basic sciences of breast cancer about molecular biology, staging, work up of breast cancer, investigations

1. BASIC SCIENCES OF BREAST CANCER
Dr D. RAMU

2. Epidemiology of breast cancer
• Most frequent cancer in women and second most frequent cause of
cancer death
• For India, for the year 2012:
• 144,937 women were newly detected wth breast cancer
• 70,218 women died of breast cancer
• 144937 / 70218 = 2.06 = round it off to 2. So roughly, in India, for
every 2 women newly diagnosed with breast cancer, one lady is
dying of it.

10. Risk factors for breast cancer
• Reproductive
• Age
• Age at menarche
• Age at first pregnancy (full term)
• Parity
• Breast feeding
• Abortion
• Age at menopause
• Exogenous harmones
• Contraceptive harmones
• Harmone replacement

11. • Genitic factors
• BRCA1/BRCA2
• P53/ Li fraumen syndrome
• PTEN/ Cowden syndrome
• Low penetrance genes
• Familial factors

12. • History of benign breast disease
• Lifestyle and dietery
• Alcohol and tobacco
• Physical activity
• Dietery
• Weight
• Radiation exposure
• Breast density

14. Magnitude of risk of known breast cancer risk
factors
Relative risk <2 Relative risk 2 – 4 Relative risk >4
Early menarche
Late menopause
Nulliparity
Estrogen plus progesterone
HRT
Alcohol use
Postmenopausal obesity
One of the first degree relative with
breast cancer
CHEK2 mutation
Age >35 y for first birth
Proliferative breast disease
Mammographic breast density
BRCA1 or BRCA2 mutation
LCIS
Atypical hyperplasia
Radiation exposure before 30

15. Risk assessment models
• Gail risk assessment model
• Claus risk assessment model
• BRCAPRO assessment model
• Adjuvantonline

16. Management of the high risk patients
Risk reducing measure Relative risk (%)
Tamoxifen 37-49
Raloxifen 56-59
Exemestane 65
Bilateral prophylactic salpingo-oophorectomy
53
Bilateral prophylactic mastectomy 85-100

17. Natural history of disease
• Natural history and prognosis vey from patient to patient
• Tumour doubling time from 44 days to 1800 with average of 212 days
• Breast lump
• Spontaneous nipple discharge
• 3% of women and 20% male breast cancer presentation
• Breast pain
• Skin changes
• Axillary lymphadenopathy
• Nipple changes
• Bone pains
• Respiratory problems

19. • Halstadian theory
• Bernard fisher theory
• Third hypothesis
• Metastasis
• Bone, liver lung, lymphnode,chestwall and brain

20. Screening of breast cancer
• Mammography
• Reduces mortality rats of breast cancer by 24%
• Annually
• Start from 40 yrs
• Life expectancy at least 3-5 yrs
• Breast physical examination
• Lack of data showing reduced risk of death from breast cancer
• Clinical breast examination
• Start from 20 yrs
• At least every three years before 40 and annually after 40
• Breast self examination
• High risk patients

21. Diagnosis and biopsy
• Mammography
• MRI
• Ultrasonography
• Biopsy
• Fine needle aspiration cytology
• Core cutting needle biopsy
• Excisional biopsy
• Staging work up
• Chest X ray
• Complete blood count and liver function test
• Bone scan

22. Mammography

23. • Diffuse or scattered distribution is typically seen in
benign entities.
Even when clusters of calcifications are scattered
throughout the breast, this favors a benign entity.
• Regional distribution according to the BI-RADS atlas
would favor a non-ductal distribution (i.e.
benignity), while
Segmental distribution would favor a ductal
distribution (i.e. malignancy).
Sometimes this differentiation can be made, but in
many cases the differentiation between 'regional'
and 'segmental' is problematic, because it is not
clear on a mammogram or MRI where the
bounderies of a segment (or a lobe) exactly are.
• Clustered calcifications are both seen in benign and
malignant disease and are of intermediate concern.
When clusters are scattered througout the breast,
this favors a benign entity.
A single cluster of calcification favors a malignant
entity.
• Linear distribution is typically seen when DCIS fills
the entire duct and its branches with calcifications.

