To address the ambiguity of different modes of human papillomavirus (HPV) transmission in breast, the immunohistochemical expression of two oncoproteins E6/E7 of HPV16 was analyzed in primary breast cancer (BC) and adjacent normal skin of 4 samples. The patients were of 35–55 years old having no previous history of cancer. The E6/E7 expressions were evident in both skin and BC. In skin, high/moderate cytoplasmic expressions of E6/E7 proteins were seen in all samples, whereas in BC, high/moderate cytoplasmic expressions of the proteins were observed in 2–3 samples. Thus, it seems that HPV infection in the breast may occur through the skin

Alam, Neyaz

Basu, Mukta

Dasgupta, Hemantika

Islam, Saimul

Mandal, Gautam Kumar

Panda, Chinmay Kumar

Roy, Anup

Roychoudhury, Susanta

English

Atharva Scientific Publications (E-Jounals)

Vol 4 | Issue 2 | Mar - Apr 2018 Indian J Case Reports 98
Case Report
Skin mediated human papillomavirus infection in breast: A report of four cases
Saimul Islam1, Hemantika Dasgupta1, Mukta Basu1, Anup Roy2, Neyaz Alam3, Gautam Kumar Mandal4, 
Susanta Roychoudhury5, Chinmay Kumar Panda1
From 1Departments of Oncogene Regulation, 3Surgical Oncology and 4Pathology, Chittaranjan National Cancer Institute, 2Department 
of Pathology, Nil Ratan Sircar Medical College and Hospital, 5Saroj Gupta Cancer Centre and Research Institute, Thakurpukur, Kolkata, 
West Bengal, India
Correspondence to: Chinmay Kumar Panda, Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. 
Mukherjee Road, Kolkata – 700 026, West Bengal, India. Tel: +91-33-2474 3922, Fax: +91-33-2475-7606. E-mail: ckpanda.cnci@gmail.com
Received - 03 January 2018 Initial Review - 31 January 2018 Published Online -  23 March 2018
Breast cancer (BC) is a frequent disease (25%) among women worldwide [1]. In India, its occurrence is about 18.5% [1]. Different etiological factors such as hormonal 
status, tobacco habits, alcohol consumption, and family history 
have been suggested to be associated with the disease [2]. In 
addition, human papillomavirus (HPV) infection has also been 
suggested to be an etiological factor in the development of the 
disease [3]. Globally, the HPV infection varies differentially 
(23.3–86%) in BC [4]. However, in India, its frequency varies 
between 0 and 26% [4,5]. Recently, our group also showed that 
HPV16 was prevalent subtype in Indian BC population [6]. 
However, there is still doubt in its possible mode of infection of 
HPV in breast tissue: (1) Transmission through blood, lymphatic 
systems or any other body fluid in patients with previous history 
of HPV-positive uterine cervical lesions, (2) transmission 
through oral sexual activity, and (3) transmission through nipple 
or microlesion of breast skin [7,8]. To address this ambiguity, 
in the present study, we have detected HPV in four primary BC 
samples and adjacent skin of the corresponding patients having 
no previous history of cervical lesions.
CASE REPORT
The four breast tumor specimens were collected during the year 
2011–2014 from Chittaranjan National Cancer Institute (CNCI), 
Kolkata, India, after appropriate approval of the Institutional 
Ethical Committee. Written informed consent was obtained from 
either of the patients or next kin of the respective patients. The 
age of the patients ranged from 35 to 55, with no previous history 
of cancer and tobacco habits (Table 1). One of the four cases has 
undergone chemotherapy before surgery. Among four cases, two 
tumors showed poorly differentiated form, whereas other two 
cases showed moderately differentiation. Depending on receptor 
status, two of four cases were belonging to luminal B, whereas 
one case falls in HER2-positive BC subtypes. After starting of 
therapy, three patients died within 5 years, but one patient is 
surviving even after 24 months. The detailed descriptions of four 
cases are given in Table 1.
Detection of HPV16 E6 and E7 Proteins Expression
The expression of HPV16 E6 and E7 proteins was analyzed in 
