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Review Article
Perineural Invasion in Cancer
A Review of the Literature
Catherine Liebig, MD1; Gustavo Ayala, MD2; Jonathan A. Wilks, MD1; David H. Berger, MD1;
and Daniel Albo, MD, PhD1
Perineural invasion (PNI) is the process of neoplastic invasion of nerves and is an under-recognized route
of metastatic spread. It is emerging as an important pathologic feature of many malignancies, including
those of the pancreas, colon and rectum, prostate, head and neck, biliary tract, and stomach. For many of
these malignancies, PNI is a marker of poor outcome and a harbinger of decreased survival. PNI is a distinct pathologic entity that can be observed in the absence of lymphatic or vascular invasion. It can be a
source of distant tumor spread well beyond the extent of any local invasion; and, for some tumors, PNI
may be the sole route of metastatic spread. Despite increasing recognition of this metastatic process, there
has been little progress in the understanding of molecular mechanisms behind PNI and, to date, no targeted treatment modalities aimed at this pathologic entity. The objectives of this review were to lay out a
clear definition of PNI to highlight its significance in those malignancies in which it has been studied best.
The authors also summarized current theories on the molecular mediators and pathogenesis of PNI and
introduced current research models that are leading to advancements in the understanding of this metaC 2009 American Cancer Society.
static process. Cancer 2009;115:3379–91. V
KEY WORDS: perineural invasion, perineural spread, neurotropic carcinoma, cancer, neurotrophins, axon
guidance molecule.
A key feature of malignant cells is their ability to dissociate from the primary tumor and to establish metastatic deposits at distant sites. Vascular and lymphatic channels are well accepted routes of metastatic
spread. They are well characterized in the literature and are the focus of much current research on tumor
biology. However, another route of tumor spread that occurs in and along nerves has been described in the
literature since the mid-1800s but has received relatively little research attention. Perineural invasion
(PNI) is the process of neoplastic invasion of nerves. It also has been called neurotropic carcinomatous
spread and perineural spread. PNI was reported first in the European literature by scientists who described
head and neck cancers that exhibited a predilection for growth along nerves as they made their way toward
the intracranial fossa.1,2 PNI has emerged since then as a key pathologic feature of many other malignancies, including those of the pancreas, colon and rectum, prostate, biliary tract, and stomach. For many of
these malignancies, PNI is a marker of poor outcome and a harbinger of decreased survival.3-7
Corresponding author: Daniel Albo, MD, PhD, Michael E. DeBakey VA Medical Center, 2002 Holcombe Boulevard, OCL 112-A, Houston, TX 77030;
Fax: (713) 794-7352; [email protected]
1
Department of Surgery, Michael E. DeBakey Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas; 2Department of Pathology,
Baylor College of Medicine, Houston, Texas
Received: September 9, 2008; Revised: December 5, 2008; Accepted: January 7, 2009
C 2009 American Cancer Society
Published online: May 29, 2009 V
DOI: 10.1002/cncr.24396, www.interscience.wiley.com
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3379
Review Article
PNI is a distinct pathologic entity that can be
observed in the absence of lymphatic or vascular invasion.
It can be a source of distant tumor spread well beyond the
extent of any local invasion; and, for some tumors, PNI
may be the sole route of metastatic spread. PNI is neither
an extension of lymphatic metastasis nor simply tumor
cell migration through a low-resistance plane. Definitive
studies have proven that there are no lymphatics within
the inner sanctum of the nerve sheath, and several layers
of collagen and basement membrane separate the inside of
the nerve from the surrounding lesion; this is not a low-resistance path.8-11
Despite increasing recognition of this metastatic
process, there has been little progress in the understanding
of molecular mechanisms behind PNI and, to date, no
targeted treatment modalities aimed at this pathologic entity. In fact, the true prevalence of PNI in various malignancies still has not been established. Lack of a concise,
universal definition for PNI across all disciplines has
resulted in significant confusion and probably is 1 reason
for the seemingly slow progress. It is the objective of this
review to lay out a clear definition of PNI and to highlight
its significance in those malignancies in which it has been
characterized best. We also aim to summarize current theories on the molecular mediators and pathogenesis of PNI
and to introduce current research models that are leading
to advancements in our understanding of this metastatic
process. It is our hypothesis that PNI is as significant in
some tumors as lymphovascular invasion. A better understanding of PNI may lend insight into tumor metastasis
and recurrence and open doors to improved staging strategies, novel treatment modalities, and perhaps even paradigm shifts in our treatment of patients with patients.
