L-14 Neoplasma II

Tumor Immunology

• Paul Ehrlich theorized in 1909 that host defenses could prevent neoplastic cells from turning into tumors.
• Lewis Thomas and Macfarlane Burnet coined the term immune surveillance in 1959-1970, suggesting the immune system constantly scans the body to destroy emerging cancer cells.
• Lymphocyte infiltration in tumors often correlates with the outcome.
• Tumors that were experimentally induced in animals are often immunogenic and can be eliminated by modulation of the immune system.
• Immunodeficient people have a higher chance of developing some cancers.
• New immunotherapies are showing impressive results in treating some cancers.

Immune Response against Tumors

• Tumor-specific CTLs migrate back to and kill tumor cells.
• Dendritic cells (DCs) phagocytose tumor cells or antigens and travel through afferent lymphatic vessels.
• DCs present tumor antigens as class I MHC-associated peptides to naive CD8+ T cells in draining lymph nodes.

Tumor Antigens

• Tumor antigens are presented on the tumor cell surface (or APC's cell surface) with Class I MHC to CD8+ cytotoxic T cells.
• The immune system has inherent tolerance to normal proteins.
• Tumor antigens also enter the class II MHC antigen-processing pathway in antigen presenting cells (APCs) and are recognized by CD4+ T cells.

Immune Evasion by Cancer

• Cancer cells are destroyed by T cell recognition of tumor antigens leading to activation.
• Escaping mechanisms involve clones outgrowth that lost tumor antigen or cannot present it with MHC class I molecules.

Immune Evasion by Cancer (Mechanisms Cont.)

• Immune checkpoints are proteins which normally transmit inhibitory signals of T-cell activation to maintain immune responses within a normal physiological range and protect the host from autoimmune reactions.
• Specific receptors like CTLA-4 and PD-1 bind to ligands B7-1 (or B7-2) and PD-L1 (or PD-L2), respectively which allows them to stop immune attacks.
• CTLA-4 and PD-1/PD-L1 pathways are used by many tumors to evade immune attack.

CTLA-4 Pathway

• Ineffective T cell response is caused by the upregulation of CTLA-4.
• CTLA-4 competes with CD28 for B7 which impedes co-stimulation needed for CD8+ T cell activation.

CTLA-4 Immunotherapies

• Blocking CTLA-4 with an antibody allows CD8+ T cells to be activated to kill tumor cells.
• Ligands for CTLA-4 include B7-1 (aka CD80) and B7-2 (aka CD86).
• Ipilimumab can now be used due to FDA approval in 2015, for metastatic cancers.

PD-1L Overexpression

• Overexpression of PD-1 on T cells, or PD-1L or PD-L2 on tumor cells are Immune Evasion Mechanisms.

PD-1L Inhibition

• Blocking the binding of PD-L1 or PD-L2 to PD-1 with antibodies allows T Cells to kill tumor cells.
• Atezolizumab , FDA approved in 2016.
• Pembrolizumab, FDA approved in 2014.

Chimeric Antigen Receptors (CARs)

• Synthetic molecules that contain
  • An extracellular domain with variable regions of antibodies binding specific tumor antigens like CD19.
  • An intracellular domain with T cell receptor that recognize tumor antigens.
• 2nd generation CAR: a single co-stimulatory domain, like 4-1BB, in addition to a CD3ζ signaling domain that makes the responses longer and more effective.
• 3rd generation CAR: contains additional costimulatory domains like CD27 for increased persistency/potency.

CAR T-Cell Therapies

• Engineered to express CARs by leukemia pheresis, then reinfused.
• First CAR T-cell therapies target CD19 to treat ALL in kids, non-Hodgkin lymphoma, and diffuse large B-cell lymphoma.
• Long-term remissions can be achieved, however "cytokine release syndrome" can occur due to CTL-released cytokines.

Pathologic and Laboratory Cancer Diagnosis

• Morphologic Methods
• Molecular Methods

Conventional Diagnosis of Solid Tumors

• Consists of signs and symptoms like:
  • Palpable lump and/or pain
  • Fever, fatigue, weight gain or loss
  • Altered metabolism
• Medical Imaging (X-rays, CT scan, MRI) can be performed
• The Gold Standard is surgical biopsy followed by pathological diagnosis that consists of direct microscopic examination.

Sampling Approaches

• Excisional biopsy (Bx) is removing the entire lesion.
• Incisional Bx - a representative sample of the tumor.
• Needle Bx is usually image-guided, with fine-needle aspiration.

Sampling Approaches (Cont.)

• Cytology (Papanicolaou) smears show;
  • Large, flat cells with small nuclei (normal)
  • Sheet of cells with nuclear pleomorphism, hyperchromatic nuclei, frequent mitosis (M), and polymorphonuclear (PMN) cells. (abnormal)

Parameters for Neoplasm Diagnosis

• Gross (macroscopic) tumor appearance.
• Microscopic examination using H&E staining.
• Immunohistochemistry for cell lineage identification or tumor type.
• Cytogenetics/molecular approaches.

Macroscopic Examination

• Considerations include adequate samples, labels on margins of incisions, open specimen sampling.
• Pathologist's note consist of :
  • Consistency, color and extent
  • Shape of the neoplastic growth

Microscopic Examination

• Uses Hematoxylin & Eosin [H&E] for classical tissue staining and Tissue Processing for Microscopic Examination, Paraffin-embedded.
• Section with a microtome, mount on slide, then stain.

Microscopic examination with H&E

• Frozen Sections (aka cryosections)
• Mostly used for intraoperative diagnosis after rapid freezing.
• Sections of ~ 1 µm are made in a cryostat (~microtome at -20 to -30°C).
• Specimen is embedded in OCT compound, providing the proper matrix.
• The process is rapid, but the quality is not as good as in paraffin-embedded specimens.

