Cancer Immunotherapy Strategies

Tumor Immunology

• Paul Ehrlich hypothesized in 1909 that host defenses could prevent neoplastic cells from developing into tumors.
• Lewis Thomas and Macfarlane Burnet coined the term immune surveillance between 1959 and 1970.
• Immune surveillance is where the immune system constantly scans the body to identify and destroy emerging cancer cells.
• Infiltration of tumors by lymphocytes often correlates with outcome.
• Tumors induced experimentally in animals are often immunogenic and can be eliminated by modulation of immune system.
• Immunodeficient individuals have increased incidence of some cancers.
• New immunotherapies are showing impressive results with some cancers.

Immune Response against Tumors

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

Tumor Antigens

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

Immune Evasion by Tumors

• Cancer cells are destroyed
• Cancer cells escape from immune mechanisms by selective outgrowth of cancer cell clones that lost their tumor antigen or cannot present it in the context of MHC class I molecules.
• It is important to recall that tumors are heterogeneous.

Immune Evasion by Cancer (cont.)

• Proteins that normally transmit inhibitory signals of T-cell activation maintain immune responses at a desired physiological range and protect the host from autoimmune reactions.
• CTLA-4 and PD-1 bind to their ligands B7-1 (or B7-2) and PD-L1 (or PD-L2), respectively.
• CTLA-4 and PD-1/PD-L1 pathways are used by many tumors to evade immune attack against them.
• CTLA-4, cytotoxic T-lymphocyte-associated protein 4; PD-1, programmed death-1; PD-L1, programmed death- ligand 1

CTLA-4 Pathway as an Immune Evasion Mechanism

• Tumors can prevent effective T cell response against them by upregulating CTLA-4.
• In the induction of the antitumor immune response in the lymph node, primed CTLs capable of killing tumor cells are the end result,
• CTLA-4 competes with CD28 for B7, impeding the co-stimulation needed for CD8+ T cell activation.
• CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; TCR, T cell receptor; MHC, major histocompatibility antigen

Immunotherapies Targeting CTLA-4

• Immunotherapies targeting CTLA-4 have improved overall survival in many metastatic cancers.
• Blocking CTLA-4 with an antibody allows CD8+ T cells to be activated and kill tumor cells.
• Ligands for CTLA-4 include B7-1 (aka CD80) and B7-2 (aka CD86).
• *E.g., advanced melanoma, non-small cell lung carcinoma, advanced renal cell carcinoma (previously, almost 100% lethal within months). In most cases, Ipilimumab (YervoyTM) used in combination therapy.
• Ipilimumab (FDA-approved: 2015)
• Example: Ipilimumab

Overexpression of PD-1

• Overexpression of PD-1 on T cells or PD-1L or PD-L2 on tumor cells contributes to immune evasion.
• Overexpression of PD-1 on T cells or PD-1L or PD-L2 on tumor cells as immune evasion mechanisms.
• An ineffective T cell response against the tumor is the end result.
• MHC, major histocompatibility antigen; CTL, cytotoxic T lymphocyte; TCR, T cell receptor; PD-1, programmed death-1; PD-L1, programmed death- ligand 1

Blocking Binding of PD-L1

• Blocking Binding of PD-L1 or PD-L2 to PD-1 with Antibodies Allows T Cells to Kill Tumor Cells
• Examples: Atezolizumab (FDA-approved: 2016)
• Pembrolizumab (FDA-approved: 2014)

Chimeric Antigen Receptors (CARs)

• Synthetic molecules (they don't exist naturally) are made up of an extracellular domain: a) variable region of an antibody that binds a specific tumor antigen (e.g., CD19*) b) intracellular domain of the T cell receptor (TCR) that recognizes that same tumor antigen

• 2nd generation CAR: a single co-stimulatory domain (e.g., 4-1BB or CD28) in addition to CD3ζ signaling domain → longer and are more effective response.

• 3rd generation CAR: additional costimulatory domain (e.g., CD27, CD28) →↑ persistence/potency.