Definition
Knowledge of the basic structure of the peripheral nerve
sheath is integral to understanding PNI (Fig. 1). The
nerve sheath is composed of 3 connective tissue layers.
From outside to in, these layers are the epineurium, the
perineurium (not to be confused with the perineurum, or
area just outside of the nerve sheath), and the endoneurium.12 The epineurium, which binds 1 or more fascicles
into a single nerve, is composed of 2 distinct layers: an
outer layer of areolar connective tissue and loosely
arranged collagen bundles and an inner layer of densely
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organized collagen fibrils and elastin fibers.13 It is within
the areolar connective tissue on the outer portion of the
epineurium that the epineurial component of the vasa
nervorum, the rich vascular network of the peripheral
nerve, and the perineural lymphatic channels reside.
According to the most recent literature, these lymphatics
do not penetrate the epineurium, although this issue historically has been the focus of considerable debate in the
PNI literature.8,14,15
Akert et al., using electron microscopy to delineate
the structural organization of the perineurial sheath of the
peripheral nerve, describe a multilamellated structure of
concentrically arranged endothelial cells.11 Each endothelial cell layer of the perineurium is flanked on either side
by basement membrane.9,11 Junctions between the endothelial cells are formed by dove-tailing, that is, overlapping and tightly fitting projections of adjacent cell
membranes.11 These junctions, or zonulae occludentes,
work together with the multiple basal laminae to confer a
highly selective barrier function to the perineurium. This
effectively separates the intrafascicular compartment of
the nerve from the surrounding epineurium.
The endoneurium, or innermost layer of the nerve
sheath, forms a matrix around individual nerve fibers and
envelops the Schwann cells and individual axons of the
nerve.14 The endothelial lining of endoneurial blood vessels is made up of tight junctions without evidence of
transendothelial channels.14 The relative impermeability
of the endoneurial blood vessels is an extension of the barrier function of the perineurium surrounding this compartment and is a key feature of the blood-nerve barrier.
Descriptions of PNI have included tumor cells
within every layer of the peripheral nerve sheath, from
abutment of tumor cells within the perineurum to well
formed cancerous glands within the perineurium or endoneurium, to small clusters of tumor cells within a nerve
that is surrounded by normal tissue well away from tumor. Which of these tumor nerve configurations are true
examples of PNI and which features are necessary for
defining PNI? In his 1985 article on neurotropic carcinomas, Batsakis offered a broad definition of PNI, characterizing it as tumor cell invasion in, around, and through the
nerves.8 The article is cited widely in the literature on PNI,
and the definition has become the generally accepted 1 for
PNI. It is sufficiently broad to cover most of the histopathologic varieties of this entity previously described in the
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Perineural Invasion in Cancer/Liebig et al
FIGURE 1. Perineural invasion: Illustrated is a tumor enveloping a peripheral nerve in cross section revealing tumor cell spread.
Molecules expressed by tumors interact with surrounding stroma as well as receptors associated with peripheral nerves.
literature and has helped to focus later research efforts by
eliminating any confusion about perineural lymphatics.
