Peritoneal carcinomatosis: Limits of diagnosis and the case for liquid biopsy

Abstract

Peritoneal Carcinomatosis (PC) is a late stage manifestation of several gastrointestinal malignancies including appendiceal, colorectal, and gastric cancer. In PC, tumors metastasize to and deposit on the peritoneal surface and often leave patients with only palliative treatment options. For colorectal PC, median survival is approximately five months, and palliative systemic therapy is able to extend this to approximately 12 months. However, cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) with a curative intent is possible in some patients with limited tumor burden. In well-selected patients undergoing complete cytoreduction, median survival has been reported as high as 63 month. Identifying patients earlier who are either at risk for, or who have recently developed PC may provide them with additional treatment options such as CRS/HIPEC. PC is diagnosed late by imaging findings or often times during an invasive procedures such as laparoscopy or laparotomy. In order to improve the outcomes of PC patients, a minimally invasive, accurate, and specific PC screening method needs to be developed. By utilizing circulating PC biomarkers in the serum of patients, a " liquid biopsy, " may be able to be generated to allow a tailored treatment plan and early intervention. Exosomes, stable patient-derived nanovesicles present in blood, urine, and many other bodily fluids, show promise as a tool for the evaluation of labile biomarkers. If liquid biopsies can be perfected in PC, manifestations of this cancer may be more effectively treated, thus offering improved survival.

Full text

Oncotarget1
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www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2017
Peritoneal carcinomatosis: limits of diagnosis and the case for
liquid biopsy
James R.W. McMullen1, Matthew Selleck2, Nathan R. Wall1 and Maheswari Senthil2
1 Department of Basic Sciences, Center for Health Disparities & Molecular Medicine, Division of Biochemistry, Loma Linda
University Medical Center, Loma Linda, CA, USA
2 Department of Surgery, Division of Surgical Oncology, Loma Linda University Medical Center, Loma Linda, CA, USA
Correspondence to: Nathan R. Wall, email: nwall@llu.edu
Correspondence to: Maheswari Senthil, email: msenthil@llu.edu
Keywords: peritoneal carcinomatosis, liquid biopsy, biomarker, exosomes
Received: January 21, 2017 Accepted: March 15, 2017 Published: March 22, 2017
ABSTRACT
Peritoneal Carcinomatosis (PC) is a late stage manifestation of several
gastrointestinal malignancies including appendiceal, colorectal, and gastric cancer.
In PC, tumors metastasize to and deposit on the peritoneal surface and often leave
patients with only palliative treatment options. For colorectal PC, median survival
is approximately ve months, and palliative systemic therapy is able to extend this
to approximately 12 months. However, cytoreductive surgery with hyperthermic
intraperitoneal chemotherapy (CRS/HIPEC) with a curative intent is possible in
some patients with limited tumor burden. In well-selected patients undergoing
complete cytoreduction, median survival has been reported as high as 63 month.
Identifying patients earlier who are either at risk for, or who have recently developed
PC may provide them with additional treatment options such as CRS/HIPEC. PC is
diagnosed late by imaging ndings or often times during an invasive procedures such
as laparoscopy or laparotomy. In order to improve the outcomes of PC patients, a
minimally invasive, accurate, and specic PC screening method needs to be developed.
By utilizing circulating PC biomarkers in the serum of patients, a “liquid biopsy,” may
be able to be generated to allow a tailored treatment plan and early intervention.
Exosomes, stable patient-derived nanovesicles present in blood, urine, and many
other bodily uids, show promise as a tool for the evaluation of labile biomarkers.
If liquid biopsies can be perfected in PC, manifestations of this cancer may be more
eectively treated, thus oering improved survival.
INTRODUCTION
Peritoneal Carcinomatosis (PC) is a late stage
manifestation of several gastrointestinal malignancies
characterized by tumor deposition across the peritoneal
surface [1]. This can be entirely asymptomatic in its early
stages, or as the disease progresses, symptoms such as
nausea, diarrhea, abdominal pain, bloating, and weight
loss may develop [1]. The disease is often discovered
when ascites or intestinal obstruction develop, occurring
generally with greater tumor burden which is more
dicult to treat [2]. Early PC detection when there is
limited tumor burden may increase the eectiveness of
current treatment options [3].
Colorectal cancer (CRC), the third most common
cancer in the world, provides a good case study of PC. In
2016 there will be an estimated 95,270 cases of colorectal
cancer in the United States and nearly 1.4 million cases
worldwide [4]. Synchronous PC is diagnosed around the
time of diagnosis of primary tumor while metachronous
PC is diagnosed at a later time, typically months to
years after the original diagnosis [5]. The incidence
of synchronous isolated peritoneal carcinomatosis in
patients with CRC varies somewhat in the literature from
4%-18% [6-9]. This may even be a low estimate given
the lack of sensitivity of imaging for PC and that it may
not be discovered until surgical exploration. Meanwhile,
metachronous PC has been reported in 5-19% of patients
following denitive treatment.

