LYMPHOMA ...an understanding?

A Riot in the Lymphocyte

The adage, “Ontogeny precedes Oncogeny,” metaphorically speaking means “physiology precedes pathology” or, understanding the normal development of cells is to know the behavioral dysfunction of cancer cells.

Keeping the above statement in view, we turn our focus to the pathology of non-Hodgkin Lymphomas in general. There is a sinister thread of complexity that has weaved itself in the fabric of our understanding lately. Back in the 1980s a duo of doctors, Barbara Collins and Robert Lukes, came up with a morphologically inspired classification of Lymphomas aptly termed Lukes and Collins Classification. They peered through the microscope and determined that lymphocytes morphologically changed their looks as they matured and this was their means of classifying the disease- a real crafty and simple way of looking at things. But then others wanted to have an impact on maturing the field of Lymphoma and created a REAL classification. Ultimately WHO (World Health Organization) got in on the game and now the unwieldly classification is a phone book of tiny molecular/genetic departures from the mainstream forms of lymphoma. But this is not about casting aspersions, just an attempt at easing the burden of understanding.

Let us review the maturation process of a Lymphocyte B cell.

When an antigen strikes the human defense, the B cell lymphocyte is activated and via a co-stimulatory influence of the T-Helper cell the B-Cell makes its way into secondary lymphoid tissues; Lymph nodes, Payer’s patches in the intestines and the Spleen, where these cells multiply with abandon creating germinal centers. These germinal centers of growth within the lymph nodes also are inhabited by T-Helper cells, Follicular Dendritic cells and macrophages. The germinal cells undergo hyper-mutation, generating point mutations, some deletions and duplications in the (V) variable regions of the immunoglobulin genes. Those mutations that are disadvantageous (premature stop codons, non-sense frame-read etc.) mark the cell for apoptosis (cell death), ultimately being consumed by the macrophages. For those cells surviving, in the germinal center “dark zone” further indoctrination towards immune surveillance takes place through positive selection via mutation and proliferation. The positively selected B-Cells then migrate to the “Light zones” where interaction with follicular Dendritic cells and T-Helper cells takes place. This migration back and forth continues until the B cells have developed a memory against the offending antigen and are now called memory B-cells. The memory B cells ultimately transform into plasma cells.

As one can see, there is a charted course of multiples of transformational gene mutations of the germinal B-cells in their act to defy the enemy (antigen). This is akin to arming the recruits with weapons and organizing a battalion in a war scenario.

From basic physiology we step into the domain of pathology. If the cell proliferation and growth is not in check and a certain extra mutation escapes the self-immolation (apoptosis) process by rogue genes then the art of war against an enemy becomes a rebellion within. Yielding to the demands of these erratic and disruptive calls, the heart of these lymphocytes, hammers against their chest walls, as they transform into the rogue warriors. From the lightening strike (antigenic assault to the signal transduction within the cell) the thunder hammers at the interior of the cell and makes it dance to the rhythm of the unseen dynamo.

To start with understanding how this train runs, look at In-Situ Follicular Nodes (ISFN) found in abnormal follicles within the lymph nodes, which have fewer chromosomal copy number abnormalities than follicular lymphomas. These abnormalities seem juxtaposed with the BCL-2 gene rearrangement expression (BCL-2 creates immortality by preventing apoptosis). Some ISFN cells transform into actual lymphomas as they acquire a myriad of genetic aberrancies with proliferation and coopting the BCL2 gene they achieve immortality. So, a benign process if allowed to fester, confronts the reality of being stabbed by venomous spear head and turned into a poisonous black widow. Do all ISFN turn malignant? No! but some do and it is the game of chance when and if the spear tip appears.

Various Lymphomas and their frequency of occurrences:

DLCBCL (Diffuse Large B-Cell Lymphoma)  = 30-40%

FL          (Follicular Lymphoma)                      = 20%

MCL       (Mantle Cell Lymphoma)                  = 5%

BL          (Burkitt Lymphoma)                          = 2%

MZL       (Mantle Zone Lymphoma)                = 5-10%

TCL        (T-Cell Lymphoma)                           = 15%

Others                                                                = 10-15%

To keep the chain of understanding going, it pays to look at the “Breast Implant lymphoma." Why look at a rare form of lymphoma first? The answer is simple…it gives us a look inside the workings of an antigenic (irritant) assault. It begins with the idea that breast implant triggers the immune cells to undergo replication and in that replication, what slings and arrows of gene mutations are thrown at it, unchecked, result in a stimulated growth pathway deviation to make the cell lose its way. There is the cautionary tale. Interestingly in the implant lymphoma, if the transformed lymphomatous cells stay in the seroma (fluid) and do not gain a foot-hold on the capsule-edge, the treatment is relatively benign; removal of the implant and all is well. If the lymphoma cells do invade the capsule, then chemotherapy is the treatment of choice with ever-decreasing limits on patient survival. Gaining the beach-head, so to speak, is a function of further mutation and aggressive nature of the beast!

The Follicular lymphomas (FL) have a relatively long biological history from inception. Yet, in a third of the cases, molecular challenges (gene mutation) can convert them into a more aggressive form of DLBCL. Therapeutic successes may be marred by lead-time bias given survival in FL may be between 10-20 years. However, Lenolidamide, an immunomodulatory agent (a derivative of Thalidomide, T-cell immune synapse enhancer and NK-cell/T-cell effector with anti-proliferative effects), is undergoing trials currently to determine action and efficacy against such conversions. What triggers the third of these FL cases into more aggressive form of lymphomagenesis is a constant source of views and news within the experts and is a subject of much discussion. The idea of using Lenolidamide as a maintenance prevention is more like cutting the vine at its base of the tree before it kills it, leaf by withering leaf.

