Approach to undifferentiated tumors

1. APPROACH TO UNDIFFERENTIATED TUMORS
by
Dr. Varughese George

2. Introduction
“Undifferentiated” tumors
• Tumors lacking evidence of lineage differentiation on
the basis of routine light microscopic morphology.
• Tumors mimicked by different types of tumors causing
diagnostic challenge
• Tumors that require an algorithmic diagnostic approach
elucidating its true nature crucial for clinical
management.

3. Diagnostic Algorithm for Workup of Undifferentiated Neoplasms

4. GENERAL APPROACH FOR
UNDIFFERENTIATED TUMORS
Categorize malignant tumors according to
morphologic appearances.
Undifferentiated tumors can be categorized
according to morphologic appearances into 4
major groups:

5. Tumors according to morphologic appearances
1. Small round cell
tumors
Heterogeneous groups of
tumors composed of
relatively small, round
to oval, closely packed
undifferentiated cells,
high nuclear to
cytoplasmic ratio, scant
cytoplasm, round nuclei
Desmoplastic small round cell tumor
The tumor cells appear monotonous and
undifferentiated and are small to intermediate
sized, round or polygonal, with small
hyperchromatic nuclei, inconspicuous nucleoli
and scant cytoplasm. Brisk mitotic activity and
foci of necrosis are frequently found.

6. Small round cell tumors
Differential Diagnosis
Childhood
• Lymphoblastic lymphoma
• Rhabdomyosarcoma, solid
alveolar type
• Wilms tumor, blastema-
predominant
• Peripheral
Neuroectodermal Tumor
/Ewing sarcoma
• Neuroblastoma
• Medulloblastoma
• Retinoblastoma
• Small cell osteosarcoma
Adulthood
• Small cell carcinoma
• Merkel cell carcinoma
• Desmoplastic small round
tumor
• Mesenchymal
chondrosarcoma

7. Tumors according to morphologic appearances
2. Epithelioid cell tumors
Tumors have large round-oval
to polygonal shape with a
nesting/sheeting
arrangement
Photomicrograph of undifferentiated
carcinomatous tumor composed of
epithelioid cells with nesting
arrangement and a desmoplastic
stroma separating tumor cell nests
(hematoxylin-eosin, original
magnification 10).

8. Epithelioid cell tumors
Differential Diagnosis
• Rhabdomyoma
• Granular cell tumor
• Epithelioid sarcoma
• Epithelioid variants of
leiomyosarcoma
• Epithelioid variant of
malignant peripheral
nerve sheath tumor
• Angiosarcoma
• Epithelioid
hemangioendothelioma
• Malignant extrarenal
rhabdoid tumor
• Pleomorphic
rhabdomyosarcoma
• Clear cell sarcoma
• Alveolar soft part
sarcoma
• Metastatic tumors

9. Tumors according to morphologic appearances
3. Spindle cell tumor
Tumors composed of
spindle-shaped
neoplastic cells with
variable patterns of
growth.
Photomicrograph of undifferentiated
sarcomatous tumor composed of spindled cells
with a diffuse arrangement with no reactive
stroma in between tumor cells.
(hematoxylin-eosin, original magnification 20).

10. Spindle Cell Tumors
Differential Diagnosis
Muscle
• Leiomyoma.
• Leiomyosarcoma.
• Rhabdomyosarcoma.
Nerve sheath
• Neurofibroma.
• Malignant Peripheral
Nerve Sheath Tumor.
Vascular
• Angiosarcoma
• Kaposi’s sarcoma
Myofibroblastic
• Fibromatosis
• Nodular Fascitis
• Inflammatory
Myofibroblastic Tumor

11. Spindle Cell Tumors
Differential Diagnosis
Fibrohistiocytic
• Dermatofibroma
• Dermatofibrosarcoma
protuberans
• Malignant Fibrous
Histiocytoma
Adipose
• Dedifferentiated
liposarcoma
Non-sarcomas
• Spindle cell carcinoma
• Spindle cell melanoma
Others
• Gastrointestinal Stromal
Tumors
• Solitary fibrous tumor
• Hemangiopericytoma
• Synovial Sarcoma

12. Tumors according to morphologic appearances
4. Pleomorphic tumor
Tumors with cellular
component showing
high-grade sarcoma-like
features with marked
cellular pleomorphism
and marked nuclear
atypia.
Highly pleomorphic tumor cells, including
multinucleated tumor giant cells haphazardly
scattered in the stroma with no distinct areas
of storiform growth pattern.