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CYTOREDUCTIVE SURGERY (CRS)
PC used to be considered a lethal disease with
no curative surgical options. However, the growing
acceptance of CRS with hyperthermic intraperitoneal
chemotherapy (HIPEC), has oered the possibility of
improved survival for carefully selected patients [3].
This method makes use of “cytoreduction” to surgically
remove gross visible tumor deposits followed by direct
contact of heated cytotoxic chemotherapy agents to
aect any residual disease. Given during surgery, this
protocol maximizes potential contact with the peritoneal
surface while minimizing systemic toxicity. Specically,
hyperthermia between 41 and 43 degrees centigrade is
combined with large molecular weight drugs that penetrate
between a few cells deep to 3mm causing cytotoxic eects
[10]. Dr. Paul Sugarbaker is credited with developing this
treatment option by combing these elements into a curative
approach to peritoneal dissemination of gastrointestinal
malignancies [11, 12]. Median survival of CRC PC
without any treatment is approximately 4-7 months, while
palliative systemic therapy may extend this to 12-23
months based on several series [13-15]. Median overall
survival with CRS/HIPEC has been reported to range
from 22 to 63 months with a 5-year survival of 40-51%
in selected patients [13, 15, 16]. The outcomes of CRS/
HIPEC are strongly inuenced by careful patient selection
and complete cytoreduction (CC-0) (see Table 1) [17].
Survival of patients with colorectal cancer who receive
less than complete cytoreduction (CC-1 or CC-2) or have
a higher burden of disease as indicated by the peritoneal
carcinomatosis index (PCI) (see Figure 1) is signicantly
diminished compared to that of a CC-0 resection [17-19].
Extensive disease burden at identication often leaves
patients with only palliative treatment options [20].
Despite the benet of CRS/HIPEC, only about 25% of
patients with PC will be eligible for this approach given
the late presentation and burden of disease. In order to
expand patient eligibility and oer treatment with a
curative intent, early detection of PC, before signicant
tumor burden develops, is essential.
IMAGING
Traditional imaging, such as CT and MRI lack
sensitivity to both detect and estimate disease burden
in PC. Classic computed tomography (CT) signs of PC
such as “omental caking,” thickening of the omentum,
and peritoneal nodules are not common radiographic
ndings in “early” disease states. Several studies designed
to determine the specicity and accuracy of CT scans in
assessing tumor burden for PC found that CT signicantly
underestimated the amount of disease present in the
peritoneal cavity [21-24]. Sensitivity for CT detection
of tumor nodules less than 0.5 cm and 1cm had been
reported to be 11% and 25-50% respectively [23]. This
is particularly important in colorectal PC, where PCI is
a critical determinant of complete cytoreduction and
Figure 1: Peritoneal Cancer Index (PCI) scoring system. PCI is a diagnostic and prognostic tool that is a sum of scores in thirteen
abdominal regions. Each receives a score of 0-3 based on the largest tumor size in each region. Scores range from 0 to 39. Higher scores
indicate more widespread and/or larger tumors in the peritoneal cavity.