However, in, around, and through has left tremendous
room for further clarification. Some authors have suggested that tumor cells must be observed inside the perineurial layer, specifically, to cite PNI.16 This definition
seems overly stringent and would exclude several examples
of clear nerve invasion, such as gland formation within the
collagen layers of the epineurium. We advocate that the
finding of tumor cells within any of the 3 layers of the
nerve sheath represents PNI. A much more frequent finding in PNI is tumor-nerve contact within the perineurum
without the finding of tumor cells inside of the sheath,
and there is wide variability among authors regarding the
degree to which this tumor-nerve contact is necessary to
call it PNI. Various growth patterns have been described,
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including complete and incomplete encirclement, concentric lamination, and tangential contact.17 For instances
in which this occurs outside of the main body of the tumor, the finding is recognized more easily as malignant
carcinomatous invasion of a neural structure. However, in
instances in which this occurs within the main body of the
tumor, it is less clear cut. For these circumstances, many
authors have proposed that at least 33% of the circumference of the nerve should be surrounded by tumor cells to
call it PNI; anything less than 33% represents focal abutment and not invasion.17-19 In summary, we advocate a
somewhat broad definition of PNI, in keeping with the
original definition by Batsakis of in, around, and through
the nerves, while incorporating many features that were
cited previously in the literature: tumor in close proximity
to nerve and involving at least 33% of its circumference or
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FIGURE 2. These photomicrographs depict perineural invasion (PNI) in human colorectal cancer specimens. Sections of human
colorectal cancers were stained with hematoxylin and eosin and were reviewed by a pathologist for PNI. Tumor cells located
within the peripheral nerve sheath either (a) in clusters or (b) forming glandular elements are clear examples of PNI. When tumor
cells are not located inside of the nerve sheath but are in close proximity to the nerve in the perineural environment, at least 33%
of the circumference of the nerve must be involved by tumor to diagnose PNI. This is true weather the nerve is located (c) within
the main body of the tumor or (d) at a site outside of the primary tumor focus.
tumor cells within any of the 3 layers of the nerve sheath
(Fig. 2).
Pathogenesis
It has become evident that PNI is not an extension of lymphatic metastasis, as once was suggested. Definitive studies have demonstrated that lymphatic channels do not
penetrate the inner sanctum of the nerve sheath.8-10,20 For
the last 40 years, the predominant theory behind the
pathogenesis of PNI has been that tumor cells spreading
along neural sheaths are privileged to a low-resistance
plane, which serves as a conduit for their migration. Once
inside of the nerve sheath, tumor cells may be in a privi3382
leged growth environment that facilitates metastasis, but
the multiple layers of collagen and basement membrane
that compose the nerve sheath make access to this path
anything but low-resistance. The reason certain carcinomas exhibit a predilection for PNI and others do not
remains unknown.
More recently, studies have demonstrated that PNI
may involve reciprocal signaling interactions between
tumor cells and nerves and that these invading tumor cells
may have acquired the ability to respond to proinvasive
signals within the peripheral nerve milieu. In a PNI model
using mouse dorsal root ganglia (DRG) cocultured in a
Matrigel matrix with prostate cancer cells, Ayala et al.
demonstrated tumor cell migration along neurites toward
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Perineural Invasion in Cancer/Liebig et al
Table 1. Neurotrophic Factors and Their Possible Functions and Expression Patterns in Cancer
Factor
Possible Function in Cancer
Expression in Cancer
References
NGF
May stimulate epithelial cancer cell growth and
mediate nerve invasion through its interaction
with trkA, an NGF-specific receptor; binding
of NGF to trkA leads to activation of the
p44/42 MAPK signaling pathway and
up-regulation of MMP-2, a proinvasive mediator
May be overexpressed by tumor cells to
promote neurite growth; stimulates tumor cell
invasion at low-to-moderate concentrations
Overexpressed in pancreas cancer and
prostate cancer cell lines; trkA is
strongly expressed on the perineurium
of peripheral nerves
Ketterer 2003,27 Okada 2004,28
Zhu 1999,31 Zhang 2005,32
Zhu 2002,33 Geldov 1997,34
Kowalski 200235
Overexpressed in pancreas cancer and
adenoid cystic carcinoma; expression
does not correlate with the presence of
PNI, suggesting that the BDNF-expressing
phenotype may appear before nerves
Overexpressed in specimens of human
neural plexi; multiple pancreatic cancer
cell lines express the RET protein
tyrosine kinase receptor for GDNF
Overexpressed in pancreas cancer
specimens
Ketterer 2003,27 Zhu 2002,33
Kowalski 2002,35
Miknyoczki 199936
BDNF
GDNF
Exhibits a chemotactic and chemokinetic effect
on tumor cells and mediates increased
MMP-9 expression and activity
NT-3
Stimulates tumor cell invasion at
low-to-moderate concentrations
Okada 1999,29 Okada 200330
Ketterer 2003,27 Miknyoczki 199936
NGF indicates nerve growth factor; trkA, tropomyosin receptor kinase A (a high-affinity catalytic receptor for nerve growth factor); MAPK, mitogen-associated
protein kinase; MMP-2, matrix metalloproteinase 2; BDNF, brain-derived neurotrophic factor; PNI, perineural invasion; GDNF, glial cell line-derived neurotrophic
factor; NT-3, neurotrophin 3.
