PTLD Panels for Copath: March 26, 2006

I am currently uploading several panels into Copath for the workup of PTLD specimens. They will be available for use with the abbreviations shown below in the near future. In the meantime, I am sending out an explanation and background of these panels along with the individual stains, so you can order them individually as appropriate if a case should come up. Feel free to use them as you see fit, I would encourage you to apply them in the algorithm I am providing in this e-mail. The information will also be uploaded to the administrative page of our website so it will be there for easy access.

There are 5 separate panels which I have named PTLDScreen, PTLDBcell, PTLDTcell, PTLDHD, and PTLDFISH. They are designed for initial screening, workup of B cell PTLD, T cell PTLD, Hodgkin's-like PTLD, and a FISH panel to be used, when tissue permits, for additional molecular workup to provide additional classification and prognostic information.

Algorithm:

The basic flow is to first use the PTLDScreen panel to help answer the question "Is it a PTLD/lymphoma?" The screen panel also provides a number of blank slides for further workup (ordered up front in order to minimize tissue waste and to allow selection of an appropriate follow-up panel depending upon initial results.

In most cases the screen panel should provide enough information to sign out the case and provide useful clinical information. The results of the additional panels, if any, can follow as addendums.

If the lesion looks like a large cell lymphoma and is positive for B cell markers, you can follow up with the PTLD B cell panel and, if enough tissue, the PTLD FISH panel. If there are occasional large ugly cells in a rather inflammatory looking or "activated" looking mononuclear background, particularly if the large cells are scattered in small numbers and are EBER positive, you would select the PTLD Hodgkin's panel and, again, optionally, the FISH panel. Finally, if the lesion appears to be a T cell proliferation, you would want to consider first if a) it is simply an inflammation, and b) if there are scattered atypical looking B cells, i.e., it could be a T cell rich B cell lymphoma. If you feel you might be dealing with a neoplastic T cell proliferation, then you would follow up with the PTLD T cell panel.

Details of each of the panels are as follows:

PTLD screening panel ( order as aPTLDScreen): CD3, CD20, CD79a, EBER, kappa, lambda, negative control, 20 blanks

This simple panel addresses the issue of whether or not EBV is present, whether it is likely to be a B cell tumor and clonal, and if it is CD20 positive and thus potentially treatable with Rituximab (anti-CD20).

There are two B cell markers, CD20 and CD79a. CD79a is included since it has a wider range of positivity throughout B cell maturation, and might be positive in some cases where CD20 is negative (such as a plasmacytoma, or “plasma cell rich PTLD”). Single kappa and lambda immunostains have a faster turnaround time than the double in situ, and are better suited for an initial screen, they will also provide evidence of B cell origin if positive. EBER is the most sensitive marker of EBV presence and is ordered as the only indicator for the virus. However, if there is a lot of necrosis you may wish to add LMP1, which is a viral protein that persists in necrotic areas when EBER becomes undetectable.

PTLD B cell panel (order as aPTLDBcell): CD5, CD10, bcl-6, mum-1, bcl-1, bcl-2, bcl-10 (the last stain to be added when developed)

The purpose of this panel is to help distinguish some of the various types of B cell lymphomas, and to provide a maturation phenotype for the usual diffuse large B cell lymphoma. This panel is presented in simplified form in the table below. It should be remembered that this is a simplification and that exceptions exist; a given case may not match the panel exactly. Reference to the WHO fascicle as well as Hematopathology is suggested to provide more sophisticated analysis. However, this approach should be useful for a large proportion of cases:

	CD5	CD10	Bcl-6	Mum-1	Bcl-1	Bcl-2	Bcl-10
DLBCL	+/-	+/-	+/-	+/-	-	+/-
MALToma	-	-	-		-	+	+
Mantle Cell	+	-	-		+	+
Burkitt	-	+	+			-
Follicular	-	+	+	+/-		+
Plasmacytoma	-	-/+ -> -	-		-/+	-
CLL/Small	+	-	-		-

As you can see, the diffuse large B cell tumors (DLBCL) are heterogeneous. Within this, several maturation phenotypes have been described, and these can be partially summarized as follows:

CD10	Bcl-6	Mum-1	Type
+	-	-	Germinal center
+	+	-	Germinal center
-	+	-	Germinal center
-	-	+	Non-GC

In standard lymphomas, it has been suggested that bcl-2 positivity in the non-GC type is an adverse prognostic sign. In our series, many of the EBV negative PTLD tend to have a germinal center phenotype.

