Seminar 1Diagnostics of haematological malignancies

”Benign ” blood disorders

Blood disorders caused by vitamin /trace element deficiencies

Malignant blood disorders

Acute leukaemiasALL, AML

Myelodysplastic syndromes (MDS)

Chronic Myeloproliferative disordersLymphoproliferative disorders,

Plasma cell disorders and amyloidosis

Other clonal and rare blood disorders

Blood disorders

REQUIRES SPECIALISED HEALTHCARE SERVICESREQUIRES BASIC HEALTHCARE SERVICES

Coagulation disorders

Hereditarytendencies for thrombosis andhaemorrhage

Acquiredtendencies for thrombosis andhaemorrhage

Hereditary red blood celldisorders and cytopenia

(Immunological) cytopenia-causingdisorders that affect bloodcomposition (e.g. AIHA, ITP,aplastic anaemia)

HEALTH CENTRE DOCTORS = GATEKEEPERS• new diagnosis

• activation of a chronic blood disorder

Problems in blood counts/ blood picture

A haematologist’s thinking process

1) Is it a blood disorder or something else?

A haematologist’s thinking process

1) Is it a blood disorder or something else?

2) Is the change malignant or benign?

A haematologist’s thinking process

1) Is it a blood disorder or something else?

2) Is the change malignant or benign?

3) Is it urgent?

A haematologist’s thinking process

1) Is it a blood disorder or something else?

2) Is the change malignant or benign?

3) Is it urgent?

Specific testsDG

DG

MEDICAL HISTORY• B symptoms (weight loss, sweating, susceptibility to infections, fever)

• Chemical/radiation/toxic (cytotoxic?) exposure

• The most common causes of reactive changes

• Family history

• Medication and lifestyle

• Long-term problems in haemostasis

STATUS• Severity of symptoms (in relation to the changes in blood count)?

NB! Acute leukaemia is almost always a very acute disease

• Skeletal pains / fractures

• Skin

• Pharynx and other mucous membranes

• Lymph node areas

• Spleen

• Bleeds or blood clots

• (Heart, breathing, BP, signs of infection, swelling)

LAB TESTS AND IMAGING• Is there one or several germline mutations?

• Is the cytopenia/cytosis mild or deep ?

• Previous blood test results? Control test sample?

• Other changes found in lab tests?

• Is the change primary or secondary?

-> bone marrow biopsy, abdominal and lymph node CT/US scans

SUPECTING/RULING OUT A MALIGNANT BLOOD DISORDER

CASE 1

• The patient is a 85 year-old male who lives at home with his wife

• Diagnosed with bladder cancer 20 years ago (treated, in remission)

• Benign prostatic hyperplasia, hypercholesterolaemia andhypertension

CASE 1

• Fell in January 2016 -> suffered from thoracic back pains allwinter and early spring

• Visited a doctor in April 2016 -> chest and thoracic spinal Xrays

Statement:

Mild rightward curvature and anteriorcalcification of the ligaments in the thoracicspine like in the old scans.

The superior endplate of the T4 is mildlycompressed. This issue has developedafter 2009 and cannot be dated moreaccurately.

Normal pedicle gap width, no bonedestruction detected. No abnormalities inother vertebrae.

-> pain management

CASE 1, continuation

•1 June: Decline in general condition, low BP ->antihypertensive medication dose was reduced

•6 June: Annual health check of persons aged 65 and aboveat a health centre (at 1:30 pm)

• The patient complains of increasing fatigue, weight loss andcough

CASE 1

COULD AN INFECTION BE THE CAUSE?

What do we know so far?

• Older male with a cancer in remission• Back pains since January 2016• Vertebral compression fracture• Respiratory infection• Anaemia• Kidney failure (and low Na levels)

COMMONDENOMINATOR?

Q2: DENOMINATOR

Cancer (relapse, a new solid tumour, a blood cancer?)

Hormonal problem?

Secondary anaemia (cause?) and (chronic) kidney failure caused byhypertension

All the mentioned are possible

DIFFERENTIALDIAGN

OSTICS

• Anaemia• vitamins• iron• haemolysis• bone marrow infiltrate

blood cancer?

could it be leukaemia?

other blood cancer?

• Hormones• Thyroid gland

• Low cortisol levels (Addison'sdisease)?