28. MRI
American Cancer Society Guidelines for Magnetic Resonance Imaging Screening
ANNUAL MRI RECOMMENDED BASED ON EVIDENCE
BRCA mutation
Untested first degree relative of BRCA carrier
Lifetime risk of breast cancer 20% to 25%
ANNUAL MRI RECOMMENDED BASED ON EXPERT OPINION
Radiation to chest between age 10 and 30
Li-Fraumeni syndrome and first-degree relatives
Cowden and Bannayan-Riley-Ruvalcaba syndromes and first-degree relatives
INSUFFICIENT EVIDENCE TO RECOMMEND FOR OR AGAINST MRI
Lifetime breast cancer risk 15% to 20%
Lobular carcinoma in situ
Atypical hyperplasia (lobular or ductal)
Extremely or heterogeneously dense breasts on mammogram
Personal history of breast cancer, including DCIS

29. • For occult breast cancers
• Differentiate scar from recurrence
• Patient with breast implantsto
• Detect chest wall involvement

30. Biopsy
FNAC
• investigation of palpable masses, regardless of whether they are
considered benign or malignant
• investigation of impalpable image-detected masses that are considered
likely to be benign or with typically malignant features
• investigation of suspected local recurrence of breast cancer, as suggested
by the presence of palpable masses, impalpable image-detected masses, or
lymph node involvement
• evaluation of cystic lesions with atypical imaging features
• confirmation of a diagnosis of breast cancer when core biopsy is not
available, not possible or contraindicated

31. • The relative advantages of FNA cytology, compared with core biopsy,
include:
• the sampling procedure for FNA cytology is quicker to perform than core
biopsy
• in most instances FNA cytology does not require local anaesthetic
• FNA cytology is generally less traumatic than core biopsy and may be more
appropriate for women taking anticoagulant medication
• FNA cytology is associated with a low complication rate
• FNA cytology results are available relatively quickly (within a few hours in
some centres); the presence of a cytopathologist may facilitate an immediate
result
• relatively inexpensive to perform.

32. • The relative disadvantages of FNA cytology, compared with core biopsy,
include:
• FNA cytology requires training in the preparation of quality smears
• considerable cytology expertise is required to interpret FNA cytology
• FNA cytology is generally inappropriate for the assessment of microcalcifications
• FNA cytology does not enable the pathologist to distinguish between DCIS and
invasive carcinoma
• Definitive diagnosis of some lesions can be difficult to make on the basis of FNA
cytology.These include atypical ductal hyperplasia (ADH), lowgrade DCIS, some
tubular carcinomas and some invasive lobular carcinomas
• FNA cytology may not be the sampling technique of choice for lesions that are
relatively hypocellular and yield scanty epithelial material.These include sclerotic
fibroadenomas, sclerosing ductal carcinoma, and infiltrating lobular carcinoma.

33. • Indications for core biopsy
• Core biopsy may be indicated in the following clinical situations:
• investigation of lesions with suspicious features identified on imaging that cannot
be identified on ultrasound
• further evaluation of a benign cytological pattern in the presence of a suspicious
lesion on imaging
• further evaluation of a lesion for which cytology results are atypical orsuspicious
• when a single surgical procedure is the desired outcome (for example wide
excision and axillary dissection). It must be noted that a core biopsy showing only
DCIS does not exclude the possibility of the presence of invasive carcinoma in the
lesion
• evaluation of microcalcifications that are radiologically indeterminate, suspicious
or typically malignant. In such cases core biopsies should be radiographed prior to
histological processing to confirm adequate sampling of the lesion
• evaluation of suspicious architectural distortion at a site of previous malignancy
• evaluation of an area that has been treated with radiation.

34. • The relative advantages of core biopsy, compared with FNA cytology, include:
• core biopsy is the investigation of choice in the evaluation of microcalcifications22
• core biopsy can be used when FNA cytology fails to correlate with clinical findings or
imaging studies, as may occur with an inadequate cytology specimen (eg fewer than three to
six epithelial groups per slide).23 If the cytological findings do not correlate with the clinical
and/or imaging findings, further investigation should be performed
• core biopsy yields tissue fragments allowing architectural features of the lesion to be
identified to determine whether DCIS or invasive carcinoma is present
• core biopsy is useful in the evaluation of lesions likely to be low histological grade and in
those presenting as architectural distortions, for which FNA cytology may fail or has failed to
provide a diagnosis
• core biopsy may be preferred when appropriate cytological expertise is not available
• compared with FNA cytology, core biopsy may achieve higher specificity and sensitivity.
Limited data suggest it may increase the probability of obtaining a satisfactory and
representative sample, particularly for imagedetected lesions
• tissue is usually available for adjunctive tests (ER, PR and Her2).

