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\begin{document}

\title{Association between genetic polymorphisms in SLC19A1, SLCO1B1 and
SLCO1B3 genes predicts survival and toxicity in North Indian lung cancer
patients undergoing platinum-based doublet chemotherapy}



\author[1]{Parul Sharma}%
\author[1]{Navneet Singh}%
\author[2]{Siddharth Sharma}%
\affil[1]{Affiliation not available}%
\affil[2]{Thapar Institute of Engineering and Technology}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Abstract Aims: SLC transporters are expressed in lungs and are essential
membrane proteins responsible for the transport of wide range of
chemotherapeutic drugs. Polymorphisms in SLC19A1, SLCO1B1, and SLCO1B3
gene in North Indian lung cancer patients are investigated. Methods: A
total of 610 lung cancer patients undergoing platinum-based chemotherapy
were recruited in the study. Polymorphisms of SLC19A1 (G80A), SLCO1B1
(A388G, T521C) and SLCO1B3 (A1683-5676G) in North Indian lung cancer
patients were assessed and statistical analysis were carried out.
Results: Our data revealed that patients harboring mutant genotype (AA)
for SLC19A1 G80A polymorphism had higher MST as compared patients with
wild type (GG) genotype (MST=9.33 versus 8.23). ADCC patients with
mutant genotype (AA) showed better survival outcomes for SLC19A1 G80A
(MST=9.4 versus 8.8, HR=0.6; p=0.04). In SCLC, SLC19A1 G80A polymorphism
revealed increased survival in the patients harboring mutant genotype
(AA) (MST=9.6 months versus 7.6 months, p=0.04). For SLCO1B3
polymorphism, patients administered with carboplatin/cisplatin and
docetaxel showed inferior survival outcomes in subjects carrying
heterozygous alleles (AG) (MST=2.9 months versus 9.6 months, p=0.006,
HR=14.01). For anemia, SLCO1B1 T521C showed that patients with
heterozygous genotype (TC) had a reduced risk of developing anemia
(OR=0.44, 95\% CI=0.20-0.96; p=0.04). Patients with SLCO1B1 A388G
polymorphism harboring AG alleles was associated with a lower incidence
of thrombocytopenia (OR=0.41, 95\% CI=0.20-0.82; p=0.01). Patients with
heterozygous (AG) genotype (OR=0.35, 95\% CI=0.17-0.72; p=0.002) for
A1683-5676G polymorphism showed lower incidence of nephrotoxicity.
Conclusion: Genotyping of SLC polymorphism is crucial for predicting
survival and toxicity in lung cancer patients undergoing platinum-based
chemotherapy.%
\end{abstract}%



\sloppy


\textbf{Association between genetic polymorphisms in \emph{SLC19A1,
SLCO1B1} and \emph{SLCO1B3} genes predicts survival and toxicity in
North Indian lung cancer patients undergoing platinum-based doublet
chemotherapy}

\textbf{Running head: SLC polymorphism and clinical outcomes in Lung
cancer patients}

Parul Sharma\textsuperscript{1}, Navneet Singh\textsuperscript{2},
Siddharth Sharma\textsuperscript{*1}

\textsuperscript{1}Department of Biotechnology, Thapar Institute of
Engineering \& Technology, Patiala, India

\textsuperscript{2} Department of Pulmonary medicine, Post Graduate
Institute of Medical Education \& Research, Chandigarh, India

Parul Sharma, Department of Biotechnology, Thapar Institute of
Engineering \& Technology, Patiala, Punjab, 147004, India. Email:
parulnancy1822@gmail.com

Navneet Singh, Department of Pulmonary medicine, Post Graduate Institute
of Medical Research, Chandigarh, 160012, India. Email:
navneetchd@hotmail.com

*Siddharth Sharma, Associate Professor, Department of Biotechnology,
Thapar Institute of Engineering \& Technology, Patiala, Punjab, 147004,
India. Email: siddharthsharma.phd@thapar.edu

*Corresponding author:

Dr. Siddharth Sharma

Thapar Institute of Engineering and Technology,

Patiala, Punjab- 147004

India

Phone: +91- 9501688366

Email: siddharthsharma.phd@thapar.edu

Number of figures: 6

No. of tables: 8

Word count: 7,309

\textbf{What is already known about this subject}

SLC transporters are known mediators of drug disposition that
facilitates the influx of substrates and various chemotherapeutic agents
into cells.

Polymorphisms in the \emph{SLC19A1} , \emph{SLCO1B1} , and\emph{SLCO1B3}
gene influence the prognosis in the cancer patients, but little is known
about their role in lung cancer in Asians.

\textbf{What this study adds}

\emph{SLC19A1, SLCO1B1} , and \emph{SLCO1B3} polymorphisms are
associated with altered activity of these transporters and
affects~overall survival as well as toxicity levels in lung cancer
patients.

Genotyping for the \emph{SLC} polymorphism could be a valuable tool for
predicting which lung cancer patients will benefit the most from
platinum-based doublet chemotherapy.

\textbf{Abstract}

\textbf{Aims:} SLC transporters are expressed in lungs and are essential
membrane proteins responsible for the transport of wide range of
chemotherapeutic drugs. Polymorphisms in \emph{SLC19A1} , \emph{SLCO1B1}
, and \emph{SLCO1B3} gene in North Indian lung cancer patients are
investigated.

\textbf{Methods:} A total of 610 lung cancer patients undergoing
platinum-based chemotherapy were recruited in the study. Polymorphisms
of \emph{SLC19A1} (\emph{G\textsuperscript{80}A),
SLCO1B1}(\emph{A\textsuperscript{388}G} , \emph{T\textsuperscript{521}C}
) and\emph{SLCO1B3} (\emph{A\textsuperscript{1683-5676}G)} in North
Indian lung cancer patients were assessed and statistical analysis were
carried out.

\textbf{Results:} Our data revealed that patients harboring mutant
genotype (\emph{AA} ) for \emph{SLC19A1
G\textsuperscript{80}A}polymorphism had higher MST as compared patients
with wild type (\emph{GG} ) genotype (MST=9.33 versus 8.23). ADCC
patients with mutant genotype (\emph{AA} ) showed better survival
outcomes for \emph{SLC19A1 G\textsuperscript{80}A} (MST=9.4 versus 8.8,
HR=0.6; p=\textbf{0.04} ). In SCLC, \emph{SLC19A1
G\textsuperscript{80}A} polymorphism revealed increased survival in the
patients harboring mutant genotype (\emph{AA} ) (MST=9.6 months versus
7.6 months, p=\textbf{0.04} ). For SLCO1B3 polymorphism, patients
administered with carboplatin/cisplatin and docetaxel showed inferior
survival outcomes in subjects carrying heterozygous alleles (\emph{AG} )
(MST=2.9 months versus 9.6 months, p=\textbf{0.006, HR=14.01} ). For
anemia, SLCO1B1 T521C showed that patients with heterozygous genotype
(\emph{TC} ) had a reduced risk of developing anemia (OR=0.44, 95\%
CI=0.20-0.96; p=\textbf{0.04} ). Patients with \emph{SLCO1B1
A\textsuperscript{388}G} polymorphism harboring \emph{AG} alleles was
associated with a lower incidence of thrombocytopenia (OR=0.41, 95\%
CI=0.20-0.82; p=\textbf{0.01} ). Patients with heterozygous (\emph{AG} )
genotype (OR=0.35, 95\% CI=0.17-0.72; p=\textbf{0.002)} for
\emph{A\textsuperscript{1683-5676}G} polymorphism showed lower incidence
of nephrotoxicity.

\textbf{Conclusion:} Genotyping of \emph{SLC} polymorphism is crucial
for predicting survival and toxicity in lung cancer patients undergoing
platinum-based chemotherapy.