13. Pleomorphic tumors
Differential Diagnosis
Skin:
• Benign fibrous histiocytoma
• Carcinoma
• Melanoma
• Atypical fibroxanthoma
Limbs:
• Unclassified pleomorphic
sarcomas (“Malignant Fibrous
Histiocytoma”)
• High-grade myxofibrosarcoma
• Leiomyosarcoma
• Liposarcoma (pleomorphic or
dedifferentiated)
• Rhabdomyosarcoma
Extremities:
• Giant cell tumor of tendon sheath
Retroperitoneum
• Dedifferentiated liposarcoma
• Leiomyosarcoma

14. General approach for undifferentiated tumors
Determine a main lineage of differentiation.
• Morphologic clues such as a specific growth pattern
and nuclear or cytoplasmic characteristics can guide
to a diagnosis in some cases of undifferentiated
tumors.

15. Lineages of tumor differentiation
Main lineages of tumor differentiation are : -
1. Epithelial tumors (carcinomas)
2. Melanocytic tumors (malignant melanoma)
3. Hematopoietic and lymphoid tumors
4. Mesenchymal tumors (sarcomas)

16. Epithelial tumors
• Poorly differentiated comprises ≈ 15% to 20% of
carcinomas.
• Cytokeratin stains are excellent marker of epithelial
differentiation  strongly & diffusely expressed in
carcinomas.
• Diagnosis of carcinoma must be seriously evaluated
when an epithelioid tumor is overwhelmingly
positive for pankeratin stains.

17. Melanocytic tumors
• A diffuse strong staining with S100, CK (-) in
undifferentiated tumor is good for a melanoma.
• S100 is not specific for melanoma as it is also expressed
by some carcinomas & sarcomas.
• Unusual variants (de-differentiated
liposarcoma/mesenchymal chondrosarcoma/malignant
peripheral nerve sheath tumor) may pose a challenge to
distinguish from melanoma.
• Requires confirmation by additional melanoma markers
such as HMB-45, Melan A, tyrosinase, or NKI/C3.

18. Hematopoietic and lymphoid tumors
• If tumor is LCA (+++) and keratin (-),further workup
is directed toward classifying the lymphoma using
pan-B, pan T-cell (CD20, CD79a, and CD3) & others.
• If LCA is ±; pan-B/pan-T cell markers are –ve with
morphology still suggestive of lymphoid neoplasm, it
could be a myeloid neoplasm.
• Myeloperoxidase, chloracetate esterase, lysozyme
stains & CD117 demonstrate myeloid lineage.
• CD43 and CD68 stains are also positive in
granulocytic sarcomas.

19. Mesenchymal tumors
• Strong vimentin expression in a non-melanocytic, non-
lymphoid neoplasm is generally an indication of being a
sarcoma.
• Sarcomas may have
– small round blue-cell tumor morphology (Ewing’s sarcoma)
– spindle and epithelioid cells (synovial sarcoma)
– pure epithelioid cells (epithelioid sarcoma)
• Not all sarcomas are negative for epithelial markers.
• Epithelioid sarcoma & synovial sarcoma shows strong
staining for epithelial markers.

20. General approach for undifferentiated tumors
Specify a diagnosis.
• Once the main lineage of tumor
differentiation is determined, one can proceed
to make a much more specific diagnosis.
• In this step, clinical correlation as well as
additional ancillary studies is needed.