the tumors and adjacent skin samples by IHC using paraffin-
embedded tissue sections [9]. About 5 μm thick sections were 
dewaxed, rehydrated, and incubated in 10 mM citrate buffer 
(pH-6.0) at 85°C for 40 min to retrieve the antigens followed 
by incubation with primary antibodies at a dilution of 1:200 at 
4°C for overnight. Goat polyclonal IgG for E6 (sc-1584) and 
mouse monoclonal IgG for E7 (sc-6981) were purchased from 
M/s Santa Cruz Biotechnology, CA, USA. HRP-conjugated 
rabbit anti-goat IgG (sc- 2768) and HRP-conjugated goat anti-
mouse IgG (sc-2005) as secondary antibodies (M/s Santa Cruz 
Biotechnology, CA, USA) for E6 and E7, respectively, were 
added at 1:500 dilutions. The slides were developed using 3-3’ 
diaminobenzidine as the chromogen and counterstained with 
hematoxylin. The staining intensity (1=weak, 2=moderate, and 
3=strong) and the percentage of positive cells (<1=0, 1–20 =1, 
20–50=2, 50–80=3, and >80=4) were detected by two observers 
ABSTRACT
To address the ambiguity of different modes of human papillomavirus (HPV) transmission in breast, the immunohistochemical 
expression of two oncoproteins E6/E7 of HPV16 was analyzed in primary breast cancer (BC) and adjacent normal skin of 4 samples. 
The patients were of 35–55 years old having no previous history of cancer. The E6/E7 expressions were evident in both skin and 
BC. In skin, high/moderate cytoplasmic expressions of E6/E7 proteins were seen in all samples, whereas in BC, high/moderate 
cytoplasmic expressions of the proteins were observed in 2–3 samples. Thus, it seems that HPV infection in the breast may occur 
through the skin.
Key words: Breast cancer, breast skin, E6/E7 oncoproteins, human papillomavirus, human papillomavirus transmission
Islam et al. Skin mediates human papillomavirus infection in breast
Vol 4 | Issue 2 | Mar - Apr 2018 Indian J Case Reports 99
Parameters Case-I Case-II Case-III Case-IV
Case description A 54-year-old woman: 
Mammography and FNAC 
were used to diagnosed in 
right-sided breast tumor
A 35-year-old woman: 
FNAC detected breast 
cancer in left sided
A 55-year-old woman: 
FNAC diagnosed 
left-sided breast cancer
A 55-year-old woman: 
USG detected solid and 
heterogynous growth in 
left-sided breast and was 
confirmed cancer by FNACC
Symptom with 
duration
Lump in right breast for 
1 year
Lump in left breast for 7 
months
A fungating mass in left 
breast for 2.3 year
Breast lumps increasing in 
size for 8 months
Local examination 
finding
A 2×3 cm hard nodule was 
palpated in breast upper and 
outer quadrant
A 7×6 cm lump was felt in 
upper and outer quadrant 
with matted axillary nodes
A fungating bleeding 
mass in upper quadrant 
in left breast along with 
axillary nodes
A 2×1.5 cm mass felt in 
upper and outer quadrant
Family history She has no previous family 
history of cancer
Previous family history of 
cancer was absent
Patients has no previous 
family history of cancer
Family history of cancer was 
absent
Previous history of 
cancer
Absent Absent Absent Absent
Patients habits’ The patient has no any 
tobacco habits
The patient was not 
habited with any type of 
tobacco
Any type of tobacco 
habit of patients was 
absent
The patient was not habited 
with any type of tobacco
Treatment sequence Surgery followed 
chemotherapy and 
radiotherapy
Neo-adjuvant 
chemotherapy followed by 
surgery then completion 
chemotherapy and 
radiotherapy
Surgery followed 
chemotherapy
Surgery followed 
chemotherapy and 
radiotherapy
Surgical description MRM MRM Toilet mastectomy MRM
Surgical specimen 
description
Mastectomy specimen of 
17.5×16×6 cm mass, in 
which skin flap of 16×17.5 
cm was present with an area 
of ulceration 1.5×1.5 cm 
near lateral margin of the 
specimen was observed. 
In addition, 3×4.5×1.7 cm 
mass was observed beneath 
the unaltered area. 11 lymph 
nodes identified, largest being 
1.5×1 cm and smallest 0.3 cm
N/A N/A Mastectomy specimen of 
18×16×6 cm mass in which 
skin flap was 14×6 cm. In 