35. • Potential disadvantages of core biopsy include the following:
• the reliability of core biopsy depends on the skill of the operator
• false negatives may result from: (i) a ‘clear miss’, that is the lesion not being sampled, or (ii)
artefactual distortion of the lesion and/or cores making definitive interpretation of the pathology
changes on excision impossible
• it is not always possible to immediately assess the adequacy of core biopsyperformed for a mass
lesion or architectural distortion. In the case of microcalcifications the specimen can be
radiographed to confirm whether representative microcalcifications are present
• compared with FNA cytology, core biopsy is associated with an increased risk of complications,
including haematoma,8 haemorrhage and needle tract implantation of tumour cells.2, 24, 25 These
are more likely to occur if a large number of core biopsies are performed
• core biopsy requires the use of a local anaesthetic
• the mammographic lesion may not be identified in subsequent open biopsy, due to complete
removal of the lesion or in the presence of inflammation and fibrosis due to biopsy
• core biopsy may interfere with the interpretation of the subsequent excision biopsy, particularly
with grading and the estimation of the size of the lesion.This is particularly relevant in the case of
small lesions
• core biopsy requires adequate fixation and processing, and generally requires a minimum of one
working day before results can be availablefootnote
• core biopsy is generally more expensive than FNA cytology.

36. • Indications for surgical biopsy after core needle biopsy
• Failure to sample calcification
• Diagnosis of atypical ductal hyperplasia
• Diagnosis of atypical lobular hyperplasia or lobular carcinoma
insitu
• Lack of concordance between imaging findings and histologic
diagnosis
• Radial scar
• Papillary lesion

37. Technique Advantages Disadvantages
Fine needle
aspiration cytology
Rapid, painless, inexpensive. No
incision prior to selection of local
therapy.
Does not distinguish invasive from in situ cancer.
Markers (ER, PR, HER-2) not routinely available.
Requires experienced cytopathologist. False
negatives and insufficient specimens occur.
Core cutting
needle biopsy
Rapid, relatively painless,
inexpensive. No incision. Can be
read by any pathologist, markers
routinely available.
False-negative results, incomplete lesion
characterization can occur.
Excisional biopsy False-negative results rare.
Complete histology before
treatment decisions. May serve as
definitive lumpectomy.
Expensive, more painful. Creates an incision to be
incorporated into definitive surgery. Unnecessary
surgery with potential for cosmetic deformity in
patients with benign abnormalities.

38. Staging • TX Primary tumor cannot be assessed
• T0 No evidence of primary tumor
• Tis Carcinoma in situ
• Tis (DCIS) Ductal carcinoma in situ
• Tis (LCIS) Lobular carcinoma in situ
• Tis (Paget’s) Paget’s disease of the nipple NOT associated with invasive carcinoma and/or carcinoma in situ (DCIS and/or LCIS) in the underlying
breast parenchyma. Carcinomas in the breast parenchyma associated with Paget’s disease are categorized based on the size and characteristics of
the parenchymal disease, although the presence of Paget’s disease should still be noted
• T1 Tumor ≤ 20 mm in greatest dimension
• T1mi Tumor ≤ 1 mm in greatest dimension
• T1a Tumor >1 mm but ≤ 5 mm in greatest dimension
• T1b Tumor >5 mm but ≤ 10 mm in greatest dimension
• T1c Tumor >10 mm but ≤ 20 mm in greatest dimension
• T2 Tumor >20 mm but ≤ 50 mm in greatest dimension
• T3 Tumor >50 mm in greatest dimension
• T4 Tumor of any size with direct extension to the chest wall and/or to the skin (ulceration
• or skin nodules).
• Note: Invasion of the dermis alone does not qualify as T4
• T4a Extension to the chest wall, not including only pectoralis muscle adherence/invasion
• T4b Ulceration and/or ipsilateral satellite nodules and/or edema (including peaud’orange) of the skin, which do not meetthe criteria for infl
ammatory carcinoma
• T4c Both T4a and T4b
• T4d Inflammatory carcinoma