21. Immunohistochemical Reagents
• Intermediate Filaments
 Cytokeratins are constituents of the intermediate filaments
(IFs) of epithelial cells.
 LMW CKs, including CK8, CK18, & CK19, recognized by
antibodies CAM 5.2 or 35BH11 & a cocktail of keratins
(pankeratin), recognized by the antibody AE1/AE3, are useful
screening markers for the recognition of epithelial
differentiation.
• Vimentin is intermediate filament characteristic of
mesenchymal cells present in virtually all sarcomas
,melanomas and lymphomas.
• Neural IFs include neurofilament proteins and GFAP.

22. Immunohistochemical Reagents
• CD45 [LCA]) is surface antigen expressed by virtually all
hematolymphoid proliferations and monoclonal antibodies.
• EMA represents complex membrane glycoprotein isolated
from milk fat globules & used for detection of epithelial
differentiation, especially in sarcomatoid carcinoma or those
undifferentiated carcinomas that are negative or only focally
positive for CKs
• MOC-31 is a 41-kDa glycoprotein that is cell membrane-
based & widely distributed in epithelial cells & tumors in
many tissue sites.
• PLAP is oncofetal antigen in germ cell tumors, some
genitourinary, gastrointestinal & pulmonary carcinomas

23. Immunohistochemical Reagents
• CEA is serologic indicator of the growth of colorectal cancer.
• CD15 is hematopoietic differentiation antigen shared by
granulocytes, monocytes, Reed-Sternberg cells, subsets of
neoplastic B-cells & T-lymphocytes.
• CD30 is relatively restricted in distribution to activated
lymphoid cells, Reed-Sternberg cells, a subset of large-cell
lymphomas & particular malignant germ cell tumors.
• Calretinin is calcium-binding protein expressed by
mesothelial cells & malignant mesotheliomas.

24. Immunohistochemical Reagents
• S100 protein is calcium-flux determinant expressed by normal
melanocytes in identification of malignant melanoma, clear-cell
sarcoma, glioma, and malignant peripheral nerve sheath tumors.
• Other Melanocyte Markers include HMB-45, antityrosinase,
MART-1 (Melan-A), and PNL2
• HMB-45 is a quite specific marker for 90-100% of conventional
primary melanomas.
• Chromogranin A (CGA) is a protein indigenous to matrices of
neurosecretory granules in neuroendocrine tissues.
• Synaptophysin is integral membrane glycoprotein of presynaptic
vesicles detectable in normal & neoplastic neuroendocrine cells.

25. Immunohistochemical Reagents
• CD56 is neural-cell adhesion molecule, expressed on
surfaces of neuroendocrine epithelia, Schwann cells ,
selected neuroepithelial elements & tumors.
• CD99 is membranocytoplasmic protein expressed in
virtually all primitive neuroectodermal tumors (PNETs) and
Ewing's sarcomas.
• FLI-1 is a protooncogene in ETS family of transcription factor
loci involved in the regulation of cell growth &
differentiation, especially in elements of the hematopoietic
system & endothelia.
• NB84 is monoclonal antibody raised against
neuroblastomatous tumor tissue.

26. Immunohistochemical Reagents
• Desmin is expressed both in smooth and striated muscle
cells with variable expression in myofibroblasts.
• Muscle-Specific Actin are confined to cells with
muscular differentiation & aids in diagnosis of
mesenchymal neoplasms.
• Smooth muscle actin (SMA), aka α-actin, is a smooth
muscle specific isoform of actin, absent in striated
muscle.

27. Immunohistochemical Reagents
• CD34 is a potential indicator of vascular differentiation
highly sensitive for endothelial differentiation &
recognizes >85% of angiosarcomas and Kaposi's
sarcomas.
• CD31 is a transmembrane glycoprotein shared by
vascular lining cells, megakaryocytes, platelets &
selected other hematopoietic elements; highly
restricted to endothelial neoplasms like angiosarcoma.