• PTH (Osteoporosis tests, Ca / vit. D)

• General condition• Sedimentation rate• Alcalic phosphatase

• Imaging tests:• Chest Xray• Abdominal US

CBC is to be takenALWAYS IF THERE IS A CHANGE IN BLOOD COUNT!

CBCcomplete blood count

Differential counting OK-> not a primary suspicionof leukaemia

THE PATIENT’S TVK

BASIC ANAEMIA TESTS AND HORMONES inthe patient

• CA-ion elevated

• PTH decreased ->malignant hypercalcaemia?

• TSH, Korsol were normal

DENOMINATOR / DIFFERENTIAL DIAGNOSIS

• Cancer (relapse, a new solid tumour, a blood cancer?)

• Hormonal problem?

• Secondary anaemia (cause?) and (chronic) kidney failure caused byhypertension

Bladder cancer: Urine samples and imaging tests Myeloma

Myeloma• A malignant blood disease, plasma cell cancer

• Cancer cells: Malignant plasma cell clones of very mature bonemarrow B cells.

• Myeloma cells only produce one type of immunoglobulin, or antibody(= is clonal) = M component / M-protein/ paraprotein

Myeloma• M component:

• usually BOTH a heavy AND light chain;• ONLY a light chain in 15–20%;• light chains: kappa or lambda

HEAVY CHAINS:• IgG myelomas account for 50% of cases• IgA myelomas account for 20% of cases• IgD or non-secretory myelomas account for 10%; plasma cell

leukaemias.

Myeloma; special notes• (Light chain) myeloma may lead to kidney failure• High M component levels in blood may cause symptoms of

hyperviscosity.• Lytic bone metastases, bone pain and pathological fractures.• Also local plasma cell tumours, namely, plasmacytomas, may occur

even outside of bone marrow• Sometimes a myeloma is accompanied by light chain amyloidosis

Do the tests reveal M components or light chains?Are there too many plasma cells in the bone marrow?

HOW DO YOU EXAMINE THIS?

Specific myeloma tests

• C a-ion• R enum• A nemia• B one

Protein fractions

Light chains

Bone marrowaspiration sample(plasma cell count andclonality)

DG START OF TREATMENT

La

Afos

The symptoms of myeloma determinewhen to start treatment – AT LEAST NOW…FUTURE??

Myeloma tests

• C a-ion• R enum• A nemia• B one

Protein fractions

Light chains

Bone marrowaspiration sample(plasma cell count andclonality)

La

Afos

These tests can be performed inhealth centres!

DIAGNOSTIC CRITERIA OF MYELOMA1. Monoclonal plasma cells in bone marrow (flow cytometry

and/or immunohistochemistry) and/or biopsy-proven monoclonalplasmacytoma

2. Monoclonal paraprotein in serum and/or urine

3. Localised organ lesion (CRAB) caused by myeloma, at leastone of the following:

Elevated calcium levels in serum (over 2.65 mmol/l or above thereference range upper limit)Kidney failure, P-Krea above 177 mol/lB-Hb below 100 g/l or more than 20 g/l below the normal valueLytic bone lesion, pathological fracture or osteoporosis.

OTHER PLASMA CELL DISORDERS

Smouldering multiple myeloma (asymptomaticmultiple myeloma)1. M component in serum >= 30 g/l and/or2. Monoclonal plasma cells in bone marrow >= 10%3. No end organ damages caused by myeloma

Monoclonal gammopathy of undetermined significance, MGUS(monitoring in health centres)M component in serum < 30 g/lProportion of clonal plasma cells in bone marrow < 10%.No indication of other B cell proliferationNo end organ damages caused by the malady

OTHER PLASMA CELL DISORDERS

Smouldering multiple myeloma (asymptomatic multiplemyeloma)M component in serum >= 30 g/l and/orMonoclonal plasma cells in bone marrow >= 10%No end organ damages caused by myeloma

Monoclonal gammopathy of undetermined significance, MGUS(monitoring in health centres)1. M component in serum < 30 g/l2. Proportion of clonal plasma cells in bone marrow < 10%.3. No indication of other B cell proliferation4. No end organ damages caused by the malady5.

SIC! In case of suspicion of MGUS, it is always indicated to examine also proBNP/Tnt and light chains in order to exclude AL-AMYLOIDOSIS.

