Monitoring of the *healthy* immune response in the sentinel lymph node of patients undergoing a prophylactic mastectomy.

With a life time risk of 12-13 percent, breast cancer is one of the most common
forms of cancer among women. For the locoregional treatment of breast cancer,
the sentinel lymph node biopsy (SLNB) has become a standard diagnostic
procedure for accurate nodal staging. Because of the typical lymphatic drainage
pattern from the breast to the axilla, the sentinel lymph node (SLN) is the
first lymph node in the axilla to drain the primary tumor and therefore the
site with the highest chance of metastatic involvement and subsequent
activation of the immune system. For this reason the SLN has become an
increasingly popular focus for tumor immunological research.
However, sometimes the immune system fails to fight off cancer cells leading to
metastasis.
Several mechanisms are responsible for this so called tumor escape. From
earlier melanoma research we now know that immune suppression is strongest in
and around the primary tumor and in the surrounding lymph nodes, especially in
the SLN1,2. Evidence suggests that dysfunctional dendritic cells and immune
suppressive regulatory T-cells (Treg) play a major role in this process3,4 In
breast cancer, it is still unknown exactly how this immune suppression is
established and how it is expressed on a cellular level.

For accurate monitoring of possible functional and quantitative immunological
changes in the SLN of breast cancer patients it is crucial to understand how a
normal immune response in SLN of healthy patients ensues. For this purpose, SLN
of patients undergoing prophylactic breast surgery, in our opinion, can provide
reliable information regarding a normal immune response. This normal immune
response can be used as baseline measurement for further immunological research
in breast cancer SLN.

Every year, The Netherlands Cancer Institute performs a large number (20-25) of
prophylactic mastectomies. This large group of patients from the NKI combined
with experience from our research group (KG/ VUmc Cancer Center) provides a
extremely suitable combination for immunological studies of SLN in healthy
patients.

Besides the fact that SLN has become a popular focus for tumour immunological
research, the increased use of the sentinel lymph node biopsy in breast cancer
has lead to a discussion concerning the discovery of false positive findings in
routine pathological examination. Although SLN biopsy allows enhanced analysis
of lymph nodes by serial sectioning and staining by immunohistochemistry (IHC),
there is controversy regarding the clinical significance of IHC-detected,
cytokeratin-positive cells in SLN, also classified as isolated tumor cells. A
number of studies describe iatrogenic epithelial cell displacement and benign
transport of epithelial cells to axillary lymphe nodes, caused by mechanical
means such as needle biopsies or even manipulation of the breast during
surgery.
IHC is performed with antibodies that are epithelial cell specific and not
breast cancer specific, therefore IHC cannot distinguish between these
potentially displaced *benign* epithelial cells and true metastases.
From this point of view, SLN from patients undergoing prophylactic mastectomy
offer a unique possibility to examine the incidence of IHC-positive SLN due to
*benign* epithelial cell displacement. Because patients undergoing prophylactic
mastectomy do not have breast cancer, we can exclude the possibility of true
metastases when IHC-positive cells are detected.


Sentinel lymphe node and lymphatic drainage pattern of the breast
In breast cancer, the primary lymphatic drainage pathway for tumor cells is to
the axilla. The existence of lymphatic drainage of the breast to extra-axillary
sites has been known for centuries. Although this may result in better staging,
the presence of these extra axillary nodes have no consequences in the standard
treatment of breast cancer patients5. In breast cancer (T1-2N0), the sentinel
lymph node biopsy (SLNB) is used as a primary lymph node staging procedure. The
SLNB is most reliable when performed by the triple technique, using
preoperative lymphoscintigraphy and periareolar injection of blue dye (Patent
blue), followed by γ-probe-guided surgery6. Serveral studies report that in
more than 95% of all patients a sentinel lymph node can be found and that in
95% of the patients this SLN can accurately predict absence or presence of
additional axillary lymph node metastasis7-11.

In case of prophylactic mastectomy the sentinel lymph node biopsy is not part
of the routine procedure. In order to avoid additional burden for patients
participating in this study a pre operative lymphoscintigraphy will not be
performed. For identification of the SLN Patent blue will be used, but unlike
the standard procedure we will inject the blue dye into the breast parenchyma,
below the subcutaneous fat, to avoid tattooing the overlying skin.
Based on the fact that also in healthy patients the typical lymph drainage
pathway is to the axilla, we believe that these SLN provide the most reliable
information regarding a normal immune response. This normal immune response can
be used as baseline measurement for further immunological research in breast
cancer SLN.


Immune respons in the SLN in breast cancer
Our immune system can play an important role in the anti-tumor response. This
anti tumor response is initiated through the same channels as a normal immune
response. Tumor infiltrating dendritic cells (DC) take up and engulf antigens
from the tumor. Once migrated to tumor draining lymph nodes (TDLN), DC present
these antigens to CD4+ T-helper (Th) and to CD8+ Cytotoxic T-Lymfocytes (CTL)
that are capable of initiating a specific anti-tumor immune response. It is
however known that tumors can escape this immune surveillance leading to
metastasis. Several mechanisms are responsible for this so called immune
escape: immunosuppressive factors in the tumor environment, dysfunctional T
cells that are incapable to penetrate the tumor, loss or downregulation of
co-stimulatory molecules on DC which causes T cell anergy or immune suppression
by suppressive regulatory T-cells (Treg)3.

