The document discusses immunotherapy and the role of pathologists in assessing tumor samples. It describes how certain tumors express PD-L1 antigens that can be recognized by the immune system, but the tumors also engage immune checkpoint pathways like PD-1 and CTLA-4 to evade the immune response. Immunotherapy drugs target these checkpoint pathways to enhance the immune response. The document outlines the FDA-approved PD-L1 immunohistochemistry assays and biomarkers used to identify cancer patients most likely to respond to immune checkpoint inhibitors for various cancer types including NSCLC, melanoma, bladder cancer, and colorectal cancer.

1. IMMUNOTHERAPY: BASICS AND
ROLE OF PATHOLOGIST
Dr.Kanwalpreet Kaur

2. IMMUNE RESPONSE TO CANCER
• Paul Ehrlich first conceived the idea that tumor cells can be
recognized as “foreign” and eliminated by the immune system

3. DISPLAY AND RECOGNITION OF ANTIGENS

4. • Transformed cells of tumors express antigens, called tumor-associated
antigens (TAAs)
• The immune system recognizes those antigens as ‘‘not self’’ and mounts
an immune response against the tumor cells

5. Tumour microenvironment
• 3 key cells involved in the immune response:
(1) ANTIGEN-PRESENTING CELLS, such as dendritic cells (DCs),
which identify and uptake foreign antigens and present them to
T cells;
(2) T LYMPHOCYTES, which become activated by antigen-
presenting cells to recognize and destroy the cells containing
foreign TAAs; and
(3) B LYMPHOCYTES, which produce antibodies specific to TAAs.
• Other immunomodulatory cells include T regulatory cells,
natural killer cells, macrophages, and myeloid-derived
suppressor cells.
• IMMUNE CELLS, + STROMAL CELLS = TUMOR
MICROENVIRONMENT immune survellience

7. TUMOR IMMUNOEDITING AND CHECKPOINT
INHIBITION

8. • Tumor cells actively inhibit tumor immunity by engaging
normal pathways of immune regulation that serve as
“checkpoints” in immune responses

9. CTLA4 CHECKPOINT: EARLY/PRIMING PHASE

10. PD-1/PDL1 EFFECTOR PHASE

11. • In addition to T cells, PD-1 receptor is also expressed on the
surface of macrophages and B cells;
• Its 2 ligands-PD-L1 and PD-L2 are expressed by many tumor
and inflammatory cells
• PD-L1 expression correlates with poor clinical outcome but
also with the likelihood of response to targeted immune-
checkpoint inhibition

13. The importance of this science has recently
been recognized by awarding 2018 NOBEL PRIZE
IN MEDICINE to JAMES ALLISON and TASUKU
HONJO for their research on CTLA-4 and PD-1

14. Programmed Death-Ligand 1 Assays and
Inhibitors
CRITERIA PEMBROLIZUMAB NIVOLUMAB DURVALUMAB ATEZOLIZUMAB
TARGET PD-1 PD-1 PD-L1 PD-L1
PDL1-IHC
ASSAY
DAKO22C3 Dako 28-8 Ventana SP263 Ventana SP142
CELL TYPE
SCORED
NSCLC- TC
UC-TC/IC
CRC-TC/IC
NSCLC-TC NSCLC-TC NSCLC-TC/IC,
UC-IC
Cutoff
criteria
(NSCLC)
TC = 1%–49%;
TC >50%
TC> 1%;
TC > 5%;
TC > 10%
TC > 25% TC or IC > 1%;
TC or IC > 5%;
TC > 50% or
IC > 10%
Cutoff
criteria (UC)
> Any TC or stromal
cell
NA NA IC 10%; IC 5%;
IC 1%
Cutoff
criteria
(MSI-H CRC
TC < 5%; TC > 5%;
IC—any positivity (%)
NA NA NA

15. UNIQUE IHC ASSAY FOE EACH IMMUNE
CHECKPOINT INHIBITORS
• Four PD-L1 IHC assays registered with the FDA, using four
different PD-L1 antibodies (22C3, 28-8, SP263, SP142), on two
different IHC platforms (Dako and Ventana)
• Definition of positive varies which biomarker assay is used
and for which malignancy

16. PDL1 expression by IHC
• Tumor proportion score (TPS) : ratio of the number of PD-L1–
expressing tumor cells to that of all tumor cells  NO ROLE
AS PREDICTIVE INDICATOR EXCEPT NSCLC for pembrolizumab
• Mononuclear immune cell density score (MIDS) : ratio of the
number of PD-L1–expressing immune cells to that of all tumor
cells
• COMBINED POSITIVE SCORE (CPS) :the ratio of the number of
all PD-L1–expressing cells (tumor cells, lymphocytes,
macrophages) to the number of all tumor cells