Remember to consult a hematologist!

OTHER PLASMA CELL DISORDERSSmouldering multiple myelomaMGUSSolitary plasmacytoma1. Individual biopsy-proven bone or soft tissue tumour with clonal plasma cells

2. Normal bone marrow finding, no clonal plasma cells

3. Normal results of skeletal x rays without contrast medium and hip MRI (exceptfor solitary plasmacytoma)

4. No end organ damages

SUMMARY OF THE PATIENT’S DIAGNOSIS

Protein fractions -> M component: IgG kappa 29.2 g/L

Light chains -> IgLcK-V 326 mg/l (6.9–25.6)

Bone marrow aspiration sample-> MGGFE: Plasma cells 25–50% of bone marrow cellularity(normally 2–3% of the cellularity)

IFT: 99% of plasma cells were clonal

• C a-ion -> 1.51 mmol/l/pH7.4 (1.16–1.3)

• R enum -> krea 123 umol/l (60–100)

• A nemia -> Hb 109g/l (134–167)

• B one -> fractures in spine

• La -> 125 (<35)

• Afos -> 112 U7l (35–105)

SUMMARY OF THE PATIENT’S DIAGNOSIS

PATIENT HAS SYMPTOMATICMYELOMA

CASE 2: LEUKOCYTOSIS OF UNKNOWN CAUSE

• The patient is a 68-year-old recently retired male with no healthissues up until now.

• No history of smoking, good BP, exercises actively, has stayed slim andin good general condition.

• Had a lentigo maligna, or melanoma, removed a year ago at the Skinand allergy hospital. No further actions planned for this.

CASE 2: LEUKOCYTOSIS OF UNKNOWN CAUSE

• During the last year the patient’s weight has dropped from 70 to 65 kg.The patient contributes this to lifestyle changes following retirement.

• The patient has mild night sweats occasionally. Hasn't really paid attentionto it.

• Good bowel movements, appetite has been normal. No additionalsusceptibility to infections.

• Visits a dentist regularly; next appointment is scheduled for next week.

• Results of laboratory tests ordered by a health centre doctor at thecontrol visit following the removal of lentigo maligna (melanoma):

CASE 2: LEUKOCYTOSIS OF UNKNOWN CAUSE

Order control tests!

PRESEMO: Q5 When?

CASE 2…The patient was asked to come to a new assessment to havesamples collected to an urgent care clinic of a hospital.-> General condition is still very good-> Clinical status as recorded by the urgent care doctor:“In good general condition. Appropriate behaviour. Breathing is calm. Even rhythmin heart auscultation, no murmurs. Normal breathing sounds in lungs. Abdomen issoft to palpation. Spleen appears potentially enlarged. Extremities are warm, noswelling. I cannot feel abnormal lymph nodes in the neck or supraclavicularfossae. There are few enlarged lymph nodes in the left side of the groin.”

-> Similar blood count (problems included). CRP <3.

What disease do you suspect?

1) Acute leukaemia

2) Chronic leukaemia

3) EBV infection

NB! Acute leukaemia cannot, however, be ruled out from the options in thedifferential diagnosis, even though a leukocyte level of over 200 and thepatient’s remarkably good condition would suggest a less aggressive disease.

Bone marrow

BloodTissues

Uncontrolled proliferation of an immature blood cell (blast)

Acute leukemias• Myeloid• Lymphoid

Myelodysplasticdiseases• Myelodysplastic syndrome (MDS)• CMML

Myeloproliferativediseases• CML

• Polysytemia vera (PV)• Essential thrombocytemia (ET)• Primary myelofibrosis (PMF)

Uncontrolled proliferation of mature or almost mature cells(stem cell originating problem, however)

LUUYDIN

VERI

Lymphoproliferations andplasma cell dis..• CLL• Myeloma• Mb Waldenströmin

P

What do we do now?

1) Order complete blood count

2) Have a bone marrow biopsy taken at the urgent care centrewithin a day

3) Consult a haematologist

4) Ask the patient to visit a health centre doctor in a week forcontrol tests

CASE 2.