• *molecule found on the surface of B-cell leukemias (as well as healthy B cells) VL, variable light chain; VH, variable heavy chain; scFv, single-chain variable fragment

CAR T-Cell Therapies

• Patient-derived CTLs are engineered outside the body (via leukapheresis) to express CARs and then reinfused into the patient.
• First CAR T-cell therapies: target CD19 to treat certain ALLs in children and young adults, non-Hodgkin lymphoma and diffuse large B-cell lymphoma (FDA approved in 2017 and 2018).
• Can lead to long-term remissions but often cause serious complications due to "cytokine release syndrome" from CTL-released cytokines.
• FDA, Food and Drug Administration; ALL, acute lymphoblastic leukemia

Pathologic and Laboratory Diagnosis of Cancer

• Morphologic Methods
• Molecular Methods

Conventional Diagnosis of Solid Tumors

• Signs and Symptoms
  • Palpable lump, pain
  • Fever, fatigue, weight gain or loss
  • Altered metabolism
• Medical Imaging (X-rays, CT scan, MRI)
• Gold Standard: surgical biopsy/pathological diagnosis
  • Direct microscopic examination

Sampling Approaches

• Excisional biopsy (Bx) - entire lesion is removed.
• Incisional Bx - a representative sample of the tumor.
• Needle Bx (usually image-guided)
  • Fine-needle aspiration (aka FNA)
  • Core needle biopsy

Sampling Approaches (cont.)

• Cytology (Papanicolaou) smears

Parameters Used in Pathologic Diagnosis of a Neoplasm

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

Gross Appearance (macroscopic examination)

• Considerations and Practices
• Adequate sample
• Label margins of excision with indelible ink
• Open specimen
• Sections collected (with a scalpel)
• Pathologist notes -
  • Consistency
  • Color
  • Shape
  • Extent of the neoplastic growth

Microscopic Examination

• Tissue Processing for Microscopic Examination (Paraffin-embedded)
• Sectioning with microtome
• Classical staining (Hematoxylin & Eosin [H&E])
• 5-10 µm-sections mounted on slide
• H&E staining: nuclei blue/purple, cytoplasm pink

Microscopic examination using classical staining H&E

• Mostly used for intraoperative diagnosis.
• Tissue is hardened through rapid freezing.
• Sections of ~ 1 µm are made in a cryostat (~microtome at -20 to -30°C).
• Specimen embedded in OCT compound (gel-like medium that provides a matrix for cryostat sectioning).
• Rapid, but quality not as good as in paraffin-embedded specimens.

When are Frozen Sections Needed?

• If there is suspicion that a tumor has spread, a sample of the potential metastasis is sent for cryosection to verify its identity.
• Mohs surgery is a method used for some skin cancer lesions where thin layers of skin are removed one at a time until the tissue viewed under the microscope shows no remaining cancer (clear margins).

When are frozen sections needed? (cont.)

• Sentinel Lymph Node Biopsy is a procedure to test if cancer (e.g., breast cancer, melanoma) has spread beyond the original tumor to lymph nodes (LNs). a radioactive substance is injected near the tumor a probe is used to follow radioactivity and detect sentinel lymph node(s) (first lymph nodes to which cancer cells are likely to spread from a primary tumor) sentinel node(s) are excised and checked for the presence of cancer cells under the microscope
• Sentinel Lymph Node Biopsies are usually done at the same time the primary tumor is removed.

Use of Immunohistochemistry (IHC) in Tumor Diagnosis

• IHC is used to detect the status (expression or not) and localization (membrane, cytoplasm, or nucleus) in cells/tissues of a specific antigen (PROTEIN) by use of specific antibodies that are then visualized by a chromogen.
• HRP, horseradish peroxidase
• DAB, diaminobenzidine

Use of IHC in Cancer

• IHC identifies specific antigens (PROTEINS) within tissue sections
• Diagnosis of malignant tumors of unknown histogenesis.
• Identification of site of origin of metastatic lesions.
• Detection of proteins with prognostic or therapeutic significance.
• “Picking the needle out of the haystack".

Tissue-Specific Markers

• Through IHC in Cancer
• Examples: Carcinoma, Sarcoma, Melanoma, Lymphoma
• CK: cytokeratin
• VIM: vimentin

Tissue-specific" Markers (cont.)

• Used in poorly differentiated cancerous lesion and metastasis

Value Of Tumor Markers

Supports diagnosis of cancer (along with other tests) Reflects the stage (extent) of the disease Reflect Prognosis through periodic evaluation Decrease in levels → Cancer is responding to treatment Increase in levels → Cancer is refractory to treatment or recurrence

Examples Of Tumor Markers

• HER2/neu (erb-B2) common use is identification (ID) of a more malignant breast cancer (BrCa) subtype; for therapeutic decision
• Estrogen and progesterone receptor (ER/PR) common use is ID of metastatic breast carcinoma; predictive of clinical response to hormonal therapy in BrCa.
• Prostate-specific antigen (PSA) common use is ID of metastatic prostate Ca.
• Carcinoembryonic antigen (CEA) common use is ID of Gl and lung adenoCa
• CA-19-9 common use is ID of Gl and pancreatic CA
• CD117 (c-Kit) common use is used for therapeutic decisions in GISTs
• Epidermal Growth Factor Receptor (EGFR) common use is Used for therapeutic decisions in Lung Ca.
• HMB-45 common use is ID of melanoma, and ID in sentinel LNs of it
• Ki-67 common use is for Proliferative status of tumors.