39. • NX Regional lymph nodes cannot be assessed (e.g., previously removed)
• N0 No regional lymph node metastases
• N1 Metastases to movable ipsilateral level I, II axillary lymph node(s)
• N2 Metastases in ipsilateral level I, II axillary lymph nodes that are clinically fi xed
or matted; or in clinically detected * ipsilateral internal mammary nodes in the
absence of clinically evident axillary lymph node metastases
• N2a Metastases in ipsilateral level I, II axillary lymph nodes fi xed to one another
(matted) or to other structures
• N2b Metastases only in clinically detected * ipsilateral internal mammary nodes and
in the absence of clinically evident level I, II axillary lymph node metastases
• N3 Metastases in ipsilateral infraclavicular (level III axillary) lymph node(s) with or
without level I, II axillary lymph node involvement; or in clinically detected *
ipsilateral internal mammary lymph node(s) with clinically evident level I, II
axillary lymph node metastases; or metastases in ipsilateral supraclavicular lymph
node(s) with or without axillary or internal mammary lymph node involvement
• N3a Metastases in ipsilateral infraclavicular lymph node(s)
• N3b Metastases in ipsilateral internal mammary lymph node(s) and axillary lymph
node(s)
• N3c Metastases in ipsilateral supraclavicular lymph node(s)

40. • pN0(i−) No regional lymph node metastases histologically, negative IHC
• pN0(i+) Malignant cells in regional lymph node(s) no greater than 0.2 mm (detected by H&E
or IHC including ITC)
• pN0(mol−) No regional lymph node metastases histologically, negative molecular findings (RT-PCR)
• pN0 (mol+) Positive molecular fi ndings (RT-PCR), ** but no regional lymph node metastases
detected by histology or IHC
• pN1 Micrometastases; or metastases in 1–3 axillary lymph nodes; and/or in internal
mammary nodes with metastases detected by sentinel lymph node biopsy but not clinically
detected
• pN1mi Micrometastases (greater than 0.2 mm and/ or more than 200 cells, but none greater
than 2.0 mm)
• pN1a Metastases in 1–3 axillary lymph nodes, at least
• one metastasis greater than 2.0 mm
• pN1b Metastases in internal mammary nodes with micrometastases or macrometastases
detected by sentinel lymph node biopsy but not clinically detected ***
• pN1c Metastases in 1–3 axillary lymph nodes and in internal mammary lymph nodes with
micrometastases or macrometastases detected by sentinel lymph node biopsy but not
clinically detected

41. • pN2 Metastases in 4–9 axillary lymph nodes; orin clinically detected **** internal
mammary lymph nodes in the absence of axillary lymph node metastases
• pN2a Metastases in 4–9 axillary lymph nodes (at least one tumor deposit greater than
2.0 mm)
• pN2b Metastases in clinically detected **** internal mammary lymph nodes in the
absence of axillary lymph node metastases
• pN3 Metastases in ten or more axillary lymph nodes; or in infraclavicular (level III axillary)
lymph nodes; or in clinically detected **** ipsilateral internal mammary lymph nodes in
the presence of one or more positive level I, II axillary lymph nodes; or in more than
three axillary lymph nodes and in internal mammary lymph nodes with micrometastases
or macrometastases detected by sentinel lymph node biopsy but not clinically detected
*** ; or in ipsilateral supraclavicular lymph nodes
• pN3a Metastases in ten or more axillary lymph nodes (at least one tumor deposit greater
than 2.0 mm); or metastases to the infraclavicular (level III axillary lymph) nodes
• pN3b Metastases in clinically detected **** ipsilateral internal mammary lymph nodes in
the presenc of one or more positive axillary lymph nodes; or in more than three axillary
lymph nodes and in internal mammary lymph nodes with micrometastases or
macrometastases detected by sentinel lymph node biopsy but not clinically detected ***
• pN3c Metastases in ipsilateral supraclavicular lymph nodes

42. • Distant Metastases (M)
• M0 No clinical or radiographic evidence of distant metastases
• cM0(i+) No clinical or radiographic evidence of distant metastases,
but deposits of molecularly or microscopically detected tumor cells in
circulating blood, bone marrow, or other nonregional nodal tissue
that are no larger than 0.2 mm in a patient without symptoms or
signs of metastases
• M1 Distant detectable metastases as determined by classic clinical
and radiographic means and/or histologically proven larger than 0.2
mm