• The urgent care doctor consulted a heamatologist due tosuspected blood disorder

• The haematologist suspected the issue to be CHRONICleukaemia, but

• A COMPLETE BLOOD COUNT is needed before furtherdecisions are made

CASE 2.• The urgent care doctor

consulted a heamatologistdue to suspected blooddisorder

• The haematologistsuspected the issue to beCHRONIC LEUKAEMIA, but

• A COMPLETE BLOOD COUNTis needed before furtherdecisions are made

WHAT IS THE DIAGNOSIS?

1) Chronic myelogenous leukaemia

2) Chronic lymphocytic leukaemia

3) Lymphoma spillover in the blood

CASE 2.

• An urgent care doctor referred the patient to a haematologist afterfirst consulting them

-> further tests were done within a week-> the patient was instructed to go to urgent care immediately if hestarts feeling worse-> adequate hydration (creatine levels have been checked to benormal)

WHAT FURTHER TEST DOES THE HAEMATOLOGIST ORDER TOCONFIRM DIAGNOSIS AND DETERMINE THE PROGNOSIS?

Test Signs to look for

BONE MARROW ASPIRATION:Morphological test (MGGFe)

Cell shape and size (Small? No indication of developing into amore aggressive macrocytic disease?)

BONE MARROW ASPIRATION:Flow cytometry test (”IFT” or ”Flow”)

Cell surface markers: Do they refer to CLL? (differentialdiagnosis for e.g. mantle cell lymphoma in leukaemic phase orhairy cell leukaemia)

BONE MARROW BIOPSY Bone marrow filtration rate and cell size

Chromosome test of blood or bone marrowlymphocytes (interfaasi-FISH)

Most common chromosomal changes; significant forprognosis(13q-, +12, 11q-, 17p-)

Abdominal US/ body CT scans Lymph node metastasis and spleen size

CASE 2. THE PATIENT WAS DIAGNOSED WITHCHRONIC LYMPHOCYTIC leukaemia (CLL)• Malignant blood disease; the most common leukaemia type in the

developed world• Disease-causing cell: Mature clonal B cells with impaired function• The disease infiltrates into bone marrow, peripheral blood and often

also lymph nodes and spleen.

• According to the WHO classification, the term small lymphocyticlymphoma (SLL) refers to those CLL cases where the tumour’smorphology and immunophenotype correspond with CLL, but nolymphocytosis are detected in peripheral blood and the patient has nocytopenias.

CLL• Median age of those diagnosed with CLL is 72, but it may also develop in

young people• Progresses calmly in most cases but can also be aggressive• Often found as an incidental finding• B symptoms often refer to an advanced disease.• The patients have often unusually frequent infections

(hypogammaglobulinemia, poor lymphocyte “quality”)• Differential diagnosis: Prolymphocytic leukaemia, lymphoplasmacytic

lymphoma, hairy cell leukaemia, mantle cell lymphoma, splenic marginal zonelymphoma, follicular lymphoma and large granular lymphocyte (LGL)leukaemia

• Treated by haematologists (like prolymphocytic leukaemia, LGL leukaemia,hairy cell leukaemia and lymphoplasmacytic lymphoma; other lymphomas aretypically treated by oncologists)

CLL DIAGNOSTIC CRITERIA

Blood lymphC

Blood morphology: lots of small and mature lymphocytes

Imunophenotype (by histology or flow cytometry):

Bone marrow ekxamination is not compulsory

PROGNOSIS OF CLL (Binet classification)Class A

Hb ≥ 100 g/l, thrombocytes ≥ 100 x 109/l and disease only in amaximum of two lymph node areas

Median life expectancy 15.5 y

Class B

Hb ≥ 100 g/l, thrombocytes ≥ 100 x 109/l and disease only inthree of more lymph node areas

Median life expectancy 5.5 y

Class C

All patients regardless of lymph node metastasis areas, with Hb< 100 g/l and/or thrombocytes < 100 x 109/l and disease onlyin three of more lymph node areas

Median life expectancy 3 y

Use palpation to determine iflymphadenopathy (a lymph node with adiameter of > 1 cm) is uni- or bilateral.

Metastasis areas: Head and neck (incl.Waldeyer's ring) covers one area, even ifseveral lymph node groups are affected.Both armpits and sides of groin alwaysform one area. Spleen and liver felt uponpalpation are significant findings.