Exmaples of Tumor Markers Detected by IHC

• Examples: Her-2-positive BrCA and Basal Cell Carcinoma

Immunophenotyping using Flow Cytometry

• Mostly used to classify leukemias and lymphomas such as T and B lymphocytes and - Mononuclear-phagocytic ceLls.
• Fluorescent*-labeled antibodies against cell surface antigens (clusters of differentiation antigens, CDs).
• DNA binding dyes can be added to assess DNA content in tumor cells.
• Fluorescein isothiocyanate (FITC); R-phycoerythrin (PE)

Cytogenetic Analysis

• Cytogenetic Analysis is used for confirmation of Dx of certain hematological malignancies and solid tumors with chromosomal abnormalities
• CML: t(9;22) → Ph chromosome (↑ Abl gene)
• Burkitt lymphoma: t(8;14) → ↑ Myc gene

In Situ Hybridization

• Technique used to detect and locate specific nucleic acid sequences in the nucleus (DNA) or cytoplasm (mRNA) on top of tissue section
• Probes - DNA segments that are complementary to specific DNA/RNA sequences - are used to identify gene amplifications, deletions, or translocations that often occur in cancer.
• Two main types of in situ hybridization with Examples.
  • Radioactive or Chromogenic or In Situ Hybridization
• Fluorescence In Situ Hybridization (FISH)

Radioactive/Chromogenic In Situ Hybridization

• Radioactive 32P labeled probes
• Photographic on top of tissue selection
• Then can be used to expose days to weeks so their signal can be detected by autoradiography

Fluorescence In Situ Hybridization (FISH)

• It is a fluorescence in situ Hybridization
• Examples: FITC or fluorescein

Fluorescence In Situ Hybridization (FISH)

Examples.

• multicolor FISH where there are two flourochromes with
• ABL CHR9 which is red fluorescence
• and BCR CHR22 which is green flurescence
• creating a fusion of the BCR ABL gene which is yellow

Polymerase Chain Reaction (PCR)

• Used to amplify a specific DNA sequence initially present in extremely small amounts to provide sufficient DNA for analysis.
• 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
• These primers serve as the starting points for DNA synthesis.

Reagents In a PCR Reaction

• Taq polymerase (Taq) - a is a heat-resistant DNA polymerase that adds new DNA bases.
• Each PCR cycle involves 3 main steps:
  • Denaturing
    • Annealing
      • Extension

Polymerase Chain Reaction (PCR) (cont.)

• Denaturing Process
• the double-stranded template DNA is heated to separate it into two single strands

Polymerase Chain Reaction (PCR) (cont.)

• Annealing Process
• temperature is lowered to allow the DNA primers designed for the "target sequence" to attach to the template DNA

Polymerase Chain Reaction (PCR) (cont.)

• Extension process is where
• Temperature is raised to ~72°C and the DNA polymerase Taq adds nucleotides to the 3' end of both the forward and reverse primers to make strands

Result

• The End Result of PCR After n cycles you have 2n (2 to the nth power) ds DNA pieces
• After 30 you would 2 to the power of 30 billion copies

DNA Microarrays

• Used to analyze the simultaneous expression changes in thousands of genes unlike nucleic acid based techniques.

Details of DNA Arrays

• It is is nucleic hybridization based techniques that expressions individual RNA is and you can
• Compare RNA from different
• Healthy vs. Diseased issue
• To detect gene mutation
• Thousands of microsco[ic DNA spots sequence complementary dna

DNA Microarrays Information

• extracted from test and reference samples treated vs untreated and. RNA
• Versus DNA.
• Reverse transcriptions rt the RNA turns into complimentary DNA cDNA
• You level cDNA with red dye with for tests CD and green for reference cDNA and then add DNA
• micro array for overnight hybridization with specific probes used to read at each spot affect relative gene expression high vs low and so
• DNA Polymerase

Next Generation Sequencing

• Enables sequencing of entire tumor genome DNA
• May aid in identifying actionable to detect it.

Liquid Biopsies

• Analysis of components in body like blood monitor
• Disease pregressoion
• Diagnostic value - Metastatic resistant prostate cancer.

Exploration of cancer immunotherapy strategies, including understanding the tumor microenvironment's influence, enhancing tumor immunogenicity, and overcoming immune evasion. Examination of advanced sampling for tumor heterogeneity and methodologies for assessing tumor cells.