\textbf{Keywords:} Lung cancer, \emph{SLC} transporter, survival,
platinum-based chemotherapy, polymorphism, Toxicity

Introduction

Lung carcinoma is the second most common type of cancer (accounting for
13\% of all occurrences) and the leading cause of carcinoma fatalities
(23\%~of the total cases).\textsuperscript{1} It only has a 15\% 5-year
survival rate, and the median survival time is even less than tenmonths.
Moreover, regardless of histological type, half of all lung cancer
patients develop metastatic or advanced malignancy (stages III and IV)
at the time of diagnosis. Patients with advanced lung cancer~are
administered~platinum-based doublet~chemotherapy. Even though cisplatin
or carboplatin is effective when coupled with non-platinum-based
chemotherapy medications such as paclitaxel, pemetrexed, irinotecan, or
gemcitabine, significant diversity in treatment response has been
reported.\textsuperscript{2} Different individuals react to the same
drug in different ways. Although many genetic and environmental factors
such as age, concurrent therapy, drug interactions, and the nature of
the disease can impact chemotherapy outcomes, there are plenty of
inter-individual differences in drug response due to sequence variants
of genes that encode drug transporters.\textsuperscript{3}

Drug transporters are known mediators of drug disposition, facilitating
the influx of substrates into cells and the efflux of drugs and
metabolites from cells.Solute carrier (SLC) transporters are the most
prominent family of the membrane proteins accountable for the uptake
transport of numerous endogenous and xenobiotic compounds.
These~transporters are~expressed widely in the human~body, but notably
in the epithelia of essential organs such as the liver, gut, kidney, and
lung.\textsuperscript{4} These transporters are also involved in
multiple physiological processes, such as the cellular uptake of
nutrients, xenobiotics, and absorption of chemotherapeutic drugs (Figure
1).\textsuperscript{5} SLC transporters are expressed~in many tumors and
differentially in malignant and non-malignant
tissues.\textsuperscript{6} Among the SLC transporter family members,
polymorphism in \emph{SLC19A1} , \emph{SLCO1B1} , and \emph{SLCO1B3} are
reported widely in different carcinomas, and outcomes have sparked
curiosity in studying the function of these transporters in cancer
progression.\textsuperscript{6}

\emph{SLC19A1} encodes reduced folate carrier protein (RFC) and
facilitates the movement of antifolate drugs used in cancer
chemotherapy. The efficacy of chemotherapeutic drugs is associated with
the activity and levels of SLC transporters in both cancer and normal
tissues.\textsuperscript{7} The RFC gene is highly polymorphic in nature
and among the various SNP variants in the \emph{SLC19A1} gene,
polymorphism \emph{G\textsuperscript{80}A} (Arg27His, rs1051266) is
usually studied in different clinical conditions. This variant has been
extensively researched for its role in transport uptake~and its
correlation~to cancer risk, treatment response, and toxicity. \emph{G to
A} polymorphism results in the substitution of arginine amino acid with
histidine and thus leads to the alteration in the
\emph{SLC19A1}transporter structure, affecting its
function\textsuperscript{8}, thus modifying drug bioavailability and
influencing the therapeutic outcome.Reports in the past have revealed a
significant association of mutant genotype (\emph{AA} ) of
\emph{G\textsuperscript{80}A} polymorphism with an elevated risk of
esophageal and gastric carcinoma, respectively.\textsuperscript{9}
Increased influx of the methotrexate drug has also been reported in
patients with mutant genotype (\emph{AA} ) because
\emph{G\textsuperscript{80}A} mutation may be responsible for the
increased transporter activity.\textsuperscript{10} Adjei \emph{et al} .
concluded that \emph{SLC19A1} seems to predict survival differences in
pemetrexed-treated NSCLC patients.\textsuperscript{11}

Solute carrier organic anion transporting polypeptides (OATP) is another
SLC drug transporter that mediates the cellular influx of various
chemotherapeutic drugs into the cell. Among these
transporters,\emph{SLCO1B1} (OATP1B1) and \emph{SLCO1B3} (OATP1B3) are
theessential proteins localized at the basolateral membrane of the
hepatocytes.\textsuperscript{12} Overexpression of \emph{SLCO1B1}
and\emph{SLCO1B3} has been reported in ovarian, prostate, breast, and
lung cancer (Sutherland et al., 2020).\textsuperscript{6} Various
genetic polymorphisms are described in the \emph{SLCO1B1} gene; among
them, themost relevant SNPs related to drug disposition
are\emph{A\textsuperscript{388}G} (rs2306283)
and\emph{T\textsuperscript{521}C} (rs4149056). Reports have suggested
some controversial results with the \emph{A\textsuperscript{388}G}
variant as some studies have reported that
\emph{A\textsuperscript{388}G} is significantly correlated with the
increased expression of\emph{SLCO1B1} , assuring increased transporter
activity, while others have demonstrated unaltered transporter
function.\textsuperscript{13-15}On the other hand, variant
\emph{T\textsuperscript{521}C} has been correlated with lower expression
of OATP1B1 protein and significantly reduced transport activity compared
to the wild type (\emph{TT} ) genotype.\textsuperscript{13-16} Another
variant of OATP is an intronic region mutation of
\emph{SLCO1B3A\textsuperscript{1683-5676}G}(rs11045585) associated with
the docetaxel-induced leukopenia.\textsuperscript{17} \emph{SLCO1B3} is
a highly polymorphic gene that displays notable allele frequencies
variations among various ethnic populations. Polymorphism in the
\emph{SLCO1B3} gene has been correlated with the altered OATP activity
that often leads to drug-associated adverse events. Yamada \emph{et al.}
reported that the increased rate of the
\emph{A\textsuperscript{1683-5676}G} variant might contribute to the
reduced \emph{SLCO1B3} function that may alter therapeutic
efficacy.\textsuperscript{18}

Our research aimed to determine the role of~\emph{SLC19A1}
,\emph{SLCO1B1} , and \emph{SLCO1B3} gene polymorphism in North Indian
lung cancer patients. The findings could help us better understand the
molecular mechanisms underlying altered OATP expression,~anticancer drug
transport,~cancer development,~and therapy efficiency and see if these
transporters can be harnessed as potential diagnostic and predictive
molecular markers. To the best of our knowledge, no such studies
evaluating the role of these polymorphisms in lung cancer have been
undertaken in the North Indian lung cancer patients.

Methods

Sample collection and study design

Patients with lung cancer who visited the authors' institute's lung
cancer clinic (Post Graduate Institute of Medical Education and
Research, PGIMER) were included in the current study. The study enrolled
newly diagnosed individuals with histologically or cytologically proven
NSCLC and SCLC for three years. The institute's ethical committee
approved the study. The inclusion criteria of the lung cancer patients
meeting all the following requirements were eligible for enrollment: (i)
Diagnosis of lung cancer confirmed by cytology or histology (ii) Stage
IIIB - IV or IIIA (not scheduled for surgery). (iii) Untreated and
intent to treat with definitive platinum-based chemotherapy {[}platinum
agent (Cisplatin or Carboplatin) in combination with either docetaxel,
paclitaxel, gemcitabine, pemetrexed, or irinotecan{]}. (iv) An Eastern
Cooperative Oncology Group (ECOG) performance status (PS) of 0-2. (v) At
least one bi-dimensionally measurable lesion, according to the RECIST
criteria. (vi) Adequate organ function, defined as absolute neutrophil
count \textgreater{} 1500/\selectlanguage{greek}µ\selectlanguage{english}l, platelet count\textgreater{}100,000/\selectlanguage{greek}µ\selectlanguage{english}l,
and levels of creatinine, liver enzyme, and alanine aminotransferase
(ALT) less than two times the upper limits of normal (ULN).(vii) Written
informed consent was obtained. The exclusion criteria included (i)
Non-Bronchogenic tumors. (ii) Primary pleural tumors -- mesothelioma
(iii) Sarcomas (iv) Metastatic tumors to lungs or pleural (v)
Early-stage (resectable) lung cancer.

All of the participants signed an informed consent form. At the
beginning of this study, the patients had renal function tests, a
complete blood count (CBC), a contrast-enhanced computed tomography (CT)
scan of the thorax (which also encompassed the upper abdomen), liver
function tests, and a chest radiograph. The demographic characteristics
of the participants (gender, age, and performance status), disease
stage, tumor histology, and smoking status were all recorded. CBC was
done tendays after the first chemotherapy session, and any additional
cycles if needed. CBC, renal function, and~liver~tests were performed
before each treatment cycle and 3-4 weeks following the last
chemotherapy round. The paper was reported using the STROBE checklist.

Chemotherapy regimen

Docetaxel (75 mg/m2),~irinotecan (100 mg/m2), pemetrexed (500 mg/m2),~or
paclitaxel (175 mg/m2)~were given as a 1-hour infusion, followed by iv.
infusion of cisplatin (70 mg/m2) for over 3 hours. Four cycles of
chemotherapy were given before a tumor response assessment, as is the
usual procedure at PGIMER. Before the end of the four cycles, the tumor
response was assessed, and if there was undesirable toxicity or
clinic-radiological symptoms of cancer development, chemotherapy was
stopped if necessary. Subjects who exhibited an objective response to
chemotherapy were given two more treatment cycles (i.e., maximum of 6
cycles). Response Evaluation Criteria in Solid Tumors (RECIST) criteria
measure tumor response. The common toxicity criteria (CTC) version 3.0
was implemented to record and classify adverse events. PGIMER's
established procedures were used to manage any negative effects. We
assessed the following hematological toxicity parameters: leukopenia,
anemia, thrombocytopenia, and neutropenia. Diarrhea, nausea, and
constipation were investigated for gastrointestinal toxicity. Patients
with febrile neutropenia or grade 3 or higher gastrointestinal adverse
effects were admitted to the hospital~if outpatient treatment was not
available or effective. Any other side effects from the chemotherapy
were also noted. Follow-up was done every three weeks during
chemotherapy and every three months for the rest of the research.
Overall survival was calculated from chemotherapy to death or the last
follow-up date.