17. COMBINED POSITIVE SCORE (CPS)
A minimum of 100 viable tumor cells must be present in the PD-L1–stained slide
(sectioned tumor biopsy or resection tissue) for the specimen to be considered
adequate for evaluation.
Tumor cells must show partial or complete membrane staining (≥1+) to be counted
as “stained,” whereas immune cells are counted if there is any staining

18. Gastric cancer specimens stained with PD-L1 immunohistochemical 22C3 pharmDx
A, Specimen with CPS < 1.
B, Specimen with CPS > 1

19. CUTANEOUS MELANOMA: NCCN GUIDELINES
• In March 2011, ipilimumab, an anti-CTLA-4, was the first ICI to
receive the US FDA approval for the treatment of
unresectable stage III and stage IV melanoma in patients who
have received prior therapy
• Now trials have shown adding pembrolizumab and nivolumab
improves response and PFS compared with chemotherapy or
implimumab immunotherapy alone

20. PD-L1 expression in melanoma: effect on treatment
• Whether PD-L1 expression marker can be predictive factor for
immunotherapy ( response to Anti PDL1) in melanoma ?
• Across trials, response rate, PFS and OS for anti PDL1
improves with increased PDL1 expression
• However, patients with little or no PDL1 expression detected
in their tumor samples also experienced durable responses
• For nivolumab, PDL1 expression alone is insufficient
biomarker to predict OS
• None of the analyses were able to identify PDL1 expression
threshold for selection of anti PDL1 agent versus other
treatment options.

21. IHC PD-L1 (22C3, 400×)
in melanoma

22. PD-1/PD-L1 IMMUNOTHERAPY IN
NSCLC : NCCN GUIDELINES
• Assessment of PD-L1 expression by IHC
before first line treatment in all patients with
metastatic NSCLC after assessing for driver
mutations EGFR, ALK

23. PD-1/PD-L1 IMMUNOTHERAPY IN
NSCLC : NCCN GUIDELINES
• For the use of immunotherapy in metastatic NSCLC AND
NEGATIVE DRIVER MUTATIONS ( EGFR/ALK/ROS and BRAF
V600E)
1) Patients with PD-L1 EXPRESSION > 50% are recommended to
receive single agent PEMBROLIZUMAB regardless of histology
as first line treatment
2) Single agent pembrolizumab can also be used as first line
therapy in patients of metastatic carcinoma and PDL1 levels
of 1-49%, regardless of histology
3) Combination of pembrolizumab and chemotherapy as first
line therapy regardless of PDL1 expression

24. • For subsequent therapy options, pembrolizumab is
recommended as therapy option in patients with metastatic
NSCLC and PDL1 expression >1%
• Nivolumab or atezolimumab can be used as subsequent
therapy regardless of PDL1 levels

25. • Among patients with a PD-L1 proportion score of at
least 50% the response rate was 45.2%; median
progression-free survival (PFS) was 6.3 months (95%
CI, 2.9 to 12.5) for all patients, 6.1 months (95% CI,
2.1 to 12.5) for previously treated patients, and 12.5
months (95% CI, 2.4 to 12.5) for previously untreated
patients

26. IMMUNOTHERAPY IN UROTHELIAL
CARCINOMA
• Pembrolizumab, atezolimumab, nivolumab, duravalumab are
prefferd second line therapy options after platunum based
therapy in metastatic cancers
• Pembrolizumab can also used as first line in patients with
metastatic disease
1) Patients are not eligible for cisplatin based chemotherapy and
whose tumour express PD-L1 as measured by combined positive
score of atleast 10
2) Patients are not eligible for any platinum based chemotherapy
regardless of PD-L1 status

27. • Atezolizumab can also used as first line in patients with
metastatic disease
1) Patients are not eligible for cisplatin based chemotherapy and
whose tumour express PD-L1 stained tumour infiltrating
immune cells covering atleast 5% of the tumour area
2) Patients are not eligible for any platinum based chemotherapy
regardless of PD-L1 status

28. PD-1/PD-L1 IMMUNOTHERAPY IN
MSI-H CRC
• MSI-H CRC ( microsatellite instable- coclorectal carcinoma)
have an active immune microenvironment infiltrated by
cytotoxic T lymphocytes and activated Th1 cells
• Greater tumor-infiltrating lymphocyte (TIL) densities
compared with MSS tumors because of the presence of
numerous neoantigens (mutated proteins) created by the high
mutational load, typically between 10 and 50 times that of
DNA repair-sufficient (MSS) tumors

