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What to understand?

- the process of diagnosis of cancer including the interplay of clinical information, morphology, immunohistochemical techniques, and genetic and molecular studies in diagnosis
- the increasing value of immunohistochemical and molecular genetic information in determining treatment
- the role of molecular techniques in changing our understanding of the mechanisms of malignant transformation
- the directions of current resarch into cancer diagnosis and management


What is the story?

family in outer eastern melbourne:
→ angela (16)
→ bradley (10)
→ carl (5)
→ denise (38) and edward (40)

- in late 2006 angela arrived at her doctor's, describing 1 month of feeling unwell, with intermittent nausea, comiting, poor appetite, and abdominal pain after eating, to the point that eventaully she couldn't tolerate any food
- on questioning she described increasing constipation, leg and hip pain, and low back ache
- had also lost about 8kg over 1 month

- presentations vary dramatically depending on what it is and where it is
- sometimes people will see tumours (e.g. skin cancer), feel them (soft tissue tumours → marble palpable in muscle)

- subcutaneous nodule over her left scapula (shoulder blade), that had grown from pea to golf vall size over a few weeks, could still be other things but more likely unrestrained proliferation, this rate of growth suggests absolutely uncontrolled proliferation
- also hard, slightly irregular lumps in left groin
- radiology: widespread tumour in pelvis and abdomen
- bone scan: several metastases
- echo: pericardial effusion


What is clinical information?

- the history is an essential part of making a diagnosis
- it is sometimes overlooked, perhaps because the technology of diagnosis is seductive
- it involves:
→ the history of the problem
→ the past history of the patient (including social, hobbies, travel, and many other components)
→ the medical history of the family

- examination of the patient is also necessary
→ masses anywheere
→ other signs
→ → anaemia
→ → hints (in this girl) of a congenital condition that may be associated with malignancy such as neurofibromatosis


What is the specimen? How do we get it?

- at present, after the history, examination, and other investigations, we need tissue in some form for diagnosis of cancer
- options will depend on various things (size, location of mass, suspected diagnoses etc) and include:
→ excisional biopsy (cutting something out, attempting to remove all of it, so both therapeutic and diagnositc)
→ incisional biopsy (cut out only a small portion of a mass for diagnosis – bot therapeutic)
→ needle biopsy (a core that preserves the architecture, just not much of it!)
→ fine needle aspiration (sich out cells, without preserving the architecture)


How do we investigate the specimen?

- morphology (the appearance of the tumour)
-- light microscopy (most common) (H&E stain)
→ histology
→ cytology
-- electron microscopy (very rarely used now for tumours)

- immunohistochemistry
-- stain tissue sections with labelled antibodies raised against antigens (membrane/cytoplasmic/nuclear) that give information about cell lineage/type


What was seen when investigating Angela?

- Angela underwent excisional biopsy of two hard lumps in her groin (likely representing lymph nodes)
- definitely a tumour: proliferation of cells, resembles no normal tissue, clonality, don't look like a differentiated section of lung/lymph node etc, irregular nuclei, lots of mitoses, lots of pyknotic/apoptotic cells, undifferentiated rapidly turning over proliferation


How do we interpret findings? What was the interpretation of the specimen?

- with many tumours, the clinical setting already shortens the list of likely/possible tumours: this is an important part of selecting what other investigations to do
→ as an extreme example, in this girl's tumour you would not bother staining for melanoma marks, breast cancer markers until other things had been exhausted, and you wouldn't bother staining for prostate cancer at all

- in this tumour, the morphology is characteristic of the commonest malignant soft tissue tumour of childhood and adolescence, but it is well recognised that the diagnosis can be difficult on morphology alone


How was diagnosis made before the late 20th century?