this specimen, 2×1.5×4 cm 
area was ulcerated which was 
1 cm away from cut margin
Histopathology of 
Breast tumor
Invasive Ductal Carcinoma 
with Grade-III
Invasive Ductal Carcinoma 
with Grade-III
Ductal 
carcinoma-Grade-II
Infiltrating Ductal Carcinoma 
with Grade-II
Auxiliary lymph 
node histopathology
4/11 lymph nodes show 
metastatic deposit
9/11 lymph nodes show 
metastatic deposit
Lymph nodes 
positive (detail not 
known)
All 11 lymph nodes were free 
of tumor
Clinical stage of 
tumor
N/A T3N1M0 T4N1Mx T2N0Mo
Tumor size in 
specimen
2×3 cm 7×6 cm N/A 2×1.9 cm
Parity (No of child 
birth)
N/A She has four ‎children. She has three ‎children. She has five ‎children. Among 
them, only two are alive
Estrogen receptor 
status
Negative N/A Positive Positive
Progesterone 
receptor status
Negative Negative Positive Positive
Her2 receptor status Positive Negative Positive Positive
Chemotherapy 4 cycles of FEC - 4 cycles of 
taxanes
FEC=5-Fuloro-uracil, 
epirubicin, and 
cyclophosphamide
Taxanes=Paclitaxel
4 cycles of FAC - 6 cycles 
of Paclitaxel and 
Cis-platin.
FAC=5-Fluro-uracil, 
adriamycin, and 
cyclophosphamide
6 cycles of FAC
FAC=5-Fluro-uracil, 
adriamycin, and 
cyclophosphamide
6 cycles of FEC
FEC=5-Fluoro-uracil, 
epirubicin, and 
cyclophosphamide
Table 1: Clinical and histopathological history of the four HPV16-infected breast cancer cases
(Contd...)
Islam et al. Skin mediates human papillomavirus infection in breast
Vol 4 | Issue 2 | Mar - Apr 2018 Indian J Case Reports 100
independently and by combining the two scores. Then, the 
final evaluation of expression was done as follows: 0–2=low, 
3–5=intermediate, and 6–7=high.
DISCUSSION
To understand the mode of HPV transmission in the breast, the 
expression of E6 and E7 oncoproteins of HPV16 was analyzed 
in primary BC and their respective adjacent normal skin tissues. 
Differential expression pattern of E6/E7 proteins was seen in BC 
and the respective skin (Table 2).
In normal breast skin, high/moderate cytoplasmic expression 
of E6 and E7 proteins was seen in basal cells with low/negative 
nuclear expression in the majority of cases (Figs. 1 and 2 and 
Table 2). This indicates that breast skin may be one of target organs 
of the HPV infection. HPV DNA was detected in superficial 
layer of nipple epidermis, breast milk, ductal lavage fluid, and 
colostrum [10,11] and normal breast tissue [12]. HPV infection was 
also reported in normal perilesional skin of ear, wrist, forehead, 
leg, and temple [13]. The prolong infection of HPV in the skin has 
been shown to be associated with carcinogenesis [13,14]. Hence, 
the presence of E6 and E7 proteins in the basal layer of skin 
indicates the route of HPV transmission as suggested in uterine 
cervical epithelium [15].
Like skin, high/moderate cytoplasmic expressions of E6/E7 
proteins were seen in 2–3 cases of BC (Figs. 1 and 2 and Table 2). 
However, high/moderate nuclear expressions of E6/E7 proteins 
were also evident in 2 cases. Similar, E6 and E7 protein expressions 
Parameters Case-I Case-II Case-III Case-IV
Radiation N/A EBRT completed in 
Co60-50Gy-25 fraction 
from 11.3.13–16.4.13.
Boost chest wall dose - 7.5 
Gy in 3 fractions
Not given (patient died) Radiation not required.
Survival 14 months after starting of 
therapy
5 months after starting of 
therapy
58 months after starting 
of therapy
Still now, she is alive during 
24 months after starting of 
therapy
FNAC: Fine-needle aspiration cytology, N/A: Not available. MRM: Modified radical mastectomy, HPV: Human papillomavirus, USG: Ultrasonography
Table 1: (Continued...)
Table 2: Expression pattern of E6 and E7 proteins of HPV16 in normal breast skin and adjacent breast cancer
Cases Normal breast skin Breast cancer
E6 E7 E6 E7
Nucleus Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm Nucleus Cytoplasm
Case-I Absent M L H L L Absent H
Case-II L H M H H L H M
Case-III L H L H M H H L
Case-IV L M L H Absent M L M
L: Low expression; M: Moderate expression; H: High expression. HPV: Human papillomavirus