Genotyping of SLC variants

The genomic DNA was extracted from 3-4 ml of blood using the
phenol-chloroform extraction technique. The genotype of variations was
determined using the PCR-PFLP method as previously
mentioned.\textsuperscript{10,19,20} For amplifying a DNA fragment for
the \emph{SLC19A1} (\emph{G\textsuperscript{80}A} , rs1051266) variant
following sets of primers were used: forward primer 5'-
AGTGTCACCTTCGTCCCCTC-3' and reverse primer 5'- CTCCCGCGTGAAGTTCTT-3'.
For \emph{SLCO1B1 A\textsuperscript{388}G} in exon 4 (rs2306283), the
set of primers used was forward primer 5'-ATAATGGTGCAAATAAAGGGG-3'and
reverse primer 5'-ACTATCTCAGGTGATGCTCTA-3' and for \emph{SLCO1B1
T\textsuperscript{521}C} (rs4149056) variant, following sets of primers
were used: forward primer 5'-TTGTCAAAGTTTGCAAAGTG-3' and reverse primer
5'-GAAGCATATTACCCATGAGC-3'. For amplifying a DNA fragment for
the\emph{SLCO1B3} (\emph{A\textsuperscript{1683-5676}G} , rs11045585)
variant following sets of primers were used: forward primer
5'-GTGGGTAAAAGGCAGGTAAATG-3' and reverse primer 5'-GAATTCAAACATCTC
ACTGTGCTC-3'. The PCR mixture of 20 \selectlanguage{greek}µ\selectlanguage{english}l comprised of 1X PCR buffer, 0.5
\selectlanguage{greek}µ\selectlanguage{english}M of forward and reverse primer, 1.5 mM MgCl\textsubscript{2}, 100
\selectlanguage{greek}µ\selectlanguage{english}g/ml bovine serum albumin (BSA), 200 \selectlanguage{greek}µ\selectlanguage{english}M dNTPs, 1U Taq polymerase
(DNAzyme, Thermo Scientific), and 200 ng of DNA. PCR conditions used for
the mixture were: 95@\selectlanguage{english}C for 5 min and 94@\selectlanguage{english}C for 30s (denaturation), 59@\selectlanguage{english}C
(\emph{G\textsuperscript{80}A} /rs1051266), 55 @\selectlanguage{english}C
(\emph{A\textsuperscript{388}G} /rs2306283), 60@\selectlanguage{english}C
(\emph{T\textsuperscript{521}C} /rs4149056) and 57 @\selectlanguage{english}C
(\emph{A\textsuperscript{1683-5676}G} /rs11045585) for 45s (annealing)
followed by 72@\selectlanguage{english}C for 30s (extension) for the 29 cycles as well as the
final extension for 5 min at 72@\selectlanguage{english}C. For \emph{SLC19A1
G\textsuperscript{80}A} , a PCR product of 230 base pairs (bp) was
digested with 5U of \emph{HhaI} restriction enzyme (New England Biolabs)
at 37@\selectlanguage{english}C. The wild-type alleles were recognized as GG (125, 68, 37 bp),
variant alleles were identified as AA (162,68 bp), and heterozygous
alleles produced both bands (162,125,68,37 bp) (\textbf{Figure2a}
).\textsuperscript{10} For\emph{A\textsuperscript{388}G} , the PCR
product of 214 bp was checked on 2.0\% agarose gel and then digested
with 5U of \emph{TaqI} restriction enzyme (New England Biolabs),
respectively at 65@\selectlanguage{english}C. The wild type allele (\emph{AA} ) yielded bands of
151, 63 bp variant allele (\emph{GG} ), were identified by 128,63,23 bp,
and heterozygous displayed all the bands\textbf{(Figure-2b)} . For
\emph{T\textsuperscript{521}C} , a PCR product of 209 bp was digested
with 5U of \emph{HhaI} restriction enzyme (New England Biolabs) at 37@\selectlanguage{english}C.
The wild-type alleles were recognized as TT (209 bp), and variant
alleles were identified as CC (189, 20 bp) (\textbf{Figure2c}
).\textsuperscript{19} For\emph{A\textsuperscript{1683-5676}G} , a PCR
product of 286 bp was digested with 5U of \emph{Rsa I} restriction
enzyme (New England Biolabs) at 37@\selectlanguage{english}C. The wild type allele (AA) yielded
bands of 262, 24 bp, variant allele (GG) were identified by 155, 107, 24
bp, and heterozygous displayed all the bands \textbf{(Figure-2d)}
.\textsuperscript{20}Digested fragments were separated on 8 \% Native-
polyacrylamide gel electrophoresis (PAGE) and were stained with the
ethidium bromide and visualized under UV trans-illuminator. Two separate
people looked into the data to eliminate any potential biases, and 20\%
of the samples were chosen at random, ensuring that the outcomes were
100\% reproducible.

Statistical analysis

The study focused on the North Indian population and included age,
gender,~and smoking behavior information. The Chi-square goodness-of-fit
test was applied to see if the cases followed Hardy-Weinberg
equilibrium. The odds of lung carcinoma risk were studied using MedCalc
Statistical Software (version 14.8.1 MedCalc Software, Ostend, Belgium).
Descriptive data are presented as median, mean \selectlanguage{ngerman}± standard deviation
(SD), or number/ percentages. The odds ratio (OR) with the 95 \%
confidence interval~(CI)~evaluated using the logistic regression
analysis for any toxicity. All the patients' overall survival (OS) was
calculated from day one of chemotherapy to death or the last follow-up
date. The univariate Kaplan-Meir method assessed OS time using log Rank
p-value and median OS time. After adjusting for other factors,
multivariate Cox regression analysis was used to assess the independent
effect of polymorphism on overall survival. In all analyses, a p-value
of \textless{}0.05 was considered~significant.

Furthermore, the recursive partitioning strategy accounted for
high-order gene interactions. This method generates the initial split by
considering the critical factor~determining the patients' overall
survival. Survival analysis trees (STREE)~application was used to
generate the decision tree (http://c2s2.yale.edu/software/stree). The
log-rank approach was used as the splitting method. The generated tree
was binary, with each terminal node representing a subset of individuals
with a specific genotypic combination, thus displaying a range of
survival times and prognosis. Multivariate cox proportional hazard
analysis was used to determine the~HRs and 95\% CIs for every terminal
node, adjusted for gender, age, histology, ECOG,~KPS, stage, and
regimen.Toxicity in the lung cancer patients was assessed according to
standard National Cancer Institution Criteria 3.0
(http://ctep.cancer.gov). Maximum attention was paid to hematological
toxicity such as neutropenia, anemia, leukopenia and thrombocytopenia,
gastrointestinal toxicity nephrotoxicity. Severe toxicity or severe
event consisted of grade 3 or 4 hematologic toxicity and grade 1--4
nephrotoxicity. With severe hematologic toxicity, the subsequent
treatment was postponed until recovery to grade 1 or grade 0. With grade
3 and 4 gastrointestinal toxicity, the doses of each drug were reduced
by 25\%. If creatinine clearance decreases to within the range of 59\selectlanguage{english}-41
ml min\selectlanguage{english}-1, platinum was reduced by 25\%. If creatinine clearance
decreases below 40 ml min\selectlanguage{english}-1, platinum was stopped. Clinical data has
been systematically recorded during treatment.