- before the late 20th century, diagnosis was made on morphology, with the aid of some histochemical stains
→ wide variety of chemical techniques to demonstrate specific cell types, cell products
→ often difficult skill to acquire
→ some are still very commonly performed, two examples are:
- Perls Prussian Blue in which iron is in its ferric state is demonstrated by releasing it from hemosiderin with hydrochloric acid, forming ferric chloride. the iron reacts with potassium ferrocyanide to form blue ferric ferrocyanide
- PAS (periodic acid Schiffs) in which periodic acid oxidises carbohydrates to produce aldehyde groups, which then condense with Schiff's reagent forming a bright red product


What has happened since the 1980s?

- immunohistochemistry has become very widely used to identify antigens that allow assessment of:
→ cell lineage in otherwise undifferentiated tumours, or confirm it in cases where you think you see morphologic evidence but aren't sure
→ whether a tumour may respond to particular treatments
- C-erbB-2/Herceptin
- immunotherapy
- BRAF V600E mutation product
- etc etc


What does the diagnosis of the girl's tumour require?

- more than morphology
→ typically a panel is ordered
→ in this example we want to cover various tumours of children and adolescents that show sheets of undifferentiated smallish cells by light microscopy
→ we would want to cover:
- lymphoid neoplasms
- soft tissue tumours
-- rhabdomyosarcoma
-- ewing family tumours
-- primitive neural tumours
- rare tumours such as Rhabdoid tumour

- antibody tagged against desmin: hallmark filament of muscle, myogenenin


What did Angela have?

- Alveolar rhabdomyosarcoma


What is Rhabdomyoscarcoma?

- a lot of information is included in the word:
→ sarcoma indicatios
-- malignant
-- non-epithelial tumour including bone, cartilage and what are called 'soft tissues' (tumours of fat, fibrous tissue, smooth muscle, skeletal muscle)
→ myo
-- showing muscle differentiation
→ rhabdo
-- showing specifically skeletal muscle differentiation

- in addition, 'alveolar' describes a particular and more aggressive subtype of rhabdomyosarcoma with specific microscopic appearances


What is rhabdomyoma?

- benign tumour showing skeletal muscle differentiation


What are leiomyoma?

- benign tumour showing smooth muscle differentiation


What is leiomyosarcoma?

- malignant tumour showing smooth muscle differentiation


So we stop there?

- no! (once, yes, but not any more)
- in many cancers, molecular and genetic studies provide information that is useful for prognosis (predicting what is likely to happen) and management
- this is particularly true of soft tissue tumours in children and adolescents (but also in many others like carcinoma of breast)


What sort of genetic 'event' is seen in ARMS?

75% have a characteristic translocation (t(2;13)(q35;q14))
creates a fusion transcript

10% have t(1;13)(p36;q14)

lots of tumours have genetic finger print


What is PAX3-FKHR?

- fuses paired homeodomain region of PAX3 gene (Chr 2) with forkhead domain gene FKHR (chr 13)
- pax3 normally involved in skeletal muscle differentiation
- translation of the novel gene results in synthesis of chimaeric proteins, which in turn contribute to the initiation or progression of the neoplastic state
- similar process occurs with t(1:13) resulting in the PAX7:FKHR fusion product


What is the pathologic diagnosis of many cancers?

- combined morphologic and molecular/genetic exercise
- e.g. medulloblastoma
- previously two types according to what was seen under a light microscope
- now four pathways: WNT, SHH, group 3, group 4
- this is happening very quickly
- now six groups
- cancer diagnosis splits into different groups increasingly diagnosed by molecular events


How will cancers be defined in the future?

- perhaps by their molecular events
- less and less by classical histology


What is grade?

- an assessment of how aggressive the tumour appears to be on microscopy
- in general the more closely a tumour resembles a tissue, the lower grade it is
- conversely, an almost undifferentiated sheet of malignant cells with very atypical nuclei, highly mitotic activity and maybe some necrosis is likely to be a high grade tumour


What is stage?

- an assessment of how far the cancer has spread at the time of diagnosis


How was Angela's cancer managed?