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long-term outcomes [25]. In a study by Koh et al looking
specically at CRC PC they determined CT signicantly
underestimated clinical PCI [23]. In fact sensitivity of
small bowel involvement in each region ranged from
8-17%. Despite this, the Fifth International Workshop on
Peritoneal Surface Malignancy in Milan, identied CT as
the principal imaging modality to assess suitability for
CRS [26]. Ultrasound also has very limited sensitivity
for detecting PC nodules [27-29]. Magnetic resonance
imaging (MRI), and particularly diusion weighted
images, has been demonstrated in prospective studies to
have increased accuracy in detection of carcinomatosis
within certain areas of the abdomen [30]. This however
carries its own limitations due to the motion artifacts
of peristalsis, cost, and the need for radiologists trained
in their interpretation and inter-observer variation.
Additionally, positron emission technology (PET) may
have increased sensitivity, but similar limitations and
absence of added clinical value often precludes its use for
determining resectability [24, 31-33]. These diculties,
especially limited sensitivity, lack of meaningful clinical
correlation and high cost, reduce the utility of non-
invasive imaging in the early detection of PC. With the
current technology, laparoscopy or exploratory surgery is
often necessary to conrm the diagnosis and extent of PC
(see Table 2) [34].
LIQUID BIOPSY
Given the narrow subset of patients who are oered
CRS/HIPEC, a potentially life-saving treatment, we are
in need of a paradigm change. Patients who are either at
risk for developing PC or who are in the earliest stage
of this disease process may signicantly benet from
expanded treatment options. The term “liquid biopsy” has
reached prolic use as large-scale investigations seek to
identify tumor markers in the serum. This usually refers to
molecular diagnostic studies that are performed on blood
or body uid as opposed to cancerous tissue itself [35].
Multiple serum tumor markers: carcinoembryonic antigen
(CEA), carbohydrate antigen CA 19-9, and CA 125, are
commonly elevated in patients with PC and the degree of
elevation tends to correlate with the extent of PC [36].
However, these serum tumor markers are inadequate for
early detection of PC. Moreover, they lack specicity to
predict the presence or risk of PC in patients with CRC.
There is a critical clinical need to identify circulating
tumor biomarkers of aggressiveness, likelihood of
recurrence, risk of metastasis such as PC, or even the
presence of a malignancy to better tailor therapy for
patients. For example, if a patient with a newly diagnosed
stage III colorectal cancer is known to be at signicant risk
for peritoneal recurrence due to the presence of a specic
set of biomarkers in their serum, they may benet from
prophylactic HIPEC. This is just one example of how this
Table 1: CC is the completeness of cytoreduction score. It indicates the size of the largest tumor that remains after
cytoreductive surgery.
Completeness of Cytoreduction scores
Score Size of largest post-surgery residual tumor
CC-0 No visible tumor
CC-1 Less than 0.25 cm
CC-2 Between 0.25 cm and 2.5 cm
CC-3 > 2.5 cm or conuent
Table 2: This table summarizes the pros and cons of each non-invasive imaging modality in assessing PC
Non-Invasive Imaging Utility in PC Detection
Image
Modality Pros Cons
Sensitivity/Specicity as
compared to surgical analysis References
Ultrasound
Inexpensive,
Eective for
ascites detection
Limited PC nodule
sensitivity, highly operator
dependent
Non-specic [26] [24, 25]
CT
Standard staging
workup
Limited small PC nodule
sensitivity, Inter-observer
variability
25-100%/78-100% with only 11-
48% sensitivity for tumors less
than 5 mm [26] [18-21]
MRI
High PC
sensitivity
Relatively Expensive, slight
peristalsis motion artifact,
inter-observer variability
90%/95.5% (diusion weighted)
[26] [28]
PET/ PET-CT
High PC
sensitivity
Relatively Expensive,
Peristalsis motion artifact 78-97%/55-90% [26] [27, 28]

