Detection of Tumour Containing Sentinel Lymph Node in Breast

Cancer by Injection of Fluorescence Tracer through “Dual Route”

in Breast Tissue and Intravenously

Darakhshan Qaiser

, Anurag Srivastava

, D. S Mehta

, A. Sharma

, Anita Dhar

, V. Seenu

K. Dalal

, S. Mathur

and S. Anand

Department of Surgery, All India Institute of Medical Sciences, Delhi, India

Department of Physics, Indian Institute of Technology, Delhi, India

Department of Biophysics, All India Institute of Medical Sciences, Delhi, India

Department of Pathology, All India Institute of Medical Sciences, Delhi, India

Department of Biomedical Engineering, All India Institute of Medical Sciences, Delhi, India

Keywords: Breast Cancer, Fluorescein, Fluorescence, Sentinel Lymph Node Metastasis.

Abstract: A new technique of identifying tumour containing sentinel lymph node has been developed. Sentinel node

biopsy is the current standard of care practice for assessing axillary nodal status in ladies with early breast

cancer. Sentinel node biopsy is performed by using a combination of a blue dye and an isotope tagged to a

large particulate matter like sulphur colloid or albumin. These tracers are injected in the breast tissue or skin

over the breast. We describe a new method of “dual route of injection” of tracers both in the breast as well

as in the systemic circulation through a peripheral vein. This method of dual route injection is expected to

detect the axillary nodes containing tumour metastasis and reduce the probability of missing a “false

negative sentinel lymph node”.

1 INTRODUCTION

Assessment of axillary nodal is necessary for proper

staging and planning adjuvant therapy and extent of

surgery in patient with breast cancer. Traditionally

this was done by full axillary node dissection.

Axillary lymph node dissection leads to morbidity

such as edema in arm, pain, and shoulder stiffness.

Hence, sentinel node biopsy has been developed as a

minimally invasive procedure for axillary lymph

node evaluation. In sentinel node biopsy surgeon

removes the first lymph node that receives lymphatic

drainage from the tumor. Therefore, sentinel node

biopsy is associated with minimal morbidity

(Schrenk, 2000). Currently, Sentinel node biopsy is

considered the “standard of care” method for

axillary nodal evaluation. Among women with early

breast cancer with a clinically negative axilla, the

sentinel node biopsy will identify about one third

women who will have sentinel lymph node

metastasis. In the largest randomized controlled trial

of sentinel node biopsy, the NSABP- B32 trial

(Krag, 2010), sentinel lymph node metastasis was

detected only in 29% women. The sentinel node

biopsy is carried out by injecting a blue dye and

isotope in the diseased breast in the skin, deep to

areola, or in and around the tumor. This route of

injection detects a sentinel node with about 95%

identification rate and a false negative node rate of

5% to 10% (Hiram 2006).

In this proposal we describe a new method of

identifying a lymph node harboring tumor deposits.

It is expected that the use of dual route of injection

of tracers will reduce the probability of missing a

false negative sentinel lymph node.

Kawada and Taketo (Kawada, 2011) have

described the mechanism of metastasis to a lymph

node. The metastasis in the lymph node is classified

as a “macrometastasis” if it measures more than 2

mm in diameter on histological section (Takeuchi

and Kitagawa). A metastasis is described as

“micrometastasis” if it measures between 0.2 mm to

2 mm in diameter. The presences of only few

tumour cells in the lymph node are called “isolated

tumour cells”. Long term studies in women with

early breast cancer have revealed that presence of

125

Qaiser D., Srivastava A., Mehta D., Sharma A., Dhar A., Seenu V., Dalal K., Mathur S. and Anand S..

Detection of Tumour Containing Sentinel Lymph Node in Breast Cancer by Injection of Fluorescence Tracer through “Dual Route” in Breast Tissue and

Intravenously.

DOI: 10.5220/0005403401250128

In Proceedings of the 3rd International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS-2015), pages 125-128

ISBN: 978-989-758-092-5

 2015 SCITEPRESS (Science and Technology Publications, Lda.)

isolated tumour cells and micrometastasis in a lymph

node do not increase the chance of locoregional or

systemic recurrence and do not adversely affect

disease free survival or overall survival. However,

presence of a “macrometastasis” in a lymph node is

determinant of increased locoregional failure. Thus,

from a treatment point of view, it is important to

detect an axillary lymph node containing a

macrometastasis of > 2 mm in diameter.