the ganglia of origin as well as focused, directional outgrowth of neurites toward cancer cell colonies.21 The
addition of stromal cells to the aforementioned in vitro
model resulted in increased neurite outgrowth and cell
colony formation.22 This suggests that the signaling
mechanisms behind PNI likely involve at least 3 different
cellular elements, including tumor cells, nerve cells, and
stromal cells, and may include autocrine and paracrine
mechanisms.
The increased neurite formation demonstrated in
the previous in vitro studies suggests that axonal migration
may be a key element of PNI. Axonal growth is a complex
process that requires neurotrophic growth factors and axonal guidance molecules.23,24 The neurotrophins are the
best characterized family of neurotrophic factors and
comprise nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and
neurotrophin 4/5 (NT-4/5).25 It is their potent effects on
neuronal growth that have made the neurotrophins prime
candidates for study in the PNI invasion pathway. There
is a growing body of literature implicating these molecules
as well as other neurotrophic factors in cancer (Table 1).
Recent evidence in prostate cancer suggests that there is an
up-regulation in neurotrophin expression by tumor cells
as an escape mechanism from dependence on paracrine
expression by stromal elements.26 Ketterer et al., by using
polymerase chain reaction analysis of microdissection
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August 1, 2009
specimens, discovered that there is an up-regulation in
neurotrophin expression by tumor cells as well as intratumoral nerves in pancreas cancer.27 Tumor cells cannot
migrate through extracellular matrix or nerve sheath without expressing proteinases. Matrix metalloproteinases
(MMPs), and, in particular, the gelatinases (MMP-2 and
MMP-9), seem to play a pivotal role in PNI. Okada et al.
reported that exogenous NGF led to a dose-dependent
increase in MMP-2 expression and tumor cell invasion in
pancreatic cancer cells.28 This effect was mediated by
binding NGF to its tropomyosin receptor kinase A (trkA)
receptor, which is expressed on the tumor cell surface,
with subsequent activation of the p44/42 mitogen-associated protein kinase signaling pathway.28 Pancreatic cancer
overexpresses glial cell line-derived neurotrophic factor
(GDNF).29 Migration of pancreatic cancer cells is
increased by GDNF-secreting glioma cells in a dose-dependent fashion, suggesting both a chemotactic effect and
a chemokinetic effect of GDNF on tumor cells.29 Furthermore, this pancreatic cancer cell migration depends
on GDNF-induced up-regulation of MMP-9 expression
and activity.30
Experimental Models
Our understanding of the pathogenesis of PNI has been
limited by a lack of effective models for this complex
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FIGURE 3. This in vitro model of perineural invasion in prostate cancer was developed by Ayala et al.21 Mouse dorsal root ganglia
(DRG) are cocultured in Matrigel with circumferentially placed prostate cancer cells. DRG exhibit directional outgrowth of neurites in response to tumor cells and tumor cell colonies reveal increased growth in (a) a brightfield image and (b) a darkfield
image (original magnification, 40).
interaction between nerve, tumor cell, and stroma. Few in
vitro models have proven capable of capturing even a single aspect of the disease process, and much of their limitations stemmed from the difficulty in culturing peripheral
nerve preparations and in replicating the neural microenvironment. In vivo models have proven more promising,
but these only recently have been developed. Below, we
outline the available, effective in vitro and in vivo models
of PNI and highlight some of the current research using
these experimental designs.