Results

Patient characteristics and clinical predictors

Demographic characteristics of the case study group consisted of gender,
age, smoking status, Tumor, node, metastasis (TNM) staging, pack-years,
and various other clinical factors that are illustrated in \textbf{Table
1.} At the time of diagnosis, the lung cancer patients had an average
age of 60.10 years (range 29--86). Among 610 patients, 496 (81.3\%)
males and 114 (18.6\%) females were present in the case group. Smokers
accounted for 79.6\% of the group, more significant than non-smokers
(20.3\%). Smokers had an average pack-year of25.58+-31.88. TNM staging
was used to classify the patients: 0.81\% had stage I lung cancer,
3.11\% had stage II lung cancer, 27.5\% had stage III lung cancer, and
63.1\% had stage IV lung carcinoma.The lung cancer patients T3 and T4
exhibited high frequencies of 20\% and 56.8\%, whereas subjects with T1
and T2 tumor size constituted 5.90\% and 8.8\%, respectively. TNM data
revealed that 44.5\% of patients had no metastatic involvement (M0),
while 50.6\% had distant metastases (M1).Regarding lymph node invasion,
12.9\% of lung carcinomasubjects had no lymph node involvement, while
N1, N2, N3, and N4 lymph node invasion accounted for 8.3, 41.8, 31.9,
and 0.16\% of all cases, respectively. Histologically, squamous cell
carcinoma (SQCC) patients made up 40.8\% of lung cancer diagnoses,
adenocarcinoma (ADCC) patients made up 40.66\%, and small-cell lung
cancer (SCLC) patients made up 17.3\%. Karnofsky's performance status
(KPS) and the Eastern Cooperative Oncology Group (ECOG), which indicated
the participants' performance status, were used to classify the cases.
There were 168 individuals (27.5\%) with KPS scores of 100-90, 314
(51.4\%) with KPS scores of 80-70, and 106 people (17.3\%) with KPS
scores of less than 60 in our study. As per the ECOG score, 43.7\% (267)
of the people had ECOG scores ranging from 0 to 1, 38.1\% (233) had ECOG
2, and 14.5\% (89\%) of the people had ECOG scores of 3-4. A total of
419 patients received cisplatin/carboplatin-based therapy, with 130
patients receiving pemetrexed, 71 patients receiving irinotecan, 87
patients receiving paclitaxel, 70 patients receiving docetaxel, 29
patients receiving gemcitabine, 15 patients receiving gefitinib, 17
patients receiving etoposide, and 21 patients receiving other regimens.

Genotypic distribution of SLC variants

For \emph{SLC19A1 G\textsuperscript{80}A} polymorphism, 195(31.9\%)
patients were homozygous for the \emph{GG} genotype, 131 (21.4\%)
patients were homozygous for the \emph{AA} genotype, whereas 284
(46.5\%) patients were heterozygous (\emph{GA} ) \textbf{(Table
2).}While for \emph{SLCO1B1 A\textsuperscript{388}G} polymorphism, 152
(24.9\%) patients were harboring homozygous \emph{AA} genotype, 297
(48.6\%) were heterozygous \emph{AG} , and 161 (26.3\%) were
homozygote\emph{GG} variant whereas, for \emph{SLCO1B1
T\textsuperscript{521}C} , 563 (92.2\%) patients were homozygous for
\emph{TT} genotype, 45 (7.3\%) patients harbored heterozygous (TC)
genotype and 2 (0.32\%) individuals had homozygous (\emph{CC} )
genotype. For the \emph{SLCO1B3 A\textsuperscript{1683-5676}G} variant,
545 (89.34\%) patients were harboring homozygous \emph{AA} genotype, 64
(10.4\%) were heterozygous\emph{AG} , and 1 (0.16\%) were homozygote
\emph{GG} variant. The genotypic frequencies for \emph{SLC19A1
G\textsuperscript{80}A} was (\selectlanguage{greek}χ\selectlanguage{english}2=2.08,df=2; p=0.14). The genotypic
frequencies of \emph{SLCO1B1 A\textsuperscript{388}G} (\selectlanguage{greek}χ\selectlanguage{english}2=0.41,df=2;
p=0.52) and \emph{SLCO1B1 T\textsuperscript{521}C} polymorphism was
(\selectlanguage{greek}χ\selectlanguage{english}2=1.13, df=2; p=0.28) whereas for \emph{SLCO1B3
A\textsuperscript{1683-5676}G} genotypic frequency was \selectlanguage{greek}χ\selectlanguage{english}2=0.38,df=2;
p=0.53. Therefore, genotypic distribution for these four polymorphic
sites followed the Hardy-Weinberg equilibrium with a
p-value\textgreater{} 0.05, as shown in \textbf{table 2.} So, these
samples could be a Mendelian population in a state of genetic
equilibrium.

Association of SLC genotype with overall survival

The correlation of the \emph{SLC} polymorphisms with survival in 593
lung cancer samples was investigated. After three years, 83.1\% (493) of
patients had died, and 16.8\% (100) had survived. The association of
the\emph{SLC} ~SNPs with lung cancer survival was investigated using
both univariate and multivariate analysis after adjusting for smoking
status, gender,~age, tumor stage, ECOG,KPS, and histology. This analysis
used four alternative models (co-dominant, dominant, recessive, and
additive) to determine the relationship between \emph{SLC} ~polymorphism
and overall survival (OS) in lung cancer patients. As demonstrated
in\textbf{Table 3} , the findings were presented as the best-fitted
model. Lung carcinoma patients who were harboring mutant genotype
(\emph{AA} ) for \emph{SLC19A1 G\textsuperscript{80}A} polymorphism had
a higher median survival time (MST) as compared to the subjects carrying
wild type (\emph{GG} ) genotype (MST=9.33 versus 8.23, p=0.4).
The\emph{SLC19A1 G\textsuperscript{80}A} polymorphism did not show any
significant difference in survival in any of the models used.

For \emph{SLCO1B1 A\textsuperscript{388}G} , in the co-dominant model,
we observed very marginal lower survival in patients carrying both
mutant (\emph{GG} ) alleles as compared to the patients who were
carrying both wild-types(\emph{AA} ) alleles (MST=7.8 versus 7.9;
Log-rank p=0.41; HR=1.10). After applying the Cox regression model,
results demonstrated poor survival outcomes in patients carrying mutant
(\emph{GG} ) alleles when compared with patients carrying wild
(\emph{AA} ) allele (HR=1.40, 95\% I=1.0-1.97; p=\textbf{0.04} ), as
shown in \textbf{figure 3.} For\emph{SLCO1B1 T}
\textsuperscript{521}\emph{C} polymorphism, we observed lower survival
in the lung cancer patients who had mutant genotype (\emph{CC} ) as
compared to the patients carrying wild-type genotype (\emph{TT} )
(MST=5.16 versus 7.97; p=0.80). However, no significant differences in
terms of survival were observed for the \emph{SLCO1B1
T\textsuperscript{521}C} variant.

For the \emph{SLCO1B3 A\textsuperscript{1683-5676}G} variant, the
patients having heterozygous allele (\emph{AG} ) showed lower median
survival in lung carcinoma patients as compared to the individuals
harboring wild type (\emph{AA} ) genotype (MST=7.6 versus 7.97; p=0.58).
However, only one patient harbored mutant genotype, so the survival
differences were not calculated for this group\textbf{.} None of the
other models showed any prognostic significance
for\emph{A\textsuperscript{1683-5676}G} polymorphism for overall
survival in lung cancer patients.

Association of SLC polymorphism with histology and overall survival

We further assessed the role of SLC variants in predicting median
survival of lung carcinoma patients based on histological subtypes. On
analysis, we noted that ADCC patients harboring mutant genotype
(\emph{AA} ) showed better survival outcomes compared to the ADCC
subjects with wild-type genotype (\emph{GG} ) for \emph{SLC19A1
G\textsuperscript{80}A} (MST=9.4 versus 8.8,Log-rank p=0.2) as shown
in\textbf{Table 4} . After applying the cox regression model, there was
a decreased hazard ratio for lung cancer patients
(HR\textsuperscript{1}=0.60, 95\%CI=0.36-1.00, \textbf{p=0.04} ).

For \emph{SLCO1B1 A\textsuperscript{388}G} , patients diagnosed with
SQCC and harboring the mutant genotype (\emph{GG} ) showed poor survival
outcomes when compared to combined wild type and heterozygous genotype
(\emph{AA+AG} ) (MST=6.8 months versus 7.5 months; log-rank p=0.42).
After multivariate analysis, the overall death risk of patients
carrying\emph{GG} genotype at \emph{A\textsuperscript{388}G} was 1.54
times higher than individuals with \emph{AA+AG} genotype (HR=1.54, 95\%
CI=0.99-2.39; p=\textbf{0.05)} . However, on analyzing the association
of\emph{SLC19A1 G\textsuperscript{80}A} polymorphism with SCLC
histology, our results depicted increased survival in the patients
harboring mutant genotype (\emph{AA} ) as compared to the SCLC subjects
having wild type genotype (\emph{GG} ) (MST=9.6 months versus 7.6
months, p=0.34). After multivariate analysis, such patients had a low
death ratio than SCLC subjects carrying the wild genotype (\emph{GG} )
(HR=0.22, 95\%CI=0.05-0.93, \textbf{p=0.04, Table 4, Figure 4} ).
Similar results were also obtained when the recessive model was applied
(HR=0.30, 95\%CI=0.11-0.84, \textbf{p=0.02)} .None of the other SCLC
patients showed prognostic significance for \emph{the SLCO1B1
A\textsuperscript{388}G, T\textsuperscript{521}C,} and \emph{SLCO1B3
A\textsuperscript{1683-5676}G} polymorphism (\textbf{Supplementary Table
1).}