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technology may be applied.
If blood-borne biomarkers for PC with high
sensitivity and specicity are discovered, patients
developing PC may be quickly identied with a blood test,
a liquid biopsy. One such type of biomarker, microRNAs
(miRs), short, non-coding RNAs that regulate mRNAs, has
demonstrated diagnostic utility by correctly identifying
several cancers of unknown primary with reasonable
accuracy [37]. The diagnostic miR prole that was used
in this study was generated from miR analysis in well
dierentiated primary tumors [37]. Several miRs, such as
miR-21, have been linked to gastrointestinal cancers as
potential diagnostic targets and prognostic indicators [38].
However these miRs and other types of RNAs are rapidly
degraded in the plasma [39-41].
EXOSOMES
Exosomes, small cell-derived vesicles (Figure 2),
can protect RNAs and miRNAs, from being degraded [42-
46]. When researchers exposed miRs to RNase, the miRs
that were in exosomes and cells were protected while the
free RNAs were degraded [42]. When exosomes were
exposed to RNase the contained RNAs were protected
from degradation while cellular RNA was degraded by the
same RNase [45]. Exosomes hold great potential for both
diagnosis and prognosis of diseases and are exceptionally
useful as cancer biomarkers [47]. When a panel of lung
cancer associated miRs was examined in solid tumors and
tumor exosomes from patient plasma, most of the miRs
were found to have highly comparable expression levels
(see Table 3 for the miRs) [48, 49]. Cervical cancer cell
line tumor derived exosomes (TEXs) contain survivin,
which contributes to cancer aggressiveness and metastatic
potential [50, 51]. In a study of ovarian cancer, greater
numbers of cancer exosomes were found in the serum
as more advanced cancer stages were examined [52].
Furthermore, approximately four times more serum
exosomes were discovered in lung cancer patients as
compared to cancer free controls and the exosomes
contained more than twice the miRs [48]. In our research,
we found increased serum exosome levels in patients with
prostate and breast cancers as compared to disease free
controls [53-55]. TEXs are prevalent in patient serum from
Table 3: These lung cancer associated miRs were discovered in both solid tumor and in tumor exosomes.
miR-17-3p miR-21 miR-106a miR-146 miR-155 miR-191
miR-192 miR-203 miR-205 miR-210 miR-212 miR-214
Table 4: Gastric Cancer PC associated exosomal miRs and their prevalence in various cancers.
Gastric Cancer Lung
Cancer
Liver
Cancer
Breast
Cancer
Prostate
Cancer
Colorectal
Cancer Adrenal Cancer
miR 1202
↔malignant ascites,
peritoneal lavage
uid, cell culture
[52]
X X
↑ serum
exosomes
[54] XX↑Ø tumor tissue [62]
miR 1207-
5p
↔malignant ascites,
peritoneal lavage
uid, cell culture
[52], ↓tNm tumor
tissue [63],
↓ tumor tissue [64]
X
↓ solid
tumor [56]
↑ serum
exosomes
[54] ↑ serum [65]
↑ solid
tumor
[66] X
miR 1225-
5p
↑tnM peritoneal
lavage uid [52],
↓ cancer tissue [53] X↓ blood
[67]
↑ serum
exosomes
[54]
↑ blood [68],
↓ solid tumor
[69]
↓ solid
tumor [70]
↓ multiple types of
solid tumors [71]
miR 320c
↔malignant ascites,
peritoneal lavage
uid, cell culture
[52]
↓ solid
tumor
[72] X X X X X
miR 4270
↔malignant ascites,
peritoneal lavage
uid, cell culture
[52]
X X
↑ serum
exosomes
[54] X X X
All data is from human studies
↑, ↓-increased/decreased expression associated with cancer
↑tnM, ↓tnM -increased/decreased expression associated with metastatic cancer
↑tNm, ↓tNm -increased/decreased expression associated with lymph node metastasis
↑ Ø, ↓Ø -increased/decreased expression associated with shorter patient survival
↔-present but no signicant dierential regulation
X -no research discovered in literature