28. Immunohistochemical Reagents
• Thrombomodulin (CD141) is a membrano-cytoplasmic
glycoprotein distributed among endothelial cells,
mesothelial cells, osteoblasts, mononuclear phagocytic cells
& selected epithelia.
• CD117 is cell-membrane determinant expressed by
gastrointestinal stromal tumors (GISTs), mast-cell neoplasms,
seminomas, small-cell carcinomas, primitive
neuroectodermal tumors, granulocytic sarcomas &
melanomas.
• Podoplanin is mucin-type glycoprotein localized to cell
membranes of endothelia, mesothelium, osteocytes,
glandular myoepithelial cells, ependyma, & reticulum-
dendritic cells of lymphoid organ.

29. Immunohistochemistry
• Epithelial origin
– Cytokeratins
• Melanoma
– S100
– HMB45
• Germ Cell Tumour
– AFP
– bHCG
– PLAP
• Neuroendocrine
– Chromogranin
– Synaptophysin
– CD56
• Lymphoma
– CD45
– CD20
– CD10
– CD3
• Thyroid
– Thyroglobulin
– TTF1
• Prostate
– PSA
• Sarcoma
– AML
– CD31
– CD34

30. Why Gene Expression Profiling Can Classify
Tumor Types
Adrenal Gland
Endometrium
Pancreas
Brain
Breast
Uterus
Esophagus
Gall Bladder
Kidney
Liver
Lung
Ovary
Skin
Bone
Stomach
Thyroid
Head & Neck
Prostate
Germ Cell
Soft Tissue
Lymph
Cervix
Bladder
GIST
Colon
Adrenal Gland
Endometrium
Pancreas
Brain
Breast
Uterus
Esophagus
Gall Bladder
Kidney
Liver
Lung
Ovary
Skin
Bone
Stomach
Thyroid
Head & Neck
Prostate
Germ Cell
Soft Tissue
Lymph
Cervix
Bladder
GIST
Colon
Adrenal Gland
Endometrium
Pancreas
Brain
Breast
Uterus
Esophagus
Gall Bladder
Kidney
Liver
Lung
Ovary
Skin
Bone
Stomach
Thyroid
Head & Neck
Prostate
Germ Cell
Soft Tissue
Lymph
Cervix
Bladder
GIST
Colon
• Tumors from different
origins are derived
from cells from
different
developmental
processes
• Gene expression is
distinct between
different
developmentally-
derived cell types

31. TO ISOLATE THE SITE
CK7 AND CK 20 4 DIFFERENT COMBINATION
Colon ca

33. Photomicrograph of urothelial carcinoma with
positive staining for CK7
(original magnifications x20).
Photomicrograph of urothelial carcinoma with
positive staining for CK20
(original magnifications x20).

34. Photomicrograph of Merkel cell carcinoma showing positive
staining for CK20 with a distinct paranuclear dot-like pattern
(original magnification x40).

35. Expression of EMA in Carcinomas
CK+ / EMA –
• HCC (Cam5.2+, AE1/AE3–,
CK903–)
• Adrenocortical neoplasms
(frequently negative for
all CKs)
• Most neuroendocrine
neoplasms
• Embryonal carcinoma,
yolk sac tumor
• Thyroid
CK– / EMA+
• Meningioma
• Perineurioma
• Plasma cell neoplasms
• Anaplastic large cell
lymphoma
• Popcorn or lymphocyte
predominant (LP) cells
[formerly L&H cells] in
• Hodgkin lymphoma
• RCC (sometimes)

36. Expression of Vimentin in Carcinomas
Vimentin +ve carcinomas
• RCC, clear cell type
• Endometrium
• Mesothelioma
• Salivary gland
• Thyroid
• Sweat gland
• Spindle cell carcinoma of
any site
Vimentin –ve carcinomas
• RCC, chromophobe type
• Endocervix
(adenocarcinoma)
• Lung carcinoma
• Breast
• Ovary
• Prostate
• Colorectum
• HCC

37. Clear cell papillary renal cell
carcinoma
Note papillary architecture and
clear cell cytoplasm
(hematoxylin-eosin, 200x)
CK7 is positive in tumor (CK7, 200x) Vimentin is positive in tumor

38. Photomicrograph of sarcomatoid carcinoma with cytokeratin
expression in both sarcomatous and carcinomatous areas
(original magnification x20).