A critical element in the metastatic cascade is the
ability of tumor cells to invade through basement membranes. These thin, specialized sheets of extracellular matrix act as barriers against cellular and macromolecular
movement, particularly across epithelial layers and endothelial-lined spaces of the vasculature. Basement membrane also lines several cell layers of the neural sheath,
making this metastatic process elemental to PNI as well.
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By using an in vitro assay that mimics tumor cell invasion
of basement membrane and correlates with in vivo metastatic potential of cells, Miknyoczki et al. and Albini
observed that BDNF and NT-3 stimulated pancreatic tumor cell invasion.36,37 In similar experiments, human
prostate cancer cell invasion was up-regulated markedly in
response to exogenous NGF and to NGF-like proteins
secreted by human prostate stromal cells.34,38 These
experiments suggest that there are complex signaling
interactions between the peripheral nerve milieu, tumor
stromal elements, and tumor cells.
In 2001, Ayala et al. described a novel in vitro PNI
model in which mouse DRG were cocultured with prostate cancer cells in a Matrigel matrix.21 The 3-dimensional Matrigel matrix suspends the ganglia and allows
for multidirectional neurite outgrowth (Fig. 3). Some
studies of neurite outgrowth have used cell suspensions of
isolated neurons rather than explanted mammalian DRG;
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Perineural Invasion in Cancer/Liebig et al
however, the influence that supportive glial cells have in
promoting neurite growth and PNI certainly is lost.39
Three phenomena, as outlined above in the discussion of
pathogenesis, were observed in the Matrigel/DRG model:
1) focused outgrowths of neurites projected into cancer
cell colonies within 24 hours of culture; 2) cancer cell colony formation increased; and, 3) cancer cells migrated
along contacted neurites toward the ganglion of origin.
The results were reproducible across 3 different prostate
cancer cell lines. This experiment effectively establishes
the presence of a reciprocal growth interaction between
cancer cells and neurites in vitro and also suggests that
actively growing nervous tissue somehow may promote tumor cell invasion. To date, there are no data supporting
this phenomenon in vivo.
Several animal models recently have been developed
for tumors that are known to metastasize through the perineural route. Pour et al. developed a Syrian hamster
model of both carcinogenesis-induced and orthotopically
transplanted pancreatic cancer.40 Both tumors demonstrated PNI in about 90% of cases. Furthermore, in this
model, those authors demonstrated not only that tumor
cells invade extrapancreatic neural plexi by this route but
that they also reach distant metastatic sites, such as lymph
nodes, through the perineural space. Eibl and Reber
developed a model for pancreatic cancer recurrence in
nude mice that underwent resection of pancreatic tumors
at 4 weeks, 6 weeks, and 8 weeks after orthotopic implantation.41 Eighty percent of pancreatic tumors that were
resected 6 weeks after orthotopic implantation recurred
with extensive invasion of retroperitoneal nerves, but
tumors that were resected at 4 weeks did not show signs of
recurrence. These findings suggest that metastasis of retroperitoneal nerves may occur later in the disease process.
This model may serve as an effective tool for studying
early recurrences and may help with elucidating the role
of nervous structures as sources of recurrence.
Other animal models used for studying PNI include
a chemically induced prostate carcinoma in Wistar rats
that demonstrates frequent PNI and a nude mouse orthotopic injection model of human head and neck squamous
cell carcinoma that leads to PNI in 100% of tumors.42,43
Both of these models result in tumors that closely mimic
their human counterparts histologically and phenotypically and, thus, serve as useful tools for studying the molecular pathogenesis of PNI.
