Case 10 - Breast Cancer notes PDF

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Case 10: Breast Cancer Case 10 – Breast Cancer Page 1 of 65 Case 10: Breast Cancer Case Introduction  Risk factors  Age over 60 years  Oestrogen exposure – early menopause, late menopause, oral contraception use, HRT  Breastfeeding and physical activity protect against breast cancer  Obesity  ...

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Case 10: Breast Cancer

Case 10 – Breast Cancer

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Case 10: Breast Cancer

Case Introduction 

Risk factors  Age over 60 years  Oestrogen exposure – early menopause, late menopause, oral contraception use, HRT  Breastfeeding and physical activity protect against breast cancer  Obesity  Alcohol  Certain occupational exposures  Family history of breast cancer  Inheritance of mutated BRCA genes confers an 80-90% lifetime risk of breast cancer

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Presentation  Patients will typically present with a palpable breast lump or a radiologically identified mass  A change in the size or shape of the breast  Dimpling of skin or thickening in the breast tissue  A nipple that’s turned in (inverted)  A rash (like eczema) on the nipple  Discharge from the nipple  Swelling or a lump in the armpit

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Screening  Breast self-examination  Mammography  UK National Breast Screening Programme screens all women aged 50-70 every 3 years  Facilitates the detection of early breast cancers reducing mortality by 20-30%

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Treatment  Surgery  Endocrine therapy  Radiotherapy  Chemotherapy  mAbs and other targeted treatments

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Case 10: Breast Cancer

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Immunohistochemistry or IHC refers to the process of detecting antigens e.g. proteins, in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues – linked to a chromophore so that expression levels can be scored based on the intensity of staining FISH (fluorescence in-situ hybridisation) – works on a similar principal but using DNA primers linked to a fluorescent marker

TNM (tumour size, lymph nodes and metastasis) staging can be used for all solid tumours, the principle is the same although the criteria for the classifications changes TNM is the most commonly used staging system for breast cancer DCIS = Ductal carcinoma in situ – proliferation of malignant cells confined to mammary ducts without invasion through the basement membrane e.g. non-invasive precancerous lesion

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Case 10: Breast Cancer

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Pathological classification  Invasive breast tumours include invasive ductal and lobular cancer  A firm, fibrous mass with sharp margins and small, glandular, duct-like cells are seen in invasive ductal tumours – they’re the worst and most invasive of the tumours as well as the most common  Invasive lobular cancer often presents bilaterally with multiple lesions in the same location  Pathologically, they present as an orderly row of cells  Fleshy, cellular lymphocytic infiltrate is seen with medullary breast carcinoma and it has a good prognosis  Inflammatory breast tumour presents with dermal lymphatic invasion and has approximately 50% survival at 5 years

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Hormone receptor positive tumours make up 65-75% of all breast cancers The cells of these tumours are largely dependent on female hormones for their growth and survival

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Prognosis and response to treatment will depend on the subtype of breast cancer involved Most breast cancers are luminal tumours – luminal tumour cells look the most like cells of the breast cancers that start in the inner (luminal) cells lining the mammary ducts

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Luminal A tumours tend to be ER+ and/or PR+, HER2- and tumour grade 1 or 2 – luminal A tumours 5tend to have the best prognosis, with fairly high survival rates and fairly low recurrence rates Since luminal A tumours tend to be ER+, treatment often includes hormonal therapy

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Luminal B tumours tend to be ER+ and/or PR+ - they are often HER2+ as well Women with luminal B tumours are often diagnosed at a younger age than those with luminal A tumours and have a poor prognosis due to poorer tumour grade, large tumour size and lymph node involvement

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Triple negative breast cancers are ER-, PR-, and HER2There are several subsets of triple negative breast cancer  Basal-like  Tumours have cells with features similar to those of the outer (basal) cells surrounding the mammary ducts  Most have mutations in p53 (tumour suppressor gene) Page 5 of 64