Several studies have indentified immunological alterations in SLN of breast
cancer patients. Some authors describe breast cancer SLN as immune competent,
with increased presence of (mature) DC, prior to the process of
metastasis12-14. Others suggest more severe immunosuppression in SLN compared
to non SLN, prior to metastasis15,16. Thus, to date, it is still unclear when
this immunosuppression in breast cancer SLN ensues, how it exactly affects the
activation state and functionality of the different DC and T-cel subsets, and
what the underlying mechanisms are. Therefore, additional immunological
research is necessary.

To our knowledge, there have been no studies describing the immune respons in
axillary SLN of healthy patients. For accurate monitoring of possible
functional and quantitative immunological changes in the SLN of breast cancer
patients it is crucial to understand how a normal immune response in SLN of
healthy patients ensues. The aim of this study is to establish this healthy
immune respons.
In another study, initiated by our research group and running parallel to this
project, the immune response in SLN of breast cancer patients will be
established by functional and quantitative immunological analyses.


Detection of isolated tumor cells
Presence of axillary metastasis is one of the most important prognostic
criteria in breast cancer. As described earlier, the SLNB is an established
method to predict axillary metastasis. Detailed examination of the sentinel
node by means of serial sectioning with optional immunohistochemical staining
permits the detection of small metastases or isolated tumor cells (ICT). In
2002, the American Joint Committee on Cancer (AJCC) revised the breast cancer
staging system and classify micro-metastasis (deposits >0.2 to <=2.0 mm) and ICT
(with deposits <=0.2 mm) as separate categories next to macro metastasis17.
Regarding the clinical significance of detected micro-metastasis, several
studies have shown that the risk of additional axillary lymph node involvement
is up to 15%. Furthermore, SLN micrometastasis are associated with a worse
prognosis18-20. Therefore, additional axillary lymph node dissection (ALND) is
warranted and adjuvant treatment with chemotherapy should be considered.
In case of isolated tumor cells detected in SLN, the clinical and prognostic
significance is still in question. Reed et al. and Cox et al. 21,22 report
that isolated tumor cells do no influence disease free survival in their series
of patients. On the contrary, authors of the recently published MIRROR study,
conclude that ICT in SLN were associated with a reduced 5-year disease free
survival rate, among women with favourable early-stage breast cancer who did
not receive adjuvant therapy. Interestingly, the observed prognostic
significance of ICT was similar to that of micro-metastasis23. The current
Dutch guidelines regarding the treatment of breast cancer do not recommend ALND
for patients with ICT positive SLN.

Pathological examination of SLN includes hematoxylin and eosin (H&E) step
sections and more enhanced pathologic analysis with immunohistochemical
stainings (IHC) for detection of micrometastasis or ICT that are not found on
H&E examination. IHC is performed with the cytokeratin antibodies CAM 5.2
and/or AE1/AE3. IHC methodology employs antibodies that are epithelial cell
specific and not breast cancer specific.
It is known in literature, that false positive findings can occur using the IHC
methodology and that ICT detected by IHC may not be true metastasis. Several
studies have suggested that IHC-detected cells in SLNs of patients with breast
carcinoma represent mechanically displaced benign or malignant epithelial cells
resulting from manipulation of the breast during mammography, SLN biopsy or
surgery. It has also been described that pre-operative needle biopsy or needle
localization, can displace epithelial fragments into nearby lymphatic channels,
which result in transportation of epithelial cells to the axillary lymph nodes.
This so called *benign transport* is thought to be responsible for *falsely
positive* detected ICT in SLN24-27. Furthermore, there seems to be evidence
that some IHC detected ICT concern tumor cells with limited malignant
potential, which likely lack outgrowth potential and therefore not clinically
relevant28.

Most of the previous studies examining the incidence of IHC-positive cells in
SLN have evaluated patients with breast cancer, making it difficult to exclude
the possibility of true metastases when cells are positive on IHC staining.
Because in this study only SLN of patients undergoing PM (without cancer) are
examined, these SLN should not contain metastasis of tumor cells. However,
owing to the surgery itself, mechanical manipulation of the breast does occur.
Thus, when IHC positive cells are detected in these SLN, we can exclude the
possibility of true metastasis and confirm the hypothesis that iatrogenic
displacement of benign epithelial cells causes false positive findings.

Primary goal of this study is to establish the *healthy* immune response in SLN
of patients undergoing a prophylactic mastectomy.
Secondary goal is to determine the incidence of IHC-positive SLN due to
*benign* epithelial cell displacement

From 20 patients undergoing a prophylactic mastectomy, the SLN will be removed
during the same surgery. Normally the sentinel node is identified by the triple
technique using preoperative lymphoscintigraphy and periareolar injection of
blue dye (Patent blue), followed by γ-probe-guided surgery. In this study
however, patients are included who undergo prophylactic mastectomy and the
sentinel lymph node biopsy is not part of the routine procedure. In order to
avoid additional burden for patients participating in this study a pre
operative lymphoscintigraphy will not be performed. For identification of the
SLN Patent blue will be used, but unlike the standard procedure we will inject
the blue dye into the breast parenchyma, below the subcutaneous fat, to avoid
tattooing the overlying skin.
The removed SLN will be bisected crosswise. Half will be used for immunological
analysis and the other half for pathological analysis by serial sectioning and
staining by immunohistochemistry (IHC).

Patient will undergo an additional sentinel lymph node procedure. This extends
the operationtime with approximately 15 minutes. Furthermore, due to the
injection of Patent Blue, which is part of the sentinel lymph node procedure,
patients might get an anafylactic reaction.