Association of SLC polymorphism with the chemotherapeutic regimen and
overall survival

The lung carcinoma patients in the study were administered
platinum-based chemotherapy along with paclitaxel, docetaxel,
pemetrexed, and irinotecan. So, we chose to investigate the modifying
effects of the \emph{SLC} ~polymorphism and its relationship to
chemotherapy, and overall patient survival, as shown
in\textbf{Supplementary Table 2.} Chemotherapy was divided into four
regimens, each given to different patient groups. All lung~malignancy
patients received platinum-based therapy (carboplatin/cisplatin) and a
second-line chemotherapy~treatment such as docetaxel,
pemetrexed,~paclitaxel, or irinotecan. For \emph{SLCO1B3
A\textsuperscript{1683-5676}G} polymorphism, our results showed that
patients who were given carboplatin/cisplatin along with the docetaxel
and carrying heterozygous alleles (\emph{AG} ) showed inferior survival
outcomes as compared to the patients having wild type genotype
(\emph{AA} ) (MST=2.9 months versus 9.6 months, Log-Rank
p=\textbf{0.006, HR=14.01} ). Upon adjusting with multivariate factors
such as age, gender, smoking, stage, histology, and performance status,
we observed a high hazard or death ratio in the patients having
heterozygous genotype (\emph{AG} ) as compared to the subjects harboring
wild type genotype (AA) (HR=3.84, 95\% CI=1.16-12.72; \textbf{p=0.02} ,
\textbf{Figure 5} ) as shown in \textbf{Supplementary Table 2} .

In the case of lung cancer subjects receiving carboplatin/cisplatin
along with irinotecan, paclitaxel, or pemetrexed, no substantial
correlation was seen between overall survival and \emph{SLC19A1,
SLCO1B1,} and \emph{SLC19A1} polymorphism \textbf{Supplementary Table 2}
.

Association of SLC polymorphisms with gender and OS

In lung carcinoma patients, 83.2\% (404) of males and 82.4\% (89) of
females were dead at the time of analysis. Males having \emph{SLC19A1
G\textsuperscript{80}A} polymorphism and possessing heterozygous
genotype (\emph{GA} ) showed reduced survival as compared to the
subjects harboring wild-type alleles (\emph{GG} ) (MST=7.13 versus 7.9
months; HR= 1.1; log-rank p=0.38). After using multivariate analysis and
adjusting for factors such as age, smoking, stage, histology, KPS, ECOG,
the death risk increased to 1.26 times in heterozygous genotype
(\emph{GA} ) as compared to the wild type (\emph{GG} ) genotype
(HR=1.26, 95\% CI=0.99-1.61; p=\textbf{0.05} ). Also,
for\emph{SLCO1B1A\textsuperscript{388}G} polymorphism, in the
co-dominant model, lower survival was observed in the males having
mutant genotype (\emph{GG} ) as compared to the subjects harboring wild
type genotype (\emph{AA} ) (MST=7.57 versus 7.7 months; HR=1.35, 95\%
CI=1.01-1.81; p=\textbf{0.04} ) as shown in \textbf{Supplementary Table
3} . However, we did not find any significant association of SLC
polymorphism in females with overall survival. We also evaluated the
survival differences in lung cancer patients according to age; however,
no significant association was found between SLC polymorphisms and
overall survival (Data not shown)

Association of SLC polymorphism and chemotherapy response

Correlation between \emph{SLC} polymorphism and the effectiveness of
response rate and clinical benefits were studied using univariate
logistic regression. \textbf{Supplementary Table 4} shows the
relationship between \emph{SLC} ~polymorphism and treatment
responses.The individuals showing stable and progressive disease were
categorized as non-responders, whereas subjects demonstrating complete
or partial response were classified as good responders. Our outcomes
showed no association between \emph{SLC} polymorphisms according to the
chemotherapeutic response to platinum-based doublet therapy. As a
result, we concluded that none of the SLC~polymorphisms might predict
chemotherapeutic response or clinical benefit in a significant way.

Association of SLCgenotype with tumor characteristics

In order to assess the correlation between \emph{SLC} variants and the
clinic-pathological parameters, the patients were bifurcated based on
cancer stage (stage III versus stage IV), lymph node involvement
(Nx+N0+N1 versus N2+N3+N4), primary tumor extension (T3 versus T4), and
metastasis (positive versus negative) as shown in \textbf{Supplementary
Table 5.} 18.7\% of lung cancer patients diagnosed with T4 tumor
extension carried mutant alleles for
SLC19A1\emph{G\textsuperscript{80}A} genotype compared to 30.3\%
patients in T3 tumor extension \textbf{(Table 5).} A decreased odds of
developing T4 was observed in mutant genotype (\emph{AA} ) as compared
to the patients categorized in T3 tumor extension (AOR=0.42, 95\%
CI=0.2-0.89; p=\textbf{0.02} ). Similarly, when the comparison was made
between the patients with T3 and T4 size in \emph{SLCO1B1
T\textsuperscript{521}C} , a decreased odds towards lung carcinoma was
observed in the heterozygous (\emph{TC} ) genotype of T4 as compared to
the patients with T3 tumor extension (AOR=0.29, 95\% CI=0.11-0.77,
p=\textbf{0.01)} . Using the dominant model, combining the two risk
genotypes (\emph{TC +CC} ), increased risk for lung cancer was not
observed in T4 compared to patients with T3 tumor extension (AOR=0.34,
95\% CI=0.13-0.88; p=\textbf{0.02} )\textbf{.} Therefore, our results
show that these genotypes did not show any relationship with the tumor
size and its progression.

High-order gene-gene interactions using STREE

Multiple interaction analyses~involving various genes implicated in SLC
transporters add a new dimension to assess the high-order parametric
interactions within them and their impact on the overall survival~of
lung cancer patients. The recursive partitioning strategy was adopted to
generate a survival tree in this study, demonstrating various SLC SNPs'
roles in prognosis. \textbf{Figure 6a} ~shows the tree generated by the
STREE software. The log-rank approach was used to generate the tree out
of all the methods available. There were a total of four-terminal nodes
found. \emph{SLC19A1G\textsuperscript{80}A} ~was the most crucial
factor~as it is a~root node influencing the prognosis of patients.
Terminal node 5 with the highest MST was used as a reference node (13.1
months). In addition, each node's HR is mentioned. The survival curves
and the MST associated with each terminal node are illustrated
in\textbf{Figure 6b} . It shows a~difference between the median survival
time of different nodes~and the reference node (log-rank p 0.18), though
a significant correlation was not observed. The findings of the Cox
regression analysis for every terminal node are summarized
in\textbf{Supplementary Table 6} . Significant differences were observed
in the median survival times when terminal node 4 was compared to the
reference node (4.8 months versus 13.1 months; HR=2.24, 95\% CI=
0.99-5.02; p=\textbf{0.05).} After adjusting with different covariates,
including age, gender, stage, smoking, histology, regimen, ECOG, and
KPS, the death risk was 1.94 times as compared to the reference node
(HR=1.94; p=\textbf{0.002} ). A similar trend was noted in all the other
nodes; however, significant differences were not achieved.

Association between SLC genotypes and toxicity

One of the current research goals was to look into the emergence of
toxicity in lung carcinoma~patients given platinum-based doublet
chemotherapy with~docetaxel, pemetrexed, irinotecan, and paclitaxel. The
odds ratio (OR) and 95 \% confidence interval (CI) for obtaining
different toxicity grades following therapy in participants with each
genotype were calculated using univariate logistic regression analysis.
The adjusted ORs were also evaluated using a multivariate regression
model with the toxicity grades as the dependent variable. Age, gender,
regimen, and~performance status, were all included in the multivariate
model~and were evaluated for~their interactions or~confounding effects.
We utilized three possible analysis methods to see how SLC polymorphisms
affected different toxicity levels. We compared patients with any
toxicity grade (grades 1-4) to no toxicity (grade 0) in the initial
assessment, individuals with intermediate/severe toxicity (grades 2-4)
to those with no/low-grade (grade 0-1) toxicity were compared in the
second analysis and the third assessment, and high-grade toxicity
(grades 3-4) versus no/low/intermediate toxicity (grades 0-2) to see if
the SLC~polymorphisms were linked to severe toxicity. In this manner,
cases were categorized into different toxicity levels.