Frozen Section Uses

• To verify identity of potential metastasis.
• Mohs surgery- to remove thin skin layer to check for remaining cancer.

Frozen Sections are useful for

• Procedure to test for cancer spread, most commonly to the lymph nodes (LN); Sentinel Lymph Node Biopsy (breast cancer, or melanoma).
  • Inject a radioactive substance, then use a probe, excise node, and check for cancer under a microscope.
  • Sentinel Lymph Node Biopsies are usually done when the primary tumor is removed.

Immunohistochemistry (IHC) in Tumor Diagnosis

• Oxidization will cause the enzyme to form a colored precipitate at the reaction site
• The IHC technique identifies,
  • Protein status expression
  • Localization in cell membranes/tissues
  • By use of specific antigens that visualize chromogen

IHC in Cancer

• Identifies specific antigens (PROTEINS) within tissue sections.
• Identifies malignant tumors of unknown histogenesis.
• Identifies origin sites of metastatic lesions.
• Detects proteins with prognostic or therapeutic significance.

IHC Cancer Markers

• The tumor type depends on:
  • Carcinoma that contain cytokeratin
  • Sarcoma that contain vimentin
  • Melanoma that contain S-100 proteins
  • Lymphoma that contain CD45

More with IHC Cancer Markers, including:

• Pan-CK positivity that can be used to trace undifferentiated tumors.
• S-100 positivity can trace melanomas.

Tumor Marker Uses Include;

• Support the diagnosis, with more tests
• Reflects the stage/extent
• Reflects the prognosis, through future evaluations

Tumor Markers detected on IHC

• HER2/neu (erb-B2) is a more malignant Breast cancer, used for its therapeutic decision.
• Estrogen and progesterone receptor (ER/PR) is for breast carcinoma used to predict the hormone therapy response.
• Prostate-specific antigen (PSA) for metastatic prostate.
• Carcinoembryonic antigen (CEA) is for Gl and lung adenoCa.
• CA-19-9 for Gl and pancreatic CA.
• CD117 (c-Kit) is for use in therapeutic decisions in GISTs.
• Epidermal Growth Factor Receptor (EGFR) uses therapeutic decisions in Lung Ca.
• HMB-45 melanoma detection and sentinel Nodes ID
• Ki-67 measures Proliferative status

Immunophenotyping

• Mostly used to classify leukemias and lymphomas:
  • T and B lymphocytes
  • Mononuclear-phagocytic cells
• Fluorescent-labeled antibodies identify clusters of differentiation antigens (CDs) on cell surfaces.
• DNA binding dyes analyze DNA content.

Cytogenetic Analysis

• Primarily to confirm the diagnosis of certain hematological malignancies and solid tumors with chromosomal abnormalities.
  • CML(↑ Abl gene): t(9;22)-> PH chromosome
  • Burkitt lymphoma(↑ Myc gene): t(8;14)

Molecular Methods

• In situ Hybridization is a technique used to detect and locate specific nucleic acid sequences in the nucleus (DNA) or cytoplasm (mRNA) on top of tissue section

In Situ Hybridization

• Uses segments that are complementary to DNA/RNA to identify amplifications, deletions, or translocations.
• Two main types of in situ hybridization:
  • Radioactive or Chromogenic
  • Fluorescence (FISH)

In Situ cont: a. Radioactive or Chromogenic

• Radioactive probes (32P).
• Photographic emulsion the signals.
• Signal (black dots) autoradio images.
• Chromogenic (biotin-labeled) probes.
• Enzymes horseradish enhance signal.
• Substrate precipitate visualization.

Molecular Diagnosis b. Fluorescence In Situ Hybridization (FISH)

• Probes labeled with a fluorophore (FITC, fluorescein) allows detections with afluorescence microscope

In Situ Hybridization (FISH)

• Multicolor FISH, 2 different fluorophores.
• Abl (Chr 9)- will show red
• Bcr can be stained by green

Molecular Diagnosis

• Using Polymerase Chain Reaction, "PCR" which is; used to amplify a sequence with small sample sizes.
  • Involve specific primers to match nucleotide sequences
  • Forward and reverse primers (short single-strands of DNA) are designed to match the nucleotide sequence of short segments on each side of the target DNA.
  • Primers can serve as the starting points for DNA synthesis
  • Heat resistant polymerase (Taq) adds new DNA bases
  • PCR cycle involves 3 main process, those are:
    • Denaturing
    • Annealing
    • extension

PCR cont - Step 1 and 2

• Denaturing is the double-stranded split into one strand at 95 C.
• Annealing is temperatures cooling to attach primers on each end

PCR cont - Step 3 Extension

• The end temperature is at 72 C add Taq polymerase, that makes new strands.
  • There are now billions of dsDNA pieces

DNA Microarrays

• Unlike northern blotting, DNA microarrays allow a simultaneous expression changes in thousands of genes.Used to compare mRNA expression profiles (gene expression analysis) between different conditions, or mutations (genotyping) Tens of thousands DNA spots Each DNA or RNA is attached to a slide.

DNA Microarrays (cont.)

• Reference samples for gene experiment are tested to extracted
• RNA is complied by a transcription
• Dyes determine amounts when scannned, red and green

Next Generation Sequencing

• Enables identification of genetic mutations and is may aid in identifying with genomic DNA, for better diagnosis & assessment

Liquid Biopsies

• Minimizes invasiveness of cancer biopsies
• Can check for genetic and molecular characteristics of patients with liquid samples to determine cancer progression