29. • Despite such a ‘‘hostile’’ microenvironment full of immune
cells, MSI-H tumor cells are not eliminated by the immune
system because of the cancer-specific upregulation of
inhibitory checkpoints, including PD-1, CTLA4, LAG3, and
indoleamine 2,3-dioxygenase (IDO)
• Potentially good candidates for checkpoint immunotherapy
• Approximately 3-5% of stage IV colorectal cancers are MSI-H

30. • What is strikingly different in MSI-H CRC from
melanoma, renal cancer, or lung cancer is that there
is little PD-L1 expressed by the tumor cells; rather,
the PD-L1 is expressed predominantly by infiltrating
immune cells, T cells, and myeloid cells

31. • FDA approval of anti-PD-1 drug pembrolizumab for the
treatment of metastatic/ refractory MSI-H CRC assessed
expression of CD8 and PD-L1 with a cutoff of 5% for PD-L1 of
tumor cells and assessed the percentage of PD-L1 TILs and
macrophages within the tumor and at the invasive fronts
• The expression of CD8 and PD-L1 was not significantly
associated with PFS or overall survival but was predictive of
response to pembrolizumab therapy

32. PD-L1 expression in microsatellite instability–
high (MSI-H) colorectal cancer (CRC)
Strong membranous expression along
tumour-stromal surface diffuse membranous expression in tumour

34. • All 6 patients (100%) with sporadic MSI-H CRC had an
objective response, whereas only 3 of 11 patients (27%) with
germline MSI-H CRC (Lynch syndrome) had treatment
responses
• Related to the differing pathogeneses (eg, methylation
patterns

35. New developments
• September 2017, FDA approval of pembrolizumab for
treatment of patients with gastric or gastroesophageal
junction adenocarcinoma that expresses PD-L1

36. New developments
• On March 8, 2019, the Food and Drug Administration granted
approval to atezolizumab in combination with paclitaxel
protein-bound for adult patients with unresectable locally
advanced or metastatic triple-negative breast cancer (TNBC)
whose tumors express PD-L1 (PD-L1 stained tumor-infiltrating
immune cells [IC] of any intensity covering ≥ 1% of the tumor
area)
• VENTANA PD-L1 (SP142) Assay  selecting TNBC patients for
atezolizumab

37. New developments
• On June 10, 2019, the Food and Drug Administration
approved pembrolizumab for the first-line treatment of
patients with metastatic or unresectable recurrent head and
neck squamous cell carcinoma (HNSCC)
• Tumors express PD-L1 (Combined Positive Score [CPS] ≥1) as
determined by an FDA-approved test (PD-L1 IHC 22C3
pharmDx kit)

38. Programmed Death-Ligand 1 Assays and
Inhibitors
CRITERIA PEMBROLIZUMAB NIVOLUMAB DURVALUMAB ATEZOLIZUMAB
TARGET PD-1 PD-1 PD-L1 PD-L1
PDL1-IHC
ASSAY
DAKO22C3 Dako 28-8 Ventana SP263 Ventana SP142
CELL TYPE
SCORED
NSCLC- TC
UC-TC/IC
CRC-TC/IC
NSCLC-TC NSCLC-TC NSCLC-TC/IC,
UC-IC
Cutoff
criteria
(NSCLC)
TC = 1%–49%;
TC >50%
TC> 1%;
TC > 5%;
TC > 10%
TC > 25% TC or IC > 1%;
TC or IC > 5%;
TC > 50% or
IC > 10%
Cutoff
criteria (UC)
> Any TC or stromal
cell
NA NA IC 10%; IC 5%;
IC 1%
Cutoff
criteria
(MSI-H CRC
TC < 5%; TC > 5%;
IC—any positivity (%)
NA NA NA

39. Take home message
• The FDA approved PD-L1 immunohistochemistry (IHC) as a
companion or complementary diagnostic tool for PD-1/PD-L1
inhibitor therapy in patients with non-small cell lung cancer or
urinary bladder cancer
• However, currently, PD-1/PD-L1 inhibitors are used in the
treatment of melanoma regardless of the specific PD-L1
expression in patients
• The absence of PDL1 expression was not an absolute
indicator of the lack of benefit from immunotherapy

40. • Pathologists have to cope with the rapid expansion of
immunotherapies, new novel targets, and therapeutic
strategies.
• The surgical pathologists’ practice must change from the
traditional morphology-based routine to a fully clinical-
oriented interaction with medical oncologists, surgeons, and
radiotherapists, that is, to become multidisciplinary

41. THANK YOU…