All chemotherapy-related adverse effects~(AEs) were recorded for each
treatment cycle. In hematological toxicities, we evaluated~anemia,
leukopenia,~absolute neutropenia, and thrombocytopenia. In anemia, a
total of 406 patients' toxicity data was available \textbf{(Table 6a)} .
The table depicts that 25.1\% of patients were categorized in grade 0
toxicity, and74.8\% individuals were categorized in grade 1-4 toxicity.
As observed for \emph{SLCO1B1 T\textsuperscript{521}C} polymorphism,
7.14\% of individuals harbored single copy mutant allele (\emph{TC}
).\textbf{Supplementary Table 8} shows that subjects with the
heterozygous genotype (\emph{TC} ) had a reduced risk of developing
anemia (OR=0.44, 95\% CI=0.20-0.96); p=\textbf{0.04} ) when compared to
the patients with a wild type genotype (\emph{TT} ). For \emph{SLC19A1
G\textsuperscript{80}A} polymorphism, on comparing individuals~with
severe toxicity (grades 3-4) versus no/low-grade (grade 0-2) toxicity,
we observed that 26.6\% of patients in grade 3-4 anemia harboring
heterozygous (\emph{GA} ) genotype showed a reduced risk of anemia
(OR=0.35, 95\% CI=0.14-1.74; p=\textbf{0.02} ) as compared to the wild
type genotype (\emph{GG} ). We did not find any association between the
SLC polymorphisms other hematological toxicities such as leukopenia and
other AEs due to chemotherapy (\textbf{Supplementary Table 7} ).

For analysis of absolute neutrophil count (ANC), we compared
intermediate/severe toxicity (grades 2-4) versus no/low-grade (grades
0-1) toxicity as shown in \textbf{Supplementary Table 9} . A total of
367 patients' toxicity data was available for ANC. For \emph{SLCO1B1
A\textsuperscript{388}G} polymorphism, 16\% (3) patients harbored the
mutant genotype (\emph{GG} ). \textbf{Supplementary Table 9} depicts
subjects with mutant genotype (\emph{GG} )
of\emph{A\textsuperscript{388}G} had a reduced risk of developing
neutropenia toxicity (OR=0.42, 95\% CI=0.10-1.76, log-rank-p=0.23) when
compared to the individuals with wild type genotype (\emph{AA} ). After
adjusting with confounding, factors like age, gender, regimen, and
performance status, the decreased risk was consistent in mutant genotype
(\emph{GG} ) as compared to wild-type genotype (\emph{AA} ) (AOR=0.17,
95\% CI=0.02-1.04; p=\textbf{0.05} ).

Chemotherapy-induced thrombocytopenia is a potentially lethal
consequence resulting in chemotherapy dosage delays, reductions, or
discontinuance. Keeping this complication in view, we have analyzed the
impact of SLC variants in causing thrombocytopenia. As shown
in\textbf{Table 7,} we compared low/intermediate/severe toxicity (grades
1-3) versus no grade (grade 0) toxicity. For thrombocytopenia, there
were 367 patients with available~toxicity data, of which 32.1\% (18)
patients with \emph{SLCO1B1 A\textsuperscript{388}G} polymorphism
harboring a single copy of mutant allele (\emph{AG} ) was associated
with a lower incidence of thrombocytopenia when compared to the patients
carrying wild type alleles (\emph{AA} ) (OR=0.41, 95\% CI=0.20-0.82;
p=\textbf{0.01} ). After adjusting for confounding variables such as
age, gender, regimen, and performance status, the reduced risk in
heterozygous genotype (\emph{AG} ) for causing thrombocytopenia was
consistent when compared to wild-type genotype (\emph{AA} ) (AOR=0.35,
95\% CI=0.14-0.85; p=\textbf{0.02} ).

Renal functions are assessed during chemotherapy as chemotherapeutic
drugs are eliminated \emph{via} the kidneys and nephrotoxic. We have
evaluated chemotherapy-induced nephrotoxicity by comparing
intermediate/severe toxicity (grades 3-5) versus no/low grade (grades
1-2) toxicity\textbf{.} As shown in \textbf{table 6a} , data of 366
patients were available to assess the associationbetween the SLC
polymorphisms and nephrotoxicity.Lung carcinoma patients with
heterozygous (\emph{AG} ) genotype
for\emph{A\textsuperscript{1683-5676}G} polymorphism showed a lower
incidence of nephrotoxicity when compared to the patients with wild type
genotype (\emph{AA} ) (OR=0.35, 95\% CI=0.17-0.72; p=\textbf{0.002)} .
When confounding characteristics such as age, gender, regimen, and
performance status were taken into account, the reduced risk of
nephrotoxicity~in heterozygous~and mutant~genotype (\emph{AG+GG} ) was
consistent when compared to wild-type genotype (\emph{AA} ) (AOR=0.38,
95\% CI=0.15-0.98; p=\textbf{0.04} ) as shown in \textbf{table 7} .

Additionally, we~investigated~gastrointestinal (GI) toxicity
by~comparing patients~with low toxicity (grade 1) grade to
individuals~with intermediate-high toxicity (grade 2). We
evaluated~diarrhea, anorexia,~constipation, and
nausea-vomiting~\textbf{(Table 6b).} Lung cancer patient with the
heterozygous genotype (\emph{AG} ) for \emph{SLC19A1
A\textsuperscript{388}G} polymorphism has a reduced risk of having
constipation as compared to the patients harboring wild-type genotype
(\emph{AA} ) (AOR=0.17, 95\% CI=0.03-0.87, p=\textbf{0.03} ) as shown
in\textbf{Table 8} . The reduced risk was consistent when we compared
the combined heterozygous and mutant (\emph{AG+GG} ) genotype with the
wild type (\emph{AA} ) genotype (AOR=0.22, 95\% CI=0.05-0.97;
p=\textbf{0.04} ). For \emph{SLCO1B1 A\textsuperscript{388}G} , lung
cancer patients with heterozygous genotype (AG) revealed a 3.87-fold
increased risk of having constipation (AOR=3.87, 95\% CI=0.91-16.48;
p=\textbf{0.04} ) as compared to the patients with wild type genotype
(\emph{AA} ).

In nausea/vomiting, the comparison was made between intermediate/ severe
toxicity (grades 2-3) versus low-grade (grade 1) toxicity. Among 116
patients, 54.3\% experienced grade 2-3 nausea/vomiting. For\emph{SLCO1B1
A\textsuperscript{388}G} polymorphism, a lower incidence of
nausea/vomiting was observed in patients with mutant genotype (\emph{GG}
) as compared to patients~with wild-type genotype (\emph{AA} ) (AOR=
0.22; 95\%CI=0.05-1.03, p=\textbf{0.05 (} T\textbf{able 8)} . We did not
find any association between the SLC polymorphisms, other
gastrointestinal toxicities such as diarrhea and anorexia, and other AEs
due to chemotherapy (\textbf{Supplementary Table 10} ).

Discussion

SLC transporters mediate the flow of chemotherapeutic drugs~across
biological membranes in various organs, and the various single
nucleotide polymorphisms alter this inflow of drugs. Genetic
polymorphisms can cause individual variations in the metabolism and
pharmacotherapy of these~transporters, which may create
population-specific disparities in~drug transport. In a population,
heterogeneity in the pharmacokinetic profile of chemotherapeutic drugs
results from multiple interactions between genetic, environmental, and
physiological factors. In the past, many theories have been put forth to
explain the observed discrepancies regarding several therapeutic
drugs~that may increase or inhibit the function or expression of solute
carrier protein, thus altering the phenotypic activity of the~SLC
transporters. Given the effect of these polymorphisms on SLC transporter
activity, it is essential to look into their role in lung cancer, as
their expression levels could control the extent and duration of
chemotherapeutic drug inflow, affecting patients' responses. Hence,
understanding and minimizing inter-individual variation in drug
responsiveness, toxicity, and sensitivity~and its critical role in
identifying the efficient treatment option for lung carcinoma~patients
is therefore imperative and clinically meaningful.

We have attempted to investigate the significance of SLC polymorphisms
in influencing survival rates and their correlation with toxicity in
lung cancer patients undergoing platinum-based chemotherapy treatment.
Our results from North Indian lung carcinoma patients suggest that
mutant genotype (\emph{AA} ) of
\emph{SLC19A1G\textsuperscript{80}A}polymorphism is correlated with
increased survival. Our results align with Collin \emph{et al.,} who
found no correlation of\emph{G\textsuperscript{80}A} polymorphism in
recessive, dominant, or additive models in prostate
cancer.\textsuperscript{21} A study by Liu\emph{et al} . did not find
any significant association for\emph{RFCvG\textsuperscript{80}A}
polymorphism with lung cancer susceptibility.\textsuperscript{22}
However, we can explain the increased survival trend in lung cancer
patients with mutant genotype (\emph{AA} ) of
\emph{SLC19A1G\textsuperscript{80}A} polymorphism. The\emph{A} allele of
\emph{G\textsuperscript{80}A} might have a greater influx propensity for
the substrates, resulting in improved bioavailability and
chemotherapeutic drug intake,\textsuperscript{23}thereby leading to
better survival outcomes in the individuals with the mutant genotype
(\emph{AA} ). On the contrary to our outcomes, Jabeen\emph{et al.} has
reported better survival in the patients with wild-type genotype
(\emph{GG} ) in comparison to the mutant genotype (\emph{AA} ) of
\emph{SLC19A1 G\textsuperscript{80}A}polymorphism.\textsuperscript{24}
These incongruent results could be attributed to the differences in
clinical characteristics and ethnicity of patients.