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Clinical Significance
Some of the earliest observations of neural involvement by
tumor were in cancers of the head and neck in which large
nerves seemed to serve as ready conduits for intracranial
extension. The incidence of PNI in head and neck cancers
varies considerably by histology but is reported most commonly in squamous cell carcinoma in which the incidence
is as high as 80%.44-46 PNI is a significant pathologic feature in head and neck cancers, heralding decreased survival, increased locoregional recurrence rates, and a
shorter time to recurrence.17,45,47,48 In 1 series of 239
patients with mucosal squamous cell carcinomas, PNI was
associated with a 23% 3-year survival rate versus 49% in
patients with stage-matched, PNI-negative tumors in univariate analysis.45 Although treatment strategies for head
and neck malignancies were beyond the scope of this
review, it is noteworthy that PNI status often significantly
affects surgical strategies and adjuvant treatments in head
and neck cancers.49-53 The rationale for changing therapy
based on PNI status is largely anecdotal without significant evidence.53 This is 1 of the only malignancies for
which assessment of PNI status is a required component
of the pathologic analysis according to the reporting protocols published by the College of American
Pathologists.54
PNI is observed in as many as 75% of resected prostate cancer specimens and in 25% of biopsies from
patients without lymph node metastases, and many
authors agree that it is the most significant route of extracapsular spread in this malignancy.55,56 In 1 series of 78
prostatectomy specimens with extracapsular invasion,
50% of specimens had tumor penetrating the capsule
solely along nerves; and, in the remaining 50% of specimens, capsule penetration through nerves was the predominant route.57 Several studies have demonstrated that
the presence of PNI on prostate needle biopsy specimens
can reliably predict capsular penetration at prostatectomy,
although this claim remains disputed.58-60 In a series of
381 patients who received for low-risk prostate cancer,
Beard et al. reported that the 5-year prostate-specific antigen (PSA) failure-free survival rate was 50% versus 80%
in patients with and without PNI in their needle biopsy
specimens, respectively.5 This association was observed
only on univariate analysis, and PNI was not an independent predictor of PSA failure on multivariate analysis, as
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were pretreatment PSA level and Gleason score. However,
there was a significant correlation between the presence of
PNI and higher Gleason scores, suggesting that PNI may
predict occult, high-grade disease in otherwise low-risk
patients. Perineural involvement of benign prostatic
glands is well recognized; PNI alone, without other signs
of neoplasia, is not always diagnostic of prostate cancer.61
In a recent systematic review of investigations of PNI in
prostate needle biopsy specimens, wide variability in study
design, PNI frequency, and statistical analyses were
observed; although the results suggested that PNI in prostate needle biopsy specimens may predict an adverse outcome after surgery or radiation, no conclusion could be
reached based on data in that review, and there is no solid
evidence that a PNI-positive biopsy should alter therapy.62 With regard to prostatectomy specimens, a study
of 1550 patients from the University of Michigan indicated that PNI was a significant predictor of higher pathologic stage and positive margins on both univariate and
multivariate analysis.63 However, investigations of PNI in
prostatectomy specimens, like in studies of needle biopsies, lack consensus regarding biochemical failure rates
and survival differences after surgery. Although there were
2 studies that revealed significantly worse outcomes associated with PNI, in the remaining studies, no statistical
difference was oberved.55,64-70
In 1 study of 90 pancreatic adenocarcinoma specimens, PNI was observed in 88 tumors (98%).71 In that
study, there was no association between PNI and tumor
size, differentiation, or lymphovascular invasion; but there
was a significant correlation between PNI and tumors
with lymph node metastases. Reports on the incidence of
PNI in pancreatic cancer cite a minimum of 70% of these
tumors, and some authors even claim that 100% of these
tumors. will exhibit PNI if enough sections are evaluated.3,72-75 Positive PNI status in pancreatic cancer predicts decreased survival, often independent of stage, but
treatment remains unchanged by PNI status.3,71,73 In a
subgroup analysis of 72 patients with lymph node-negative disease (stage I and stage II), the 5-year survival rate of
patients without PNI was 75% versus 29% for those with
PNI in their tumor specimens (P < .02).3 The pancreas is
richly innervated by the autonomic nervous system, primarily through plexi from the celiac and superior mesenteric artery ganglia. Studies of extratumoral PNI in
pancreatic cancer have focused on these plexi as likely sites
3386
of micrometastatic spread and sources for retroperitoneal
recurrence, although these possibilities remain to be confirmed. Up to 72% of PNI-positive pancreatic adenocarcinomas demonstrate extrapancreatic nerve plexus
involvement at the time of resection.71,76,77 The association between intratumoral PNI and extrapancreatic plexus
involvement seems to correlate with the degree of invasion
observed in the primary tumor and with the presence of
PNI within the pancreas but outside of the main body of
the tumor.71,76,77
PNI has been recognized in many series as a prevalent pathologic feature of colorectal cancer and is reported
in up to 33% of these tumors at the time of resection.78-81
Several studies have demonstrated a significant correlation
between the presence of PNI in colorectal tumors and
increased locoregional recurrence rates, a 5-year survival
rates, and an increased likelihood of finding metastatic
disease at the time of resection.78,80,82-84 In 1 study of 563
rectal and rectosigmoid cancers, the presence of PNI was
associated with a 27% cancer-specific 5-year survival rate
versus a 78% 5-year survival rate in PNI-negative tumors
(P < .001).4 Similar results were reported by Krasna et al.