CASE 2. SUMMARY OF DIAGNOSIS• Patient is in good general condition, receives treatment regularly• No B symptoms• Leuk > 200, but the cells were small• Hb and tromb over 100• BLOOD IFT: Lymphocytes CD19&5&20+ lambda clonal• BONE MARROW:

PAD: Infiltration rate 75%Aspiration and PAD: no indication of Richter’s transformationKr 13q-

• Spleen 17 cm (normally approx. 10 cm)• No enlarged lymph nodes detected

WHAT IF THE BLOOD COUNT HAD BEEN LIKETHIS?

WHAT IF THE BLOOD COUNT HAD BEEN LIKETHIS?

(40–75%)(20–45%)(1–11%)(0–7%)(0–1%)

Would the disease in question then havebeen

1) Acute myelogenous leukaemia

2) Chronic myelogenous leukaemia

3) Polycythemia vera?

WHAT IS CML?• Chronic and malignant disease of haematopoietic stem cells.

• Chronic stage -> acceleration -> blast crisis• Poor prognosis in the past; with modern treatments, most patients have a

good prognosis if the condition is detected at its chronic stage• Usually diagnosed at the chronic stage as an incidental finding in further

tests following an abnormal blood count• Treatment of CML is based on targeted treatment: inhibitor therapy that

targets tyrosine kinase, the cause of the disease

KMLVeritaudit, Kustannus Oy Duodecim, 2016)

CML diagnosis

BCR-ABL1 fusion genedemonstrationFISH

BONE MARROW SIGNS TO LOOK FORAspiration samplemorphology

Morphology: chronic,acceleration of blast crisisphase

KromHem 9;22; other changes?Fusion gene test (PCR-based)

BCR-ABL1 fusion geneproportion

Chromosome test: 9;22

RADIOLOGY SIGNS TO LOOKFOR

Abdominal US scan: Spleen size

Risk stratification of CML

CASE 4. What if the blood count had been likethis?

CASE 4.

• The patient is a young female (16yo)

• Dm1, no complications

• Tonsillitis and a fever for one week

• Visited an ENT doctor who ordered a blood count test

PRESEMO 11WHAT DO YOU SUSPECT? (Leukocytosis, deepanaemia and thrombocytopenia)

WHAT DO YOU SUSPECT? (Leukocytosis, deepanaemia and thrombocytopenia)

1) Aplastic anaemia

2) Severe EBV infection

3) Acute leukaemiaWHAT LAB TESTS TO ORDER NEXT?

CBCMonoAb rapid test negALAT, Krea normal

CRP 23AFOS 126 (35–125, 16-18yo females)

LD 1142 (115–230)

Diff:Neut 2%Lymph 4%Blasts 94%

Different acute leukaemias

• TWO MAJOR SUBTYPES:• ALL (lymphoblastic)• AML (myelogenous)

• Divided into smaller subgroupsaccording to cytomoleculargenetic etiology

AML• AML• APL• Ak. monosytäärinen leukemia

B-ALL T-ALL

LYMPHOID OR MYELOID?

FURTHER TESTS NEEDED URGENTLY!

If left untreated, ALL and AML may lead to rapid death of thepatient

Diff:Neut 2%Lymph 4%Blasts 94%

Average Number of New Cases Per Year and Age-Specific Incidence Rates per 100,000 Population, UK

Source:cruk.org/cancerstats

58.8% <20v75% <45v25% > 45v

n. 60% <20v

Wang and Bailey: Arch Pathol Lab Med 2015

ALL AML

Inaba H, Greaves M, Mullighan CG. Acute lymphoblastic leukaemia. Lancet. 2013;381(9881):1943–1955.Diagnosis and management of acute myeloid leukemia in adults:recommendations from an international expert panel, on behalf ofthe European LeukemiaNet.Blood 2010, Vol. 115, No. 3, pp. 453-474.

Different (?) acute leukaemias

In all age groups:ALL more common in childrenAML more common in adults

Molecular “background profile”focuses on different changes indifferent age groups

B-ALL T-ALL

AML• AML• APL• Ak. monosytäärinen leukemia

Hunger et al NEJM, Oct 2015

1. Predisposing factor in stem cells• either the type polymorphism (more common but ”lighter”)• or germline mutation (rare but strong)

2. Initiating mutationchromosomal aberration, usually translocation, which leads to

• merging of oncogene and regulatory region, whichincreases oncogenic transcription• the creation of fusion gene (e.g. BCR-ABL1 or TCF-PBX1)

3. Secondary leukaemia-causing mutationsequence mutation or a change affecting the structure of the gene

• Often prevents the maturation of lymphoid cells• cell cycle control/ signal transduction genes / transcription factors

etc.