Figure 1: Immunohistochemical detection of E6 oncoprotein of human papillomavirus (HPV16)-positive breast carcinoma tissue and adjacent 
HPV16-positive normal skin: (a) IHC staining showed E6 protein expression in both normal skin and tumor tissue of the same BC sample. 
(b and c) IHC staining of E6 in HPV16-positive normal breast skin showed high cytoplasmic and low nuclear expression of E6 protein. (e and 
f) IHC of HPV16 E6 in BC showed high cytoplasmic and moderate nuclear expression of E6 protein. (d and g) IHC without primary antibody 
representing negative control [Magnification (a) is ×4, (b and e) are ×10, whereas (c and f) are ×40, scale bars=50 μm]
a
b c d
e f g
Islam et al. Skin mediates human papillomavirus infection in breast
Vol 4 | Issue 2 | Mar - Apr 2018 Indian J Case Reports 101
were also detected in BC by Lawson et al.[16]. It was evident that 
the E6 and E7 oncoproteins of HPV16 could immortalize human 
mammary epithelial cells, indicating their importance in cellular 
transformation [17]. In addition, hrHPV DNA and its transcripts 
of E6 and E7 were also previously detected in BC though having a 
low copy number. Interestingly, reports pointed out that such low 
copy number of HPV16 is sufficient to bring about carcinogenesis 
as seen in HPV16-positive cervical squamous cell carcinoma cell 
line, SiHa [4,16,18].
CONCLUSION
The present report of the similar expression pattern of E6/E7 
proteins in skin and adjacent BC indicates that HPV transmission 
in breast might have occurred through nipple or microlesion of 
breast skin in these cases. Although more samples need to be 
analyzed, it may perhaps be extrapolated to a model for plausible 
mode of transmission of HPV in breast tissue. This will be further 
useful to understand the HPV association in breast carcinogenesis 
along with its importance in prognosis and therapeutic implication 
in disease.
ACKNOWLEDGMENT
The authors thank the Director, CNCI, Kolkata, India for 
his encouragement. They thank Dr. Partha Sarathi Dasgupta, 
Emeritus Scientist, CNCI, for his valuable suggestions during 
the study. We would also like to extend our appreciation to Mr. 
Anirban Roychowdhury, CNCI, Kolkata, India, for his valuable 
help in revising this case report. Financial support for this work 
was provided by UGC-NET Fellowship grant F.2-3/2000 (SA-
I) (Sr. No. 2061030813, Ref. No.: 20-06/2010(i) EU-IV dated 
22.10.2010) to Mrs. H. Dasgupta, UGC-NET Fellowship grant 
(Sr. No. 2061430780, Ref No.: 22/06/2014(i)EU-V) to Mukta 
Basu and grants from Council of Scientific and Industrial 
Research, Government of India (No. 60[0111]/14/EMR-II of dt 
03/11/2014) to Dr. C. K. Panda.
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Figure 2: Immunohistochemical detection of E7 protein of human papillomavirus (HPV16) in breast cancer tissue and adjacent HPV16-
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HPV16 E7 in BC showed moderate cytoplasmic and high nuclear expression of E6 protein. (d),(g) IHC staining without primary antibody 
representing negative control [Magnification (a) is ×4, (b and e) are ×10, whereas (c and f) are ×40, scale bars=50 μm]
a
b c d
e f g
Islam et al. Skin mediates human papillomavirus infection in breast
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Funding: None; Conflict of Interest: None Stated.
How to cite this article: Islam S, Dasgupta H, Basu M, Roy A, 
Alam N, Mandal GK, Roychoudhury S, Panda CK. Skin mediated human 
papillomavirus infection in breast: A report of four cases. Indian J Case 
Reports. 2018;4(2):98-102.
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16 in esophageal squamous cell carcinoma. J Clin Virol 2005;32:19-23.


Skin mediated human papillomavirus infection in breast: A report of four cases

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Skin mediated human papillomavirus infection in breast: A report of four cases

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