Studies on animal and human tumors have

demonstrated that tumor growth is dependent on

angiogenesis (Kahlert 2014) . After, initial growth to

a diameter of 0.2-2 mm, Tumor cells begins to

secrete “Vascular endothelial growth factors” -

VEGF family (VEGF-A, B, C & D) (Robert 2007).

The sentinel lymph node containing the metastasis

of more than 1 mm will possess angiogenesis

induced by the malignant cells. This neovascular

network of blood vessels will increase the blood

flow in the lymph node containing a tumour deposit

of >1 mm diameter. This increased blood flow can

be detected by Doppler ultrasound flowmetry. The

increased blood flow in these lymph nodes will

allow higher concentration of intravenously injected

fluorescein or other tracer.

2 RESEARCH HYPOTHESIS

1. Macro-metastasis in a lymph node is associated

with tumor induced angiogenesis.

2. This angiogenesis would result in increased

blood flow to the node harboring a macrometastasis.

3. Increased blood flow can be detected by Color

Duplex Ultrasound scan with Power Pulsed Doppler

flow-metry and intravenous injection of Fluorescein.

4. Fluorescein containing lymph node can be

detected by fluorescence when viewed with blue

light during sentinel node biopsy.

2.1 Background

One of the authors (Anurag Srivastava) had earlier

described the presence of increased blood flow in

skin melanomas of thickness greater than one

millimeter (Srivastava, 1988). The characteristics of

“Doppler frequency shift signals” are predictive of

15 year outcome of patient with skin melanoma

(Srivastava, 2012). One advantage of this method of

detection of sentinel lymph node would be that it

will only detect nodes harboring a macro-metastasis.

Since, angiogenesis sets in at a size of 1-2 mm tumor

size, smaller micro-metastasis and isolated tumor

cells will not be detected by this method. The

presence of micro-metastasis and isolated tumor

cells in the sentinel lymph node does not adversely

effect the disease free survival and does not

necessitate any further axillary therapy (axillary

lymph node dissection or axillary radiotherapy).

Fluorescein angiography has been successfully

utilized in the detection of tumor metastasis

(Aalders, 2001) in ovarian tumours.

3 PROPOSED METHOD

Patients presenting with early breast cancer with a

no palpable nodes in the axilla, will be requested to

take part in this study.

Preoperative sonographic imaging axilla will be

carried out to detect any suspicious node which will

be biopsied. Women with a positive node biopsy

will be treated with axillary lymph node dissection

along with breast surgery. The patients without a

sonographically suspicious node and those with a

negative node biopsy report, will be offered a

sentinel node biopsy and will be selected for the

present study.

3.1 Method of Sentinel Node Biopsy

and Fluorescein

3ml of 20% fluorescein sodium will be injected

intravenously about 20-25 minutes before the

sentinel node biopsy. It is assumed that fluorescein

will reach the tissues and lymph node in this period

and sentinel lymph node containing the metastasis of

more than 1 mm would demonstrate higher

concentration of fluorescein.

Usual sentinel node biopsy will also be carried

out employing technitium tagged sulphur colloid and

indocyanine green intradermal injection in

periareolar skin of breast. Indocyanine green has the

advantage of depicting the lymphatics and

fluorescent node through the skin; hence a very

small incision will be sufficient for location of

sentinel lymph node. Indocyanine green induced

fluorescence will be examined with a Near Infrared

light (of 800 nm wavelength).

Sentinel Node Biopsy will be carried out under

general anesthesia. Fluorescent and radioactive hot

nodes will be removed and sent for histological

examination. Intravenous Fluorescein should

demonstrate lymph nodes containing tumour cells

with a deposit of greater than 1mm. These

fluorescein containing nodes will be sent separately

for histological examination.

The fluorescent nodes and primary breast tumour

PHOTOPTICS2015-InternationalConferenceonPhotonics,OpticsandLaserTechnology

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after removal during surgery are being bisected and

smears are being prepared on a glass slide for

imprint cytology. The smears are also being

prepared on "Half reflecting slides " and being

examined under the "Interference Microscope" at

Department of Optics; Indian Institute of

Technology -Delhi .

3.2 Observations

We applied this technique on two patients. In these

patients we injected fluorescein sodium 3ml

intravenous injection (IV) 20 min before incision.