39. Photomicrograph of rhabdomyosarcoma showing a diffuse
strong nuclear staining for myogenin
(original magnification x10).

40. Photomicrograph of lung adenocarcinoma with
diffuse nuclear staining for thyroid
transcription factor 1
(original magnification x10)
Photomicrograph of epithelioid mesothelioma
with nuclear and cytoplasmic staining for
calretinin
(original magnification x20)

41. Tumors showing
Coexpression of Cytokeratin and Vimentin
Carcinomas that frequently express
both Cytokeratin and Vimentin
• Anaplastic thyroid carcinoma
• Endometrial carcinoma
• Mesothelioma
• Metaplastic breast carcinoma
• Myoepithelial carcinoma
• Renal cell carcinoma
• Sarcomatoid carcinoma
• Thyroid carcinomas
Mesenchymal tumors that frequently
express both Cytokeratin and
Vimentin
• Adamantinoma
• Chordoma
• DPSRCT
• Epithelioid angiosarcoma
• Epithelioid sarcoma
• Leiomyosarcoma
• Malignant rhabdoid tumor
• Synovial sarcoma
Carcinomas that rarely express both Cytokeratin and Vimentin
•Breast carcinoma
•GI carcinoma
•Lung, non–small cell carcinoma
•Ovarian carcinoma
•Prostatic carcinoma
•Small cell carcinoma

43. Algorithmic immunohistologic diagnosis of
malignant small round cell tumors

44. Algorithmic immunohistochemical diagnosis of
malignant epithelioid cell tumors

45. Algorithmic immunohistologic diagnosis of
malignant spindle cell tumors of skin and soft
tissues

46. Algorithmic immunohistologic diagnosis of
malignant pleomorphic tumors

47. Photomicrograph of epithelioid sarcoma
showing positive staining for vimentin
(original magnification x20).
Photomicrograph of epithelioid sarcoma
showing positive staining for CD34
(original magnifications x20).

48. Photomicrograph of epithelioid angiosarcoma
showing positive nuclear staining for FLI-1
protein (original magnification x20).
Photomicrograph of seminoma, showing a
diffuse strong nuclear staining for OCT3/4
(original magnification x20).

49. Photomicrograph of desmoplastic small round cell tumor (hematoxylin-eosin, original
magnifications x10 [a] and x20 [b]), showing coexpression of cytokeratin (original magnification
x20 [c]) and desmin (original magnification x20 [d]).
a b
c d

50. 11A through D, A metastatic prostatic adenocarcinoma in the pleura at
initial presentation. Note the prostatic adenocarcinoma with clear cell
features on hematoxylin-eosin stain (11A), nuclear staining for NKX3.1
(11B), cytoplasmic PSA staining (11C), and cytoplasmic PSAP staining (11D).

51. 7A through F, An epithelioid angiosarcoma/ hemangioendothelioma from the left femur of a 76-
year-old woman. Note that the tumor cells are positive for cytokeratin (AE1/3 and CAM 5.2 [C]),
ERG (D), CD31 (E), and CD34 (F). Abbreviations: CD, cluster of differentiation; ERG, ETS related
gene (original magnification3400 [A through F]).

52. Conclusion
• Classification of a tumor beyond its apparent undifferentiated
morphology is possible in majority of cases.
• Ancillary techniques must always be correlated with H&E LIGHT
MICROSCOPIC FINDINGS.
• Never use irrelevant non-specific markers.
• Always progress from preliminary marker to most specific marker.
• Ancillary techniques helps only in guiding the diagnosis but the
overall diagnosis is based on morphology.

53. References
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