Case 10: Breast Cancer

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 Tend to occur more often in younger and African-American women  Most BRCA1 breast cancers are both triple negative and basal like Triple negative/basal-like tumours are often aggressive and have a poorer prognosis These tumours are usually treated with some combination of surgery, radiation therapy and chemotherapy The molecular subtype of HER2 type is not the same as HER2+ and isn’t used to guide treatment HER2 type tumours have a fairly poor prognosis and are prone to early and frequent recurrence and metastases Women with HER2 type tumours appear to be diagnosed at a younger age than those with luminal A and luminal B tumours HER2+ tumours can be treated with drug trastuzumab (Herceptin) Factors to take into consideration for undertaking chemotherapy  Gender and age  Personal and family history  Pathological stage of tumour  Biological characteristics of tumours Early stage breast cancers can be completely cut-out by surgery Over time however, the disease may come back – the risk of recurrence can be reduced using adjuvant chemotherapy Molecular assays such as Oncotype DX uses real time PCR to assess expression of a panel of 21 genes related to tumour proliferation It gives a validated recurrence score indicating the patients 10-year recurrence risk Gives a validated prediction as to whether the patient will have additional benefit from chemotherapy compared to tamoxifen alone Adjuvant  Adjuvant = after primary surgery  Neo-adjuvant = before surgery – reduces invasive surgery  Locally advanced tumours  Inflammatory tumours  For larger tumours or those with large amounts of nodal involvement or inflammatory component, neoadjuvant chemotherapy may be used to shrink the tumour before surgery to improve the outcome and preserve remnant breast tissue

Cell Cycle Control Page 6 of 64

Case 10: Breast Cancer

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The cell cycle is the process by which a single other cell gives rise to 2 identical daughter cells

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Events of the cell cycle  Mitosis and cytokinesis (known as M-phase) are easily observed under a microscope, but only occupy a small fraction of the cell cycle  The remaining, longer part of the cycle is known as interphase

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cycle control  Cells can be senescent at any time – most cells have a finite capacity to divide  Senescent, an antitumor agent, stops cells proliferating and ending up with tumours  Senescent cells sit within the tissue and change the plasticity of the tissue – the longer they sit around, Page 7 of 64

Cell

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can be promoting a cancer (oxidative damage can build up in the cell, change the phenotype and attract cytokines to the site) IL-6 Cell cycle control allows us to know that we have inappropriate cell division – can think about the ability to target (use nanoparticles or drug target) Can also look at the diagnostic side – we can use our understanding of receptors to tag and target Drugs may be good for blocking the receptors, although it may aggravate the gene There are different places within the progression of the cancer that we may want to facilitate As it progresses it can break through the basal membrane

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Events of the cell cycle are tightly controlled  At the point of entry into the cell cycle – start restriction point  At several critical checkpoints

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Regulation of animal cell cycle entry by growth factors  The availability of growth factors controls the animal cell cycle at a point in late G1 called the restriction point  If growth factors are not available during G1, the cells enter a quiescent stage of the cycle called G0 (inability to proliferate)  If then the cells have all the signal and everything thing it needs including growth factors, it can then move on into G1  However, some cells may have growth factors available but there is damage to the cell – the cell can’t move as it doesn’t meet all the requirements Page 8 of 64

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Cell cycle checkpoints  Several checkpoints function to ensure that complete genomes are transmitted to daughter cells  May replicate the damage  May not have all the signals to go through the checkpoint

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Cell cycle control is exerted by:  Cyclin-dependent kinases (cdks) – reform of kinases  Mechanisms of cdk regulation (kinases mediate phosphorylation, which can activate or deactivate other proteins) Cell cycle progression is controlled by cdks  Protein kinases whose activity rises and falls during cell cycle  Like other protein kinases, cdks phosphorylate protein targets  These targets go on to initiate the various events of the cell cycle  Cdks are activated by cyclins – cyclin dinds to cdk and partially activates it  Cdk 4 and 6 are important to help the cell get through restriction points and then another set of cyclins take over after that Cyclins undergo synthesis and degradation in each cycle  Cdk levels are constant  The cyclins were identified as proteins that accumulate throughout interphase and are rapidly degraded towards the end of mitosis  Cyclin levels are controlled at the level of transcription and by proteolysis