We found highly fluorescent nodes in the axilla

of these patients. Histologically these nodes were

found to contain metastasis from breast cancer.

Fluorescent node in the axilla can be seen in Figures

1 and Figure 2 shows fluorescent node separately.

Figure 1: Axilla of breast cancer patient showing

fluorescent node.

Figure 2: Fluorescent Node with Fluorescein injected IV.

4 DISCUSSION

Tumor induced angiogenesis has been demonstrated

in many tumors. The onset of angiogenesis ushers in

a phase of rapid growth, invasion into tissues and

metastasis. Since most sentinel Nodes have been

shown to harbor macrometastases (larger than 2

mm) or micrometastases (0.2 to 2mm), the growth of

cancer cells inside a lymph node should be

associated with neo-vascularisation. Our work in

skin melanoma (Srivatava, 1988) has demonstrated

that neovascularisation in tumour growth is an early

event and can be detected in as early as 1mm thick

melanoma. This was the first demonstration of

angiogenesis in a clinical setting and Dr Judha

Folkman wrote an editorial on this finding

(Folkman, 1987). The tumour vasculature differs

from normal vessels in being devoid of smooth

muscles and precapillary sphincters and having large

diameter vascular network with many arteriovenous

communications. The blood flows through this

vascular network at a high velocity with low

impedance.

In this paper we propose the application of

systemic administration of Fluorescein sodium

intravenously, 20 to 25 min before incision. This

will facilitate detection of tracer in the primary

tumour as well as in the metastatic lymph node. It is

hypothesized that the increased blood flow through a

leaky vascular network in the tumour and metastatic

node would enable high concentration of fluorescein

or other tracer.

Current techniques of sentinel node biopsy

involve injection of a blue dye and an isotope tagged

to a large particulate material viz. sulphur colloid or

albumbin in the breast tissue or skin over the breast.

These tracers pass through the lymphatics of the skin

or breast parenchyma and reach the lymph node (s)

in the axilla. Sometimes, the lymphatics may get

blocked by tumour cells or fibrosis due to prior

surgery, inflammation or radiotherapy. In these

patients the tracer does not reach the lymph node

containing tumour metastasis. Instead, the tracer dye

spreads to some “other lymph node” in the axilla,

which does not contain tumour cells. If these “other

lymph node(s)” are removed and sampled as

“sentinel lymph node”, they are reported by

pathologist as negative for cancer metastasis.

However, the cancer metastasis containing lymph

node is not identified because its lymphatics are

blocked. Such a lymph node missed by present

technique of injecting dye in breast is called the

“False Negative sentinel lymph node”. The

occurrence of “false negative sentinel lymph node”

is reported in 5 % to 10% of patient with early breast

cancer. In these patients this “false negative sentinel

lymph node” is left behind in the axilla and may

grow to present as an axillary lymph node

recurrence later in life.

Therefore, we need to develop a method of

detecting this “false negative sentinel lymph node”.

Our proposed method of injection of tracers

through the “dual route” both through the tissue in

breast as well as intravenously will detect the

tumour containing lymph node and reduce the

probability of a missing “false negative sentinel

lymph node”.

Primary tumour in the breast is treated by wide

DetectionofTumourContainingSentinelLymphNodeinBreastCancerbyInjectionofFluorescenceTracerthrough"Dual

Route"inBreastTissueandIntravenously

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local excision. If tumour is resected with minimal

tissue around it there is chance of leaving tumour

cells in the breast. This may result in recurrence in

the breast. Therefore, it is mandatory to remove the

tumour with negative margins. Our proposed method

will demonstrate a zone of increase fluorescence

around the tumour because tumour vasculature is

most abundant at the periphery of the tumour.

Hence, an excision of tumour including the zone of

increase fluorescence would accomplish complete

tumour ablation.

5 CONCLUSIONS AND FUTURE

DIRECTION

The proposed method is expected to detect cancer

containing sentinel node thus reducing the

probability of false negative rate. If the proposed

methodology is able to detect positive lymph node

with very high accuracy then we may refine the

technique of sentinel node biopsy only to

intravenous injection of the tracer because our

objective is to detect malignant lymph node.

ACKNOWLEDGEMENTS

One of the authors (Darakhshan Qaiser)

acknowledges University Grant Commission of

India (UGC) for granting research fellowship.

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