44. • HISTOPATHOLOGIC TYPES
• In situ Carcinomas
• NOS (not otherwise specifi ed)
• Intraductal
• Paget’s disease and intraductal
• Invasive Carcinomas
• NOS
• Ductal
• Infl ammatory
• Medullary, NOS
• Medullary with lymphoid stroma
• Mucinous
• Papillary (predominantly micropapillary pattern)
• Tubular
• Lobular
• Paget’s disease and infi ltrating
• Undifferentiated
• Squamous cell
• Adenoid cystic
• Secretory
• Cribriform

45. Pathology of breast cancer
• Most of tumours arise from terminal duct lobular unit
• Infiltratinn ductal carcinoma not other specified
• 75%
• Hrad on palpation
• Gritty on transection
• Associated with various degree of fibrosis
• Commnly metastasis to axilla
• Prognosis is poor
• Distance metastasis to bone , lung liver and brain
• Tumour with mixed histologic types behave as NOS type

46. • Infiltrating lobular carcinoma
• 5-10%
• Clinically ill defined thickening in breast
• Microscopically single or indian file pattern
• Multicentricity and bilaterality
• Metastasis to unusual sites like meninges and serosal surface
• Tubular carcinoma
• 2%
• >75% tumour showing tubule formation
• Nodal metastasis uncommon
• Prognosis is good

47. • Medullary carcinoma
• 5-7%
• Poorly differentiated nuclei , syncytial growth pattern
• Intense infiltration with mall lymphocytes and plasma cells
• Prognosis is favourable
• Mucinous or colloid carcinoma
• 3%
• Abundant accumulation of mucine surrounding cluster of tumour cells
• Slow growing, tend to be bulky
• Prognosis favourable

48. Grading of breast carcinoma
• Nottingham combined histologic grading (elston ellis modification of scarf bloom
Richardson grading system)
• Tubule formation
• Percent of the tumor forms normal duct structures
• 1 point: tubular formation in more than 75% of the tumor
• 2 points: tubular formation in 10 to 75% of the tumor
• 3 points: tubular formation in less than 10% of the tumor
• Nuclear pleomorphism
• 1 point: nuclei with minimal variation in size and shape
• 2 points: nuclei with moderate variation in size and shape
• 3 points: nuclei with marked variation in size and shape
• Mitotic count
• 1 point: 0-9 mitotic counts per 10 fields under X25 objective
• 2 points: 10-19 mitotic counts per 10 fields under X25 objective
• 3 points: Over 19 mitotic counts per 10 fields under X25 objective
• Overall grade
• 3-5 Grade 1 tumor (well-differentiated). Best prognosis
• 6-7 Grade 2 tumor (moderately differentiated). Medium prognosis.
• 8-9 Grade 3 tumor (poorly differentiated). Worst prognosis.

49. Molecular classification of breast carcinoma
• Luminal A
• Express cytokeratin 8 and 18
• Highest level of ER expression, low expression of proliferative markers
• Low grade
• Respond to endocrine therapy
• Prognosis favourable
• Luminal B
• Low epression of ER and ER related genes, high proliferation markers
• Prognosis poor
• Normal like breast cancer
• Geneexpression profile reminiscent of normal breast epithelium
• Prognosis similar to luminal B tumour

50. • HER2 amplified
• Amplification of HER2 gene on 17q chromosome
• IHC staining 3+(uniform intense membrane staining >30% of tumour cells)
• FISH >6 gene copies per nucleus or FISH ratio (HER2 gene signal to chromosome 17
signal) >2.2
• Decreased expression of ER&PR
• Upregulation of vascular endothelial factor
• Prognosis is poor
• Basal like
• Triple negative ER&PR negative, HER2 negative
• Cytokeratine 5,6 and 17positive
• Prognosis poor

51. Genetics of breast cancer

52. • BRCA1
• 17q21
• Breast cancer risk 60-85%
• Breast, ovarian, prostate, colon, liver and bone cancer
• Younger age, more aggressive, high grade, high proliferative rate, triple negative and
basal like . Mostly medullary histology
• BRCA2
• 13q12.3
• Breast cancer risk 60 to 85%
• Male breast, ovary, pancrease, gall blader, pharynx, stomach, melanoma, fanconies
anemia
• Histology similar to sporadic breast cancer