Toxic/chemical/radiation exposure

Secondary leukaemias: The patient has had a cancer in the past (solidtumours or a treated haematological malignancy); the administeredcytostatic and/or radiation therapy

Congenital or hereditary predisposition to haematologicalmalignancies (less than 10% of acute leukaemia cases?)

E.g.Down's syndromeFanconi anaemiaDyskeratosis congenita…

(Viruses?)

Risk factors of acute leukaemias:

The patient has a different chronic blood disease thatmay develop into acute leukaemia

• Ph-negative myeloproliferative neoplasms: PV, ET, MF• MDS• NOTE! CML may develop into a blast crisis that is similar

to acute leukaemia, but CLL (Chronic lymphocyticleukaemia) does not develop into acute leukaemia! (Itcan, however, develop into a macrocystic disease)

Acute leukaemia is often suspected afterABNORMALITIES ARE FOUND IN THE BLOODCOUNT OF A (SYMPTOMATIC) PATIENT

• Mutations in several germlines (but not always)

• Can be leukocytosis or leukopenia

• Very often neutropenia (NB! Risk of infection!)

• WBC differential reveals blasts (or several promyelo-Cytes; cells may contain Auer rods)

NB! A COMPLETE BLOOD COUNT IS ALWAYS NEEDED TO CONFIRM THE DIAGNOSIS!

Symptoms of acute leukemia- may be present, but not all and not always

• Hematomas, general bleeding tenedency• Thrombocytopenia, anemia, consumption

coagulopathy• Fever, infection

• neutropenia, disturebed immunology• Weak general condition

• anemia,hyperviscosity• Pain (bones, joints)

• Lymphadenopathy,• Tissue infiltrates (spleen, liver, lungs, skin, gingiva…)• CNS affision (likvor)- > neurological problems, numbness

usually

more rarely

Diagnostics of Acute Leukemia

• Blood• CBC, basic chemistry (liver, kidney, couagulation,

CRP, LDH, uric acid….).

• Bone marrow aspiration• Morphology, hematol. chromosome analysis,typical fusion genes, typical genetic mutations

• Spinal tap in ALL and in patients with CNS symptoms• Radiology:

• Abdomibnal ultrasound• Thorax• (CT) Final DX usually made in hospital

Differential diagnostics of acute leukemia

• Myelodysplastic syndrome

• Myelofibrosis

• Chronic myeloid leukemia in blast crisis

• Spread lymphoma

• Plasma cell leukemia

• Bone marrow infiltrate of a solid tumor disease (breast cancer,prostate cancer…)

• Leukemoid reaction in conjunction with a severe infection

Diagnostic tools of acute leukaemia are the same used for othermalignant blood diseases:

BONE MARROW ASPIRATION SAMPLE

1) Microscopic analysis (morphology,MGGFe)

• Proportion of malignant cells(leukaemic blasts)?• AL DG: over 20%; exceptions exist

• Morphological characterisation of cells(Granulation? Nucleus form? Auerrods? Vacuolation? etc.)

BONE MARROW ASPIRATION SAMPLE

1) Microscopic analysis (morphology,MGGFe)

2) Flow cytometry test (surface markers ofmalignant cells; marker antigens of earlycells, aberrant expressions)

AML M2CD7+

Monitoringmorphologicremission0.7% CD7+

Bone marrow aspirate, or suction sample

BONE MARROW ASPIRATIONSAMPLE1) Microscopic analysis(morphology, MGGFe)

2) Flow cytometry test (surfacemarkers of malignant cells;marker antigens of early cells,aberrant expressions)

3) Chromosomal and genetictests (type changes; mutations,fusion genes, deletions…)

• Hematological chromosomes• FISH• RQ-PCR (fusion genes, point mutations)

CYTOMOLECULAR TESTS

Sequencing exome / transcriptome / wholegenome from tumour tissue• Soon to be available for clinical use on a large scale

• Replaces a significant amount of other diagnostic tools

• Creates a basis for targeted and individual treatment

• Price comes down every year

In the future:• Micro chips• “collectors of all data”