in their review of 77 patients with colorectal carcinoma.
In that series, not only was survival lower in PNI-positive
patients, but those patients were almost 3 times more
likely to have metastatic disease at the time of diagnosis
(27% vs 73%; P < .01).84 These results suggest a correlation between PNI status and advanced tumor stage, a relation that also has been demonstrated in other studies. For
instance, in a review of 373 patients with rectal cancer
who underwent curative resection, the incidence of PNI
in patients with stage III disease was 20%, which was
twice the incidence reported in the overall study population (10%).85 Perhaps more noteworthy is the prognostic
significance of PNI in lymph node-negative disease, particularly stage II, in which it has been demonstrated that
chemotherapy has no survival benefit. In an analysis of
124 patients with lymph node-negative colorectal cancer
who underwent curative resection, the 5-year survival rate
was 87% for patients with PNI-negative tumors versus
57% for patients with PNI-positive tumors (P < .006).86
Other studies similarly have indicated that positive PNI
status portends a worse outcome in patients with lymph
node-negative colorectal tumors.87-89 This suggests that
PNI indicates a more aggressive tumor phenotype and
may be useful in selecting a subgroup of lymph nodeCancer
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Perineural Invasion in Cancer/Liebig et al
negative patients who could benefit from adjuvant therapy. Some patients with stage II disease currently are
offered adjuvant therapy after resection of PNI-positive
tumors, but outcomes data are not yet available.90
There is scant information on PNI in malignancies
other than those discussed above. The reported incidence
of PNI in cholangiocarcinoma is approximately 75% to
85%, and at least 2 studies have noted a significant correlation between PNI and decreased survival in patients
with this malignancy.6,91,92 One series of 354 gastric cancer specimens cited a PNI-positive rate of 60% and a significant correlation with disease progression and overall
survival.7 Reported rates of PNI in breast cancer range
from 3% to 38%.93-95 PNI is reported often in conjunction with lymphovascular invasion as 1 pathologic feature
of breast cancer, confounding its significance as an independent prognostic variable. Some studies have demonstrated a correlation between lymphovascular invasion/
PNI and decreased survival in breast cancer, whereas other
studies have failed to demonstrate a correlation between
PNI and outcome.96-99
Underreporting of PNI remains an obstacle to gaining an adequate understanding of its true prognostic significance. First, several factors make nerve invasion very
difficult to recognize. Inflammatory cells or large, mucinous pools may obscure the presence of tumor cells
around nerves. Microscopic foci of nerve invasion are
common and also may escape detection.72 It has been
demonstrated that evaluating specimens after staining for
nerve-specific antigens markedly increases PNI detection
rates compared with standard hematoxylin and eosin
staining techniques.46,79 On a re-review of slides from 40
patients with head and neck squamous cell carcinoma, the
rate of detection of PNI almost tripled from 30% to 82%
when specimens were stained for protein S100.46 It is conceivable that nerve-specific staining may become a routine
part of the pathologic evaluation of malignancies in which
PNI status affects treatment.
