time of approximately 3 months. 
Control group: Patients will not receive probiotics or prebiotics, only standard nutritional 
guidance. 
To evaluate response to treatment, blood and imaging tests with chest and abdomen 
tomography and pelvic resonance imaging, PET-CT 18-FDG scan, will be performed 
prior to the start of treatment and at 6 to 8 weeks post-completion of chemo-
radiotherapy; blood tests, CT scans and MRI scans are already part of the AC 
Camargo Cancer Center routine. 
The PET-CT scan will be done for this study. The PET-CT scan is a safe examination 
that is used in the routine treatment of various types of tumors and evaluates the whole 
body for the presence of tumor (s). We believe this test to be helpful in assessing the 
location of your tumor and also how much it decreases after conventional 
chemoradiotherapy treatment. 
We will also collect samples and blood, feces and secretion from the intestine and anus 
at three times, and HPV virus research on the biopsy material that you have already 
done. The purpose of these collections is to further study the effects of this dietary 
supplement on the chance of cure of anal cancer and to understand more about the 
origin of anal cancer, that is, because some people develop this tumor and others do 
not, and because some heal and some don’t. 
 
3 – List of study procedures (out of routine) and how they are performed  
 
Prebiotics and probiotics (group A only): a sachet dissolved in a glass of 
water once a day, starting one week before chemo-radiotherapy, during 
chemo-radiotherapy, and for 6 to 8 weeks after chemotherapy, in total 
approximately 3 months. 
 
Blood test with approximately 10 ml (2 tablespoons) of blood withdrawn per 
arm vein puncture before treatment started at 6 to 8 weeks and at 6 months 
after chemoradiotherapy. 
In these blood samples, we will investigate the profile of tumor mutations, inflammatory 
cells and hormones to better understand anal cancer 
 
Anal swab examination (collection of swab secretion) and stool collection 
before treatment started 6 to 8 weeks and 6 months after the end of chemo-
radiotherapy 
In this analysis, in the future, we will investigate the composition of the bacteria in your 
gut and whether this helps us to identify reasons why some patients are not cured. 
 
PET-CT scan 18-FDG scan: before treatment begins and 6 to 8 weeks after 
chemoradiotherapy termination.   
The PET-CT scan will assess the location and presence of metastases (tumor 
spread to other organs) and also the response to chemo-radiotherapy treatment 
(tumor reduction). 
 
4 – Description of the discomforts and risks expected in the procedures; 
The risks associated with treatment will be described below. 
 
Prebiotics and probiotics (Group A): Since these compounds are dietary supplements, 
the risks of using them are minimal. 

 
Common (> 10%): flatus enlargement, colic / abdominal discomfort, abdominal 
distension 
 
Uncommon or rare (<10%): diarrhea, intestinal infection, allergic reaction 
PET-CT scan 18-FDG 
 
Image examination that is already used routinely for patients with other tumors; as 
it requires intravenous contrast, some patients may have allergic reactions. Tell 
your doctor about any allergies you may have experienced in the past. 
5 – Risks related to pregnancy: 
Female patients of childbearing age will be advised not to become pregnant during the 
study. For them, a pregnancy test will be done before the start of treatment. If you 
become pregnant during your study participation, you should notify the study doctor 
immediately.  
 
6 – Benefits for the participant 
The hypothesis of the study is that the use of prebiotics and probiotics increases the 
chance of cure and improves the side effects of chemo-radiotherapy. This may or may 
not happen. PET-CT scanning may be useful for assessing tumor extension. 
Regardless of the results of this study, other patients in the future may benefit from the 
information obtained here. 
7 – Alternative Treatments 
There are no alternative treatments to the use of prebiotics or probiotics. 
8- Payment to research subject 
Participation in this study will not incur any additional cost to you and no payment will 
be made if you agree to participate in this study. You are entitled to damages if you 
incur damages associated with the study. 
9 – Voluntary participation / Study discontinuation 
Participation in this study is entirely voluntary (you decide whether you want to be a 
part or not). Even if you decide to participate in the study, you can leave it at any time, 
without giving explanations for it, and may even refuse to publish data collected about 
you. If this occurs the doctors no longer have collected the data about you, but may 
publish non-personal information collected before the cancellation. This decision will 
not affect your future medical treatment in any way. 
The study doctor may also remove you from this study if you feel that this is in your 
best interest after discussing and explaining the reasons to you, or in case the study is 
interrupted earlier than planned because it is considered unsafe. 
 
10 – Confidentiality 
If you choose to participate in this study, your health information and registration of 
your participation will be kept confidential and confidential. Researchers will identify 
you through a unique number and through the initials of your name (not using your full 

name). A copy of this informed consent will be filed in your AC Camargo Cancer Center 
medical record. However, there is a risk of loss of confidentiality; we will take all 
possible steps to prevent this from happening. 
11 – Access Guarantee 
Questions about procedures should be directed directly to the researchers listed at the 
end of this consent form. 
12 – Signatures 
I confirm that I read the Term of Free and Informed Consent and had the opportunity to 
clarify all my doubts related to this study. I understand that if I have any additional 
questions in the future regarding the study or my participation in it, I may contact the 
telephone 11- 2189-5000 
 
Through my signature, I agree to participate in this study as a volunteer. I received a 
copy of this Informed Consent Form. 
------------------------------------------------- 
Name of Participant (letter of form)         Date         /       /        
 
-------------------------------------------------- 
Signature of Participant 
 
-------------------------------------------------- 
Name of Investigator (letter of form)                     Date         /       /        
 
-------------------------------------------------- 
Signature of the Investigator 
 
If witness or legal representative is required: 
 
--------------------------------------------------  
Signature of witness / legal representative 
    Date         /       /        
 
-------------------------------------------------- 
Signature of witness / legal representative 
 
Contact with researchers: 
AC Camargo Cancer Center 
Rua Professor Antonio Prudente n 211, São Paulo-SP, Phone - +55(11) 2189-5000.