In patients with \emph{SLCO1B1A\textsuperscript{388}G} polymorphism, our
results demonstrated that the patients with mutant genotype (\emph{GG} )
had the same median survival time as the wild-type genotype \emph{(AA)}
. Our results are in concordance with \emph{Lee et al.,} who put forth
that \emph{SLCO1B1 A\textsuperscript{388}G} is associated with an
unaltered transport function, so there would be no modification in the
drug influx, which could be the likely cause for no difference in median
survival time between mutant \emph{(GG)} and wild type
genotypes\emph{(AA)} .\textsuperscript{25} On the contrary, Sissung
\emph{et al.}stated that \emph{A\textsuperscript{388}G} polymorphism is
correlated with the altered SLCO1B1 transport function, leading to
modifications in the transmembrane domain structure.\textsuperscript{26}
While Liutkevicie \emph{et al.} put forth that the \emph{G} allele
of\emph{A\textsuperscript{388}G} is associated with the increased
SLCO1B1 function, proposing increased functional activity of the
transporter.\textsuperscript{27} \emph{SLCO1B1} polymorphism has been
reported by numerous researchers, who have linked
the\emph{A\textsuperscript{388}G} mutation to an increase, decrease, or
no effect on the transporter activity.\textsuperscript{28} These
disparate results could be ascribed to ethnicity and clinical
characteristics of patients, such as the methodology used to detect the
expression of SLC and heterogeneity in tumor histology, all of which
could play a decisive role.

Our results for \emph{SLCO1B1 T\textsuperscript{521}C} polymorphism
showed reduced survival in patients with mutant genotype (\emph{CC} ) as
compared to both homozygous (\emph{TT} ) and heterozygous genotype
(\emph{TC} ), though the substantial significance
between\emph{T\textsuperscript{521}C} polymorphism and overall survival
was not achieved. Studies in the past have shown that the \emph{C}
allele of\emph{T\textsuperscript{521}C} is correlated with decreased
activity of SLCO1B1.\textsuperscript{29} Due to this reduced activity of
the transporter, patients harboring the variant allele (\emph{C} )
of\emph{T\textsuperscript{521}C} polymorphismmay have reduced inflow of
drugs, which can explain the lower survival in lung cancer patients. On
the contrary, Feng \emph{et al.} has shown better survival in gastric
cancer patients having mutant \emph{(CC)} and heterozygous
\emph{(TC)}genotype.\textsuperscript{30}

We have also assessed the impact of SLC polymorphism on overall survival
based on histology. Our findings demonstrated that ADCC patients with
the mutant genotype \emph{(AA)} of the \emph{SLC19A1
G\textsuperscript{80}A} polymorphism had a better prognosis
(\textbf{p=0.04} ). Mechanistically, the RFC1 protein is encoded by the
main influx transporter \emph{SLC19A1. I} t mediates folate uptake in
the cell and transports antifolate chemotherapeutic agents. RFC1 is
substantially expressed in lung cancer patients.\textsuperscript{31} It
has been reported that~in adenocarcinoma cells, RFC1 proteins are
overexpressed.\textsuperscript{32}

Furthermore, studies have linked downregulation of the RFC1 protein to
impaired drug transport, developing
resistance.\textsuperscript{24,33}The overexpression of RFC1 in ADCC
patients explains our results with better survival in the mutant
genotype \emph{(AA)} of \emph{SLC19A1 G\textsuperscript{80}A}
polymorphism. Even though \emph{SLC19A1} is the most common antifolate
uptake transporter, our results did not show any association of
\emph{G\textsuperscript{80}A} polymorphism with the survival of the
patients undergoing pemetrexed and cisplatin/carboplatin treatment. Our
results align with the various studies on NSCLC.\textsuperscript{34-36}
Our findings also revealed that patients with SCLC who had the mutant
genotype \emph{(AA)} of \emph{SLC19A1 G\textsuperscript{80}A}
polymorphism had a better prognosis (p=0.04). To the best of
our~knowledge, none of the~previous studies have looked at the role of
the \emph{SLC19A G\textsuperscript{80}A} polymorphism in SCLC, as~most
of the research has focused on its impact on NSCLC. Based on our
findings, we believe that the \emph{SLC19A1 G\textsuperscript{80}A}
polymorphism may improve overall survival in SCLC patients.

As per our results, patients who were given docetaxel along with
cisplatin/ carboplatin and were heterozygous carriers \emph{(AG)} for
the \emph{SLCO1B3 (A\textsuperscript{1683-5676}G)} polymorphism had a
significantly shorter survival time as compared to the wild
type\emph{(AA)} genotype. No supporting studies were found because the
pharmacogenetics of \emph{SLCO1B3} is little understood, and
previous\emph{in vivo} and \emph{in vitro} studies gave inconsistent
results regarding the functional effects of the \emph{SLCO1B3}
polymorphisms. Chew \emph{et al.} revealed that the \emph{SLCO1B3
(A\textsuperscript{1683-5676}G)} variant allele is related to altered
docetaxel disposition.\textsuperscript{37} However,~our investigation
did not find any patients with mutant genotype \emph{(GG)}
for\emph{SLCO1B3 (A\textsuperscript{1683-5676}G)} polymorphism
administered docetaxel and cisplatin/carboplatin therapy, and we could
not assess the role of its variant genotype on the survival of lung
cancer patients. Chew \emph{et al.} have mentioned that~inter-individual
variability in docetaxel disposition is influenced by
\emph{SLCO1B3}pharmacogenetics.\textsuperscript{38} However, more
research is needed to investigate the functional characterization
of\emph{SLCO1B3} -associated docetaxel transport, which may help
researchers better understand the molecular basis for docetaxel disposal
disposition.

We have also evaluated whether \emph{SLC} polymorphisms were associated
with toxicity among platinum-based chemotherapy-treated lung cancer
patients. Our results showed that patients with at least one mutant
allele \emph{(AG)} in \emph{SLCO1B1 A\textsuperscript{388}G} had~a
significantly lower risk of developing thrombocytopenia. Leiri \emph{et
al.} have reported that the presence of the \emph{G} allele at the codon
388 can modulate the activity of SLCO1B1.\textsuperscript{39} The
systemic concentration of the chemotherapeutic drug is strongly
dependent on the activity of SLCO1B1. An increase in the SLCO1B1
activity and subsequent increase in the hepatic clearance of the
chemotherapeutic drug may lead to reduced systemic exposure and hence
less toxicity.\textsuperscript{40} This may explain the reduced risk of
developing thrombocytopenia in the heterozygous \emph{(AG)} genotype
of\emph{A\textsuperscript{388}G,} as the occurrence of
chemotherapy-induced thrombocytopenia varies according to the treatment
used. Our findings showed a significant association of \emph{SLC19A1
G\textsuperscript{80}A} polymorphism with a reduced risk of anemia. The
heterozygous genotype \emph{(GA)} of
\emph{G\textsuperscript{80}A}polymorphism showed a substantial
protective effect from hematological toxicity-anemia. Generally, folate
is required for RBC production and is a typical target of
chemotherapeutic agents.\textsuperscript{41} These drugs impede folate
metabolism, and a deficiency of folate causes DNA synthesis
abnormalities, which leads to genomic instability leading to the
hypothesis that a lack of folic acid inhibits RBC maturation, resulting
in anemia. However, our results suggest that the heterozygous genotype
\emph{(GA)} of \emph{G\textsuperscript{80}A} polymorphism is associated
with a reduced risk of developing anemia in lung cancer patients.