In addition to problems with detection, to date,
there are no concrete guidelines on the reporting of PNI
in malignancies other than head and neck cancers. This
contributes further to the underreporting of PNI. According to the latest protocols published by the College of
American Pathologists, evaluation of PNI is not a required
element in pancreatic, colorectal, or prostate cancer pathology reports.100
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Conclusion
Since PNI first was described over a century ago, there has
been a paradigm shift in our understanding of its pathogenesis. Initially, it was believed that PNI represented lymphatic spread of tumor into nerves; however, studies
performed in the mid-1900s called this theory into question. By the late 1900s, the predominant theory became
that of the nerve sheath as a low-resistance path for tumor
spread. Although the ‘‘path of low resistance’’ theory still
persists in the literature today, evidence is emerging indicating that the PNI phenomenon is more like invasion
than simple diffusion. New models have been developed
that provide strong evidence for signaling between the
nerves and invading tumor cells. Stromal elements, including fibroblasts, also seem to play a key role in the complex
signaling interactions driving PNI. Studies involving neurotrophins and axonal guidance molecules have implicated
a few of the molecular mediators involved in the process,
although we have only just begun to scratch the surface.
We have highlighted the clinical significance of PNI
in those malignancies in which it has been studied best.
These include cancers of the head and neck, pancreas, colon and rectum, and prostate. Overall, PNI portends a significantly lower 5-year survival rate and signifies more
advanced disease. There are many malignancies, however,
for which we have only begun to recognize the significance of PNI. Breast cancer and hepatobiliary cancers are
among these malignancies; however, preliminary evidence
suggests that, 1 day, PNI also will be recognized as a significant pathologic feature in these and other tumors.
With a better understanding of the pathogenesis and
through work using animal models, we can begin to target
PNI in our treatment strategies against those malignances
for which PNI is significant. Gene profiling already has
been used to identify an expression profile predictive
of PNI in biliary tract cancers.101 These expression
profiles are being evaluated for use in risk stratification
and in guiding the extent of surgical resection; however,
1 day, the candidate genes or their downstream molecular pathways could serve as therapeutic targets. Several neurotrophins, including NGF, BDNF, and NT-3,
have been implicated in promoting tumor cell invasion
and may be key mediators in the pathogenesis of
PNI.34,36,38 Researchers have begun searching for viable
therapeutic targets among these neurotrophins and their
3387
Review Article
receptors.36,102-104 Both anti-NGF antibodies and small
interfering RNA against NGF have demonstrated efficacy
against cell proliferation and angiogenesis in a breast cancer murine model.104 Intratumoral and peritumoral injection with antibodies against receptors for NGF and NT-3
have produced significant growth inhibition in both
human pancreatic cancer and prostate cancer xenografts
in a nude mouse model.105
Although much of our research has focused on tumor cell invasion of nerves, the PNI story is beginning to
include axonal growth and possibly nerve ‘‘invasion’’ of tumor. A study performed by Maru et al. indicated that PNI
diameter, measured with an ocular micrometer and
defined as the largest focus of PNI in a tumor, was a better
predictor of outcome in prostate cancer than PNI status
alone.55 There is no clear explanation why this is the case.
It is possible that tumors with larger foci of PNI are simply
more advanced cancers, which we would expect to have
worse outcomes; or perhaps there is nerve enlargement
within these foci of PNI as a result of a cancer cell-promoted nerve growth phenomenon during PNI. Clearly,
this aspect of PNI needs to be explored further; however,
in vitro evidence certainly suggests that there is a reciprocal
growth interaction occurring between nerves and tumors.
It seems that progress in understanding PNI has
been stymied by the lack of a concise definition for this
pathologic process. We conclude that the definition
offered by Batsakis in 1985—tumor cell invasion in,
around, and through the nerves—leaves too much room for
interpretation and is not precise enough to promote uniformity among researchers. After a thorough review of the
literature, we have compiled criteria for the identification
of PNI in a histologic specimen. Finding tumor cells
within any of the 3 layers of the nerve sheath or tumor foci
outside of the nerve with involvement of 33% of the
nerve’s circumference are sufficient features for calling
PNI. It is likely that, as our understanding of the pathogenesis of PNI evolves, so too will this definition.
2.
Neumann E. Secondare cancroid infiltration des nervus
mentalis bei einem. Arch Pathol Anat. 1862;24:201-201.
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Conflict of Interest Disclosures
16. Veness MJ. Perineural spread in head and neck skin cancer.
Australas J Dermatol. 2000;41:117-119.
The authors made no disclosures.
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