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Degradation as it enters mitosis – it’s this that controls cdks

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The availability of cyclins controls the activity of cdks and promotes cell cycle progression  There are specific cdk/cyclin complexes for different cell cycle phases  When complexed with M-phase cyclin, the cdk triggers the mitosis machinery  When complexed with S-phase cyclin, the cdk triggers DNA replication  Cdks are available all the time but not activated all the time – as they are regulated by cyclins

Tagged to breakdown, to move onto the next stage and then sends a signal for the next cyclin to attach and progress to the next stage of the cycle

Case 10: Breast Cancer

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Summary of complexes  G1 = cdk2,4,6/cyclin D  G1/S = cdk2/cyclin E  S = cdk2/cyclin A  M/G2 = cdk1/cyclin B1  Cdk1 is the only on required to drive through the cell stages  Cyclin B1 and A2 are essential for normal cell cycle

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Mechanisms of cdk regulation  cyclins and

Degradation of CKIs through ubiquitination – ubiquitin to the tag it for

attaching protein to destruction

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The structural activation by cyclin and CAK (cdk-activating kinase) at threonine 160

basis of cdk

CAK helps modulate the phosphorylation and phosphorylates P at 160

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Controls of cdks by proteolysis of cyclins and CKIs  SFC and APC (anaphase promoting complex – part of what we need in order to get separation of the sister chromatids and enter the last stage of mitosis) are responsible ubiquitylating CKIs and cyclins, marking them for proteolysis  Proteolytic tagging – tagged with ubiquitin to get rid of it and then signals for the nest stage to go on

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DNA damage checkpoints – if you don’t have this, can get dysregulation within the cell cycle Cell cycle entry/passage through the restriction point is controlled in the mammalian cells by cdk4/cyclin D and cdk6/cyclin D- want to be able to go past G1 and pass the restriction point in G1 Cyclin D levels themselves to be affected by extracellular signals in the form of mitogens, growth factors and survival factors. These are detected by extracellular receptors and cyclin D transcription is affected through an intracellular cell signalling cascade Cyclin D allows the activation of retinoblastoma (cancer that develops in the immature cells of the retina)

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Cell signalling

Only active when the growth factor is in Lead to activation of cyclin D – allow more machinery processes for cell progression

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Most RTKs (receptor tyrosine kinases) activate Ras, a small GTP-binding protein mutated in many human cancers Dysregulating Ras will start the signalling cascade

Growth factor-induced signalling via Ras and a MAP kinase pathway  Polypeptide growth factors interact with growth factor receptors  Elements of intracellular signalling pathways are activated  Raf is the first kinase activated in a chain of phosphorylation events leading eventually to the transcription of Fos  Fos itself is a transcription factor which induced transcription of cyclin D1 Putting MapKinase signalling and cdks together

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Thr-183 and Tyr185

early gene

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Early regulatory proteins trigger the transcription of cyclin D  These early proteins are regulatory proteins such as transcription factors, Myc, Fos and Jun  They in turn cause the synthesis of D-family cyclins and other regulatory proteins  As these are synthesised later, this is called delayed response gene expression

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The main target of cdk4 and 6/cyclin D is Rb protein 

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Phosphor ylation of Rb

(retinoblastoma protein) frees E2F to act as a transcription factor on many S-phase genes leading to cell cycle entry Many cancers try to overthrow Rb – this can lead to uncontrollable cell proliferation

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Mitogen-stimulated signalling via myc

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Like fos, myc also stimulates cyclin D synthesis It also stimulates E2F synthesis directly, as well as p27 degradation by increasing transcription of SCF

Seen in many cancers dysregulated

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Non-mutagenic replication of every nucleotide once per cycle is essential – only want to make one copy and cause no mutations Requires for a re-replication block – can’t re-replicate the nucleotide before it has finished one cycle Control relies on an interaction between nuclear and cytoplasmic components

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Events of mitosis

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Taxol causes disarray of the spindles and stops the mitosis process

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The events of mitosis are controlled by the M-phase cdk complex: cdc2/cyclin B 