Our results showed a reduced risk of developing nephrotoxicity in
patients harboring heterozygous genotype (\emph{AG} ) on comparing
severe toxicity (grade 3-5) with the absence of any/ intermediate
toxicity (grade 1-2). Mechanistically, the kidney is the main organ
involved in eliminating platinum-based chemotherapeutic
drugs.\textsuperscript{42}Many drugs, particularly those with a
propensity for nephrotoxicity, are eliminated in the urine by active
tubular secretion in addition to glomerular
filtration.\textsuperscript{43} \emph{In vitro} studies have shown
that~\emph{OATP1B1} and \emph{OATP1B3} transport~cisplatin and
carboplatin, and the toxicity of these drugs in human tumor cells was
enhanced with increased \emph{OATP1B3} mRNA
expression.\textsuperscript{4} Hilgendorf \emph{et al.,} have reported
low mRNA expression of \emph{SLCO1B3} in the
kidney.\textsuperscript{44}Also, it has been reported that the
\emph{A\textsuperscript{1683-5676}G}variant may lead to the reduced
activity of \emph{SLCO1B3} that might vary the therapeutic
efficacy.\textsuperscript{18} These findings explain that \emph{SLCO1B3}
has reduced expression in the kidney, leading to reduced drug uptake and
thereby showing a reduced risk of causing nephrotoxicity.

Gastrointestinal (GI) toxicity is a typical adverse effect of
chemotherapy treatments in lung cancer patients. We observed that
heterozygous genotype of \emph{SLC19A1 G\textsuperscript{80}A}
and\emph{SLCO1B1 A\textsuperscript{388}G} polymorphisms were associated
with protective effects from gastrointestinal toxicity.Due to a scarcity
of research, determining the actual incidence rate of
chemotherapy-induced constipation across all cancer patients is
challenging. Some studies state that \emph{SLCO1B1} polymorphisms are
correlated with the increased risk of gastrointestinal toxicity in acute
lymphoblastic leukemia.\textsuperscript{45} In the case of\emph{SLC19A1}
, \emph{Lima et al.} found that \emph{G} carriers
of\emph{G\textsuperscript{80}A} polymorphism were linked to
chemotherapy-associated~gastrointestinal toxicity \textsuperscript{10},
whereas various other studies did not find any correlations with the
toxicity.\textsuperscript{46,47} Several factors, including the
patient's characteristics, dose schedule, the type of chemotherapy, and
regimen employed, influence the severity and extent of
chemotherapy-induced toxicity. Therefore, these conflicting data
of\emph{SLC} polymorphism and chemotherapy-related gastrointestinal
toxicity need to be clarified.

It is essential to mention~that the present study has certain
limitations~since carcinoma is often detected at a late~stage; our study
only includes patients with advanced lung cancer. This could be because
of public awareness or its~similarity to other diseases like
tuberculosis.Many patients who receive chemotherapy at an advanced stage
of cancer cannot achieve the treatment's endpoint, weakening the
research's scope on chemotherapy response.The majority of the people who
came to PGIMER for treatment were from rural areas in Northern
India.Since it was difficult for them to commute from their homes, many
patients in remote areas did not regularly visit the hospital. Some
patients left the clinic~before or after six chemotherapy cycles, while
others did~not.

Due to the shortage of clinical data could not conduct progression-free
survival analysis on the current study~group.On the other hand, our
study has several advantages, including enrollingmany lung cancer
patients.Second, patients on platinum-based chemotherapy regimens were
included, and everyone was treated at the same hospital.Third, patients
were enrolled, and clinical parameter data was obtained separately,
regardless of the information of SLC polymorphism.

Conclusion

In the present study, we have investigated the significance of four SLC
polymorphisms on the survival of North Indian lung carcinoma~patients
undergoing platinum-based chemotherapy regimens. Briefly, we used
genotyping analysis to see if \emph{SLC} polymorphism causes differences
in patient clinical outcomes. Our findings imply that SLC polymorphisms
are essential in regulating~SLC gene expression, altering
the~transporter activity, and affecting~overall survival in lung cancer
patients. To the best of our knowledge, this is the first study to
evaluate the role of \emph{SLC} polymorphism in North Indian lung cancer
patients.According to our findings, genotyping for the
\emph{SLC}polymorphism could be a valuable tool for predicting which
lung cancer patients will benefit the most from platinum-based
chemotherapy.However, a review of previous papers reveals a plethora of
contradictory findings of the activity of these transporters.
Nonetheless, more research into the role of SLC~polymorphisms is needed
before a meaningful conclusion can be reached.

When adopting this technique in the clinic, significant caution should
be exercised to better and personalize therapeutic choices to predict
prognosis and therapy outcomes. Our study evaluates the survival
differences in lung cancer patients with \emph{SLC} polymorphisms.
Surprisingly, our findings demonstrate a link between
\emph{SLC}polymorphism and a lower risk of toxicity in~north Indian lung
cancer patients.It is well understood that individual toxicities are
determined by the platinum-based chemotherapy regimen used. In the
future, further studies should be explored to analyze the impact of
these SLC polymorphisms on the activity of SLC~transporter in order to
identify the involvement of a specific polymorphism in the
pharmacokinetics,~pharmacological activity,~and toxicity of
chemotherapeutic agents. Such insights could lead to pharmacogenetically
enhanced and individualized drug dosing, correspondingto pharmacokinetic
discrepancies in chemotherapeutic drugs between individuals.

\textbf{Competing interests}

All of the authors of this manuscript report that they have no conflict
of interest

\textbf{Funding sources}

This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for profit sectors.

\textbf{Author contributions}

P.S. collected information, performed molecular and statistical
analyses, and wrote the first paper draft; S.S. assisted molecular
analyses and reviewed the final version of the paper and acted as the
corresponding author; N.S. provided with the blood samples and clinical
data of the patients from PGIMER, Chandigarh.

\textbf{Data availability statement}

All the data generated or analyzed during this study are included in
this article. Further queries can be directed to corresponding author.

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\textbf{Figure legends}

\textbf{Figure 1: Schematic model of SLC transporters in~organs
necessary for drug distribution, absorption and elimination}

\textbf{Figure 2a: 6.0\% Native-PAGE of PCR products for the detection
of SLC19A1 G80A polymorphism. Lane 1: Marker (M) (100 bp); Lane 2:
Undigested PCR product; Lane 3, 6, 9: Homozygous wild (GG) genotype;
Lane 4,7,10: Heterozygous (GA) genotype; Lane 5,8: Homozygous variant
(AA) genotype}

\textbf{Figure 2b: 6.0\% Native-PAGE of PCR products for the detection
of \emph{SLCO1B1} A388G polymorphism. Lane 1: Marker (M) (100 bp); Lane
2: Undigested PCR product; Lane 5, 8: Homozygous wild (AA) genotype;
Lane 4,7,10: Heterozygous (AG) genotype; Lane 3,6,9: Homozygous variant
(GG) genotype}

\textbf{Figure 2c: 6.0\% Native-PAGE of PCR products for the detection
of \emph{SLCO1B1} T521C polymorphism. Lane 1: Marker (M) (100 bp); Lane
2,3,4: Homozygous wild (TT) genotype; Lane 5,8: Heterozygous (TC)
genotype; Lane 6: Homozygous variant (GG) genotype}

\textbf{Figure 2d: 6.0\% Native-PAGE of PCR products for the detection
of \emph{SLCO1B3} A1683-5676G polymorphism. Lane 1: Marker (M) (100 bp);
Lane 2: Undigested PCR product; Lane 3,5,10: Homozygous wild (AA)
genotype; Lane 4,6,9: Heterozygous (AG) genotype; Lane 7: Homozygous
variant (GG) genotype}

\textbf{Figure 3: Kaplan Meier curves illustrating the association
between overall survival in different genotypes of \emph{SLCO1B1
(A388G)} polymorphism in lung cancer patients. (Note: AA represents
wild-type genotype, AG represents heterozygous genotype, and GG
represents homozygous for mutant genotype)}

\textbf{Figure 4: Kaplan Meier curves illustrating the association
between overall survival in different genotypes of \emph{SLC19A1
(G80A)}polymorphism in SCLC patients. (Note: GG represents wild-type
genotype, GA represents heterozygous genotype, and AA represents
homozygous for mutant genotype)}

\textbf{Figure 5: Kaplan Meier curves illustrating the association
between overall survival in different genotypes of \emph{SLCO1B3
(A1683-5676G)} polymorphism in patients administered with docetaxel
cisplatin/carboplatin. (Note: AA represents wild-type genotype and AG
represents heterozygous genotype)}

\textbf{Figure 6a: Recursive partitioning method (STREE) explaining
correlation of \emph{SLC} SNPs with overall survival in lung carcinoma
patients}

\textbf{Figure 6b: Overall survival according to different nodes
identified by survival tree analysis.}

\textbf{HR, hazard ratio; MST, median survival time}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2a/Figure-2a}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2b/Figure-2b}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2c/Figure-2c}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2d/Figure-2d}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\textbf{Hosted file}

\verb`Tables.docx`  available at \url{https://authorea.com/users/740631/articles/713409-association-between-genetic-polymorphisms-in-slc19a1-slco1b1-and-slco1b3-genes-predicts-survival-and-toxicity-in-north-indian-lung-cancer-patients-undergoing-platinum-based-doublet-chemotherapy}

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