A few other comments- Bcl10 is not available at present; hopefully we can incorporate it in the future. It has been suggested as a marker that can pick up MALTomas with underlying genetic abnormalities.

PTLD FISH panel (order as aPTLDFISH): FISH for c-myc, bcl-2, and bcl-6

(These stains are done in Dr. Urvashi Surti’s laboratory at Magee. At present, order 4 blanks and one H&E, map out the area of interest, and request the tests by getting a lab requisition (I have given this to Joyce) and filling in the tests. We are working on automating this procedure).

This optional panel contains markers with prognostic implications for B cell lymphomas, and it is suggested to use this on B cell PTLD when tissue permits. A c-myc translocation would support a diagnosis of Burkitt lymphoma, but it can be seen in other conditions as well, so correlation with the histology, etc. is needed. However, our early studies showed that the presence of this translocation was a bad prognostic indicator in PTLD. Bcl-2 translocation corresponds to the t(14;18)(q32;q21) translocation seen most commonly in follicular lymphomas, but not specific for this. Since bcl-2 is often upregulated by EBV, immunohistochemical detection of this protein may arise from the virus infection alone. Demonstration of upregulation in association with an underlying translocation would suggest a different pathogenesis, more similar to standard lymphoma. Similarly, bcl-6 expression may simply reflect a "germinal center" level of maturation, but expression in association with translocation of the gene would imply a different condition and in the case of standard lymphoma has been associated with a worse outcome in some reports.

PTLD Hodgkin's-like (order as aPTLDHD): CD15, CD30, CD45, Pax5, perforin/granzyme B, ALK

The relationship of Hodgkin's like PTLD to true Hodgkin's disease is problematic. This panel is designed to help differentiate the immunophenotypes that are typically expressed by classical Hodgkin's disease, anaplastic large T-cell lymphoma, and T cell rich B cell lymphoma. It should also be noted that the phenotype of T cell rich B cell lymphoma is essentially the same as that of nodular lymphocyte predominant HD. A simplified version of the immunophenotypes is listed below, with the qualification that reference should be made to more detailed sources of expertise when interpreting individual cases.

	TCRBCL	Classic HD	ALCL
CD15	-	+ (most)	- (most)
CD30	- (most)	+	+
CD45	+	-	+/- (most +)
Pax5	+	+ (most)	-
Granzyme B	-	- (most)	+ (most)
ALK	-	-	+/- (most +)
EMA	+ (most)	-	+ (most)

PTLD T cell (order as aPTLDTcell): TCR alpha beta and TCR gamma delta rearrangement, IGH rearrangement, EBV clonality 10 blanks

(These assays are done by Dr. Kant’s laboratory, and Joyce has the requisition sheets for this lab).

The presumption here is that a paraffin sample is available, and there is no evidence of B cell presence, yet the cells look atypical and a T cell neoplasm is suspected.

These lesions are very rare, and the most significant marker of a neoplastic process would be to show rearrangement of the T cell receptor. Therefore, this followup panel is designed to evaluate the existence of alpha-beta or gamma-delta T cell receptor rearrangements. Immunoglobulin heavy chain gene rearrangement is included both as a (presumably negative) control and to exclude any small clonal B cell component. EBV gene rearrangement is also included as an additional marker of clonality, since the virus may show a clonal component, and in some cases this may be the only evidence of clonality (FYI, this can also be applied if you should happen to have an EBV positive non-hematopoietic tumor such as smooth muscle tumor). If no evidence of virus has been shown by prior studies, you should delete this last test. Additional blank slides are ordered up front to further evaluate the phenotype (tissue permitting), if desired, based on the outcome of the molecular genetic studies. Very rare NK cell PTLD have been described and it should be recalled that these tumors will not show rearrangements of the T cell receptor genes.

The immunophenotypes of T cell/NK lymphomas are heterogeneous, so a single immunostain panel is not practical. A simplified table (separate Excel spreadsheet) is included as a guide if you are confronted with such a case. As always, you should consult more detailed sources when working up a case.

Good luck!