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The cdc2/cyclin B complex catalyses multiple phosphorylation events, initiating several events required during mitosis Also called MPF (maturation promotion factor) M-phase cdk levels are controlled on multiple levels

To make sure it activated for a short period of time 

The control of chromatid separation – proteolysis  A cohesin complex binds sister chromatids together  Cohesins are cleaved by separase  Separase is kept inactive by securing until securing is degraded by proteolysis via APC  APC itself is activated by cdc20 binding facilitated by phosphorylation from the M-cdk

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Unattached chromosomes invoke the spindle attachment checkpoint  Cell doesn’t commit itself to anaphase until it is fully prepared  Sensor mechanism monitors state of kinetochore  Improperly attached kinetochores send negative signal, blocking cdc20-APC activation

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The nature of the signal isn’t clear but several proteins, including Mad2, are recruited to unattached kinetochores

Control of DNA damage checkpoints  The G1 checkpoint is mediated by a transcription factor called p53  In response to DNA damage, p53 is activated  Similarly, damaged DNA in G2 sends a signal to a series of protein kinases that phosphorylate and inactivate cdc25  This blocks the dephosphorylation and activation of m-cdk, thereby blocking entry into mitosis  When the damaged DNA is repaired, the inhibitory signal is turned off and cell cycle progression resumes DNA damage and p53  p53 causes the transcription of p21, a cdk inhibitor  Cdk inhibition prevents cell cycle progression  P21 also binds PCNA, a component of the DNA replication machinery, preventing its activity  p53 is also a tumour suppressor – loss of function of it can lead to loss of control of proliferation  The core of the cell cycle control system consists of a series of cyclin-cdk complexes  The activity of each complex is influenced by various inhibitory checkpoint mechanisms which provides information about the extracellular environment, cell damage and incomplete cell cycle events

Radiochemistry: Non-Invasive Radiochemical Imaging 

Causes of nuclear instability  Size  Imbalance of ideal proton: neutron ratio

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Modes of decay  Alpha

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Beta

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Fusion

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Radiochemical dating relies on the fact that the amount of 14C in living tissue is constant (0.227 Bq gC-1), which starts decaying without being replenished upon death

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Ultrasound: non-ionising and non-invasive  UHF sound waves  Ultrasonic waves are emitted by the transducer and travel through human tissues at a velocity of 1540m/s  When the waves reach an object or surface with a different texture, a wave is reflected back  Advantages  Lack of ionising radiation  Portability of equipment  Low cost  Real time images – 3D also available  Limitations  Cannot penetrate gas (lesions lying behind can’t be visualised)  Therefore, it can’t be used for lung and bowel – gas may obscure e.g. the pancreas and renal arteries  Can’t penetrate bone so it can’t be used to look at bone lesions or the inside of the skull  Difficult to use in patients with a high body fat percentage  Applications  Useful for all solid organs of the body e.g. liver, kidneys, spleen, pancreas, pelvic organs  Smaller, high frequency probes are used for breast, thyroid, testes as well as certain parts of the musculoskeletal system such as the shoulder joint  Most muscles and tendons can be examines for rupture, inflammation, tumours etc.  Used as a guide for biopsy and drainage Non-invasive imaging – X-rays  X-rays blacken photographic film, so more dense structures appear white, while less dense structure appear grey or black  Plain film radiography (normal X-rays) rely on the natural contrast between the four basic radiographic densities:  Air/gas – black e.g. lung, bowel, stomach  Fat – dark grey e.g. SC tissue layer  Soft tissues/water – light grey e.g. solid organs, heart, blood vessels, muscle, bladder  Bone – off white  Soft X-rays have lower frequency and wavelength – less radioactive damage  Can differentiate between hard and soft tissue

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X-ray Computer Tomography  X-rays are produced by a high potential difference between a cathode and an anode in a vacuum  Electrons are boiled off the cathode, accelerated through a high potential and strike and tungsten anode to produce x-rays Page 18 of 64

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X-rays images result from attenuation of the X-rays by the material through which they pass Attenuation is the removal of X-rays from a beam by absorption and scatter Generally, the grea...