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multiple cancer types and protect labile biomarkers from
degradation [42, 45, 48, 53-55].
Exosomes have not been extensively studied in
PC diagnostics. Andre and colleagues examined ascites
exosomes from patients with PC and found that the tumor
specic markers Her2/Neu, TRP1, and Mart1 were present
in ascites tumor exosomes [56]. Tokuhisa and colleagues
have identied several RNAs present in exosomes within
peritoneal ascites, peritoneal lavage, and PC metastatic
cell lines [57]. After exosome miR screening, 5 miRNAs
were selected as a panel of signicantly dierentially
regulated RNAs: miR 1202, 1207-5p, 1225-5p, 320c, and
4270 [57]. miR 21 had the strongest signal intensity in
malignant ascites [57]. Exosomes from peritoneal lavage
were probed for miR 21, 1225-5p, and 320c; miR 21 and
1225-5p were found to be upregulated in later stages of
gastric cancer and correlated with serosal invasion [57]. In
a study on primary gastric cancer tissue, miR 1255-5p was
generally downregulated and was found to inhibit cancer
cell growth, motility, as well as cancer invasion [58]. This
apparently contradictory result suggests that miR 1225-
5p in peritoneal lavage either is being released by non-
cancerous tissue in an attempt to stop abnormal growths
or is being used by cancers in order to facilitate better
attachment to the peritoneum. In apparently contradictory
results to the above study, in gastric cancer cell lines and
in vivo, miR 1255-5p was generally downregulated and
was found to inhibit cell growth and motility as well as
cancer invasion [58].
The prospective gastric PC miR biomarkers
described in the study by Tokuhisa and colleagues [57]
have been found to be associated with other cancers as
well (see Table 4). Notably, miR 1202, 1207-5p, 1225-5p,
and 4270, were found circulating in the blood of breast
cancer patients [59]. In human bladder cancer tissue
samples, miR 320c was signicantly downregulated [60].
In hepatocellular carcinoma tissue samples, miR 1207-
5p was found to be signicantly downregulated [61].
In adrenocortical carcinoma tissue samples, increased
miR 1202 expression was found to be associated with
signicantly reduced patient lifespan [62]. The gaps in our
knowledge of RNA signaling in cancer are immediately
apparent from Table 4. The observation that these miRs,
either individually or in a group, are associated with
multiple types of cancer and are found in exosomes from
the peritoneal cavity suggest the potential of exosomal
diagnosis of PC.
RNAS AND GASTRIC CANCER
RNAs are found to be globally downregulated in
cancer [37]. Upregulated RNAs and miRs are likely to
be related to cancer growth and function or the body’s
response to cancer. miR 320c inhibited cell growth and
motility in bladder cancer [60]. miR 1207-5p functioned
to inhibit cell growth and invasion in liver cancer but
functions to increase stemness in colorectal cancer [61,
63]. Since these exosomal-associated miRs have known
functions in tumors, the possibility emerges of tailoring
PC treatment based on what biomarkers are discovered
in a patient’s exosomes. miR 320c inhibited cell growth
and motility and miR 1207-5p functioned to inhibit cell
Figure 2: Tumor Cells release nanovesicles called exosomes which carry RNAs, including microRNAs and messenger
RNAs, and proteins.

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growth and invasion. The observation that these exosomal-
associated miRs have known functions in tumors opens
up the possibility of utilizing these biomarkers to tailor an
individual patient’s treatment. [60, 61]
FUTURE DIRECTIONS
Additional research is needed to identify biomarkers
in peritoneal carcinomatosis. Distinguishing metastatic
disease of peritoneal origin from solid organ metastases
should be both biologically feasible and clinically
useful. As we look to exosomes to provide insight into
the eld, shared challenges in nanovesicle isolation and
validation will need to be addressed. Reliable and ecient
methods as well as recognized standards will need to be
established for clinical use. Future clinical work in this
eld should include the prospective collection of samples
for retrospective investigation. This will be instrumental
in establishing clinical validity and utility. We are hopeful
that the shared work of many will continue to yield
advances in reducing the burden of life lost from this
aggressive malady.
CONCLUSIONS
Recently, TEXs have been implicated in facilitating
metastasis. TEXs can be taken up by multiple cell types,
including endothelial cells, bone marrow progenitor
cells, and other cancer cells [64-67]. These exosomes
demonstrated the ability to deliver functional RNAs
and proteins to recipient cells, modifying their growth
patterns to be pro-oncogenic [65, 66]. Further, TEXs have
demonstrated the ability to greatly increase metastatic
tumor burden in a mouse model [64]. Studying TEXs
in the context of metastasis is a promising eld. PC is
currently dicult to detect at its onset. Late PC detection
usually leaves the disease incurable. PC must be detected
sooner for better patient outcomes. Non-invasive imaging
is impractical for early PC detection. Detection of PC by
means of markers within a patient’s biouids, such as a
serum “liquid biopsy” would be ideal. Both serum and
ascites contain biomarkers released by PC. Exosomes
are released into serum by multiple types of cancers and
protect their contents from degradation in the blood. These
provide us with a likely source of a sensitive and specic
diagnostic modality to detect PC in its earliest most
treatable from.
ACKNOWLEDGMENTS
The authors would like to thank the Center for
Health Disparities & Molecular Medicine for its support.
They would also like to thank members of the Wall
Laboratory for careful review of this manuscript.
FUNDING
Research reported in this publication was supported
by NIH award P20MD006988 (NRW).
CONFLICTS OF INTEREST
The Authors declare no conicts of interest.
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