- chemotherapy
→ still largely unspecific
→ devastates bone marrow
→ kills most rapidly proliferating cells e.g. bone marrow, hair follices
- 2 weeks later re-admitted with evidence of toxicity due to the chemotherapy
→ nausea, vomiting, diarrhoea and abdominal pain
→ radiology – suggested gut toxicty (ileus, dilated bowel loops)
→ improved



- this teenage girl developed an aggressive malignant neoplasm
- no cause for it is identified (in the sense of anything she inherited, or an environmental carcinogen)
- by the time of diagnosis was not possible (nor very useful) to sort out exactly where it came from – the diagnosis of rhabdomyosarcoma relates to the differentiation the tumour is showing (with atypical skeletal muscle cells and desmin expression)
- in a previous era (or today in very many parts of the world) she would have died within weeks
- following diagnosis and staging, she was treated with chemotherrapy and responded, but it is likely she will die of the disease, partly to the aggressive nature of the particular cancer she has, and partly to the late stage at diagnosis
- remember that many cancers in our society can be cured, including many paediatric cancers
- had she been diagnosed with other variants of rhabdomyosarcoma, with early stage neuroblastoma, with retinoblastoma or with different types of lymphoma, complete cure would be likely


What kind of disease is cancer?

- cancer is a genetic disease: the cancer cell has abnormalities of gene sequence or expression that may be due to exposure to environmental carcinogens, inherited abnormalities (germline mutations), random events or interactions between the above


What can translocations indicate?

- characteristic translocations when identified can provide information about the possible mechanisms of malignant transformation


Is cancer inherited?

- not necessarily
- only a small % of cancer patients have a germline mutation or other inherited abnormality
- but the basic abnormality of cancer does reside in its genes, and specifically in genes encoding critical components that regulate cell growth, differentiation and death


What is the effect on cells of cancer?

- result is abnormal regulation of cell growth, death and differentiation, so that a population has a growth advantage and proliferates without responding to normal controls


What can now be identified in tumours ?

- in many tumours we can now identify these specific genetic and molecular abnormalities, and in some cases they are diagnostic of a particular tumour
- they also increase our understanding of the molecular mechanisms of carcinogenesis


On what does management depend?

- will depend on the tumour type
- aggressiveness (grade) and how far it has progressed (stage)
- girl's tumour was high grade and in an advanced stage


Is this story usual? What is a usual story?

probably not
- this is an unusual cancer story in some ways
- almost all cancers in our society occur in adults and most of these are epithelial
- so a much more common story would involve:
→ an adult with a malignant neoplasm of epithelium - a carcinoma such as Ca. of lung, breast, or colon, often due to some carcinogen such as cigarette smoke or other less clearly defined environmental agents
→ abnormalities of multiple genes encoding various growth factors, their receptors, second messengers, apoptosis regulators, etc
→ no association with a clearly defined abnormality such as the 2;13 translocation


What is the multi-step theory of carcinogenesis?

- in many of the common carcinomas of adulthood, there is strong evidence for the multistep theory of carcinogenesis
- abnormal cells progressively acquiring mutations that alter their appearance and their capabilities
- bowel cancer is a good example of this
- soft tissue tumours (such as this one) have not yet shown such convincing evidence of a progression with sequential acquisition of mutations and many are characterised by one or two well defined mutations (or other genetic abnormalities such as the t (2:13) translocation in ARMS)


What are clearly defined avoidable or modifiable risk factors for common malignancies of adulthood?

- cigarette smoking
- UV light
- dietary factors
- others (many...)

many of the rarer tumours, including paediatric cancers, are not associated with such environmental risks as far as we know at present


What is the future of biology?

- many many questions
- why are sarcomas arising in adolescents and young adults often more aggressive than those arising in very young children?
- why do some late stage apparently aggressive neuroblastomas suddenly regress?
- what event or events cause the genetic abnormality in a tumour such as rhabdomyosarcoma?


What is the future of diagnosis and treatment?

- will we still need tissue to make the final diagnosis?
→ proteomics
→ gene expression arrays

- how can we treat malignant neoplasms more effectively, with less toxicity?

etc etc