53. • TP53
• 17p13.1
• Breast cancer risk 50 to89%
• Cowdens syndrome
• PTEN-10q233.3
• 25-50% risk

54. BRCA testing
Factors Suggestive of BRCA1 or BRCA2 Mutation
NON-ASHKENAZIC JEWISH WOMEN
Two first-degree relatives with breast cancer, one diagnosed < 50 years
Three or more first- or second-degree relatives with breast cancer, any age
Breast and ovarian cancer among first- and second-degree relatives
First-degree relative with bilateral breast cancer
Breast cancer in a male relative
Two or more first- or second-degree relatives with ovarian cancer
ASHKENAZIC JEWISH WOMEN
First-degree relative with breast or ovarian cancer
Two second-degree relatives with breast or ovarian cancer

55. Prognostic and predictive factors
• TNM classification
• Nodal involvement
• Tumour size
• Metastasis
• Inflamatory carcinoma
• Histological subtype
• Histological grade
• ER/PR status
• HER2/neu status
• LVI
• Proliferative index
• DNA content
• Response to neoadjuvent chemotherapy
• Gene expression profile

59. Prognostic indices of breast cancer
• Oncotype DX® 21-Gene Recurrence Score (RS) Assay
• Mamma printing
• Adjuvant online assay

60. Oncotype DX® 21-Gene Recurrence Score (RS) Assay
16 Cancer and 5 Reference Genes From 3 Studies
PROLIFERATION
Ki-67
STK15
Survivin
Cyclin B1
MYBL2
ESTROGEN
ER
PR
Bcl2
SCUBE2
INVASION
Stromelysin 3
Cathepsin L2
HER2
GRB7
HER2
RS = + 0.47 x HER2 Group Score
GSTM1 BAG1
CD68
REFERENCE
Beta-actin
GAPDH
RPLPO
GUS
TFRC
- 0.34 x ER Group Score
+ 1.04 x Proliferation Group Score
+ 0.10 x Invasion Group Score
+ 0.05 x CD68
- 0.08 x GSTM1
- 0.07 x BAG1
Category RS (0 -100)
Low risk RS <18
Int risk RS 18 - 30
High risk RS ≥ 31
Paik et al. N Engl J Med. 2004;351:2817-2826.

61. • The Oncotype DX® Recurrence Score assay predicts the likelihood of
adjuvant chemotherapy benefit
• It also is a prognostic assay for the risk of distant recurrence at ten
years assuming five years of adjuvant tamoxifen treatment
• Oncotype DX® Recurrence Score assay shows consistent results across
multiple independent studies
Trial Assigning IndividuaLized Options for Treatment (Rx) (TAILORx)
NSABP B20
The Kaiser Permanente Study

62. • The MammaPrint test by Agendia analyzes 70 genes from an early-stage
breast cancer tissue sample to figure out if the cancer has a low
or high risk of coming back (recurrence) within 10 years after
diagnosis.
• MammaPrint can only be used to analyze early-stage breast cancers.
In the United States, MammaPrint can be used on cancers that are:
• stage I or stage II invasive
• smaller than 5 centimeters
• estrogen-receptor-positive or -negative
• Uses
• A low-risk test result means that the cancer has a 10% risk of coming back
within 10 years without any additional treatments after surgery. With
hormonal therapy alone this risk can be reduced to 5%.
• A high-risk test result means that the cancer has a 29% risk of coming back
within 10 years without any additional treatments after surgery.

63. • Mammostrat Test
• measures the levels of five genes in breast cancer cells. These genes can affect how
the breast cancer will behave and respond to certain treatments. These
measurements are used to calculate a risk index score. The higher the risk index
score, the more likely the cancer is to come back (recur).
• Women in the low risk category have a 7.6% risk of the cancer coming back within 10
years.
• Women in the moderate risk category have a 16.3% risk of the cancer coming back
within 10 years.
• Women in the high risk category have a 20.9% risk of the cancer coming back within
10 years.
• Women in the high risk category are more likely to benefit from chemotherapy in
addition to hormonal therapy after surgery. Women in the low risk category may be
able to avoid chemotherapy that probably won’t give them any additional benefits.