Addressing the Diffuse Large B Cell Lymphoma or DLBCL is also a subject of many opinions, equally for the efficacy of therapy and for the aggressiveness of the disease. Molecular underpinnings of DLBCL show 3 subtypes: GCB (Germinal Center B Cell) DLBCL, ABC (Activated B-Cell) DLBCL and PMBL (Primary Mediastinal B Cell Lymphoma). The 30%-40% of GCB-DLBCL have a t(14:18) chromosomal translocation while 1/3 have c-rel amplification, 1 in 5 have EZH2 mutations (EZH2 is a master regulator of the GCB phenotype and cooperates with BCL6 to mediate lymphomagenesis in GCB DLBCL) and 1 in 10 have PTEN deletions.  

Interestingly the ABC DLBCL has an activated NFkB signaling pathway (its diagnostic hallmark) and thus is more aggressive and less responsive to therapeutic interventions, while in the GCB DLBCL and PMBL a locus on the short arm of chromosome 2 is amplified and these types are rendered more stable and amenable to interventions. Also noted in DLBCL is an amplification of the long arm of chromosome 18 (MYC gene) and overexpression of the anti-apoptotic BCL-2 gene. A dual expression of MYC and BCL-2 in 1 in 5 cases of DLBCL with concomitant inactivation of the TP-53, yield a very poor prognosis.

Logically this takes us to the argument posed in the beginning; a cumulative burden of genetic mutations change the morphology as it does behavior within the transformed cell and leads to different manifestation of the disease and differing responses to therapy due to a multiplicity of upregulated pathways. Addressing a single pathway in therapy is a model for failure in improving survival or cure. Addressing multiple pathways is difficult because of lack of data about cross talk between pathways and other epigenetic forces in play. Interestingly, a select version of DLBCL with IFR4 gene rearrangement seem to affect children and young adults only. (The suggestion here might be timing related to active genes in early phases of human growth). It appears that the driver mutations direct the path and discourse of the traffic. Therapy in regionally limited DLBCL has changed from 3 cycles of chemotherapy with Involved Field Radiation Therapy (IFRT) due to relapses in 7-9 years in areas outside of the IFRT region, to a more aggressive chemotherapy (R-CHOP) duration. The best chance of cure in relapsed patients today is high dose chemotherapy followed by a Stem Cell Transplant. DLBCL is a remarkable Janus-like face with many evils lurking within.

Another Lymphoma that originates in the Germinal Center is the Burkitt’s Lymphoma (BL). The difference is the molecular switch. In BL, the c-myc translocation t(8:14) is the key oncogenic event. The c-myc protein and its target genes are the hallmark of this entity. This underscores the many molecular insults and their varied influence on the lymphocytic cell as it transitions through the germinal center and sometimes ping-pongs back and forth with the mantle zone acquiring wayward behaviors. Mutations in the transcription factor TCF3 or its negative regulator ID3 occur in about 70% of sporadic BL and 40% of endemic cases. (TCF3 promotes survival and proliferation in lymphoid cells by activating the B-cell receptor/phosphatidylinositol 3-kinase signaling pathways and modulating the expression of cyclin D3, which is also mutated in 30% of BL). What is even more interesting is that the standard R-CHOP-like regimen (used extensively in DLCBL therapy) are not as effective as more intensive regimens.

Genetic Overlap

Mantle Cell Lymphoma by the very term represent lymphocytes that are transformed in the mantle zone of the lymphoid tissue. Transformed this way, they are SOX11+ and more aggressive in behavior. However, if these cells undergo a somatic hyper-mutated state in the Germinal Center (via ping pong), they are SOX11- and are much more stable with longer duration of survivals. However as is common to other lymphomas , SOX11- can develop molecular rearrangement and lead an aggressive path in growth and curtail human survival.

Back to the old classification espoused by Lukes and Collins, the transformational process makes sense. Lymphomas are transformed lymphocytes via accumulating driver or passenger mutations, and these impart varying degrees of biological behaviors and life spans. As the lymphocyte continues the path of growth, it once started, the continuously building burden of mutations that avoid self-kill, lives for want of more mutation to become angrier, harder to control killer cell!

Not mentioning the latest greatest endeavor against the Non-Hodgkin Lymphoma would leave us all unsatiated, me especially. First let us talk about the therapy itself.
What is CAR-T therapy?
The idea being to utilize the various antigens expressed on the cancer cells surface and by virtue of antagonizing and stimulating the killer T-Cells creating an armada of immune cells against the cancer.

Chimeric Antigen Receptors are recombinant receptor constructs with an extracellular exposed single chain variable fragment (scFv) joined together with a hinge-spacer peptide and a transmembrane domain. The latter is attached to a intracellular T-Cell signaling and co-stimulatory domains (to excite the stimulatory process) to develop cytotoxic memory against the exposed cancer antigen. Imagine the whole construct as a fork sticking through a piece of flesh exposing its points on the (outside) and the handle on the inside.

The 2016 debacle for Juno ( http://blogs.sciencemag.org/pipeline/archives/2016/11/23/more-juno-car-t-deaths ) (Juno's construct typically uses a single chain variable fragment, or scFv, to recognize a protein of interest-CD-19 in this case).and the hit with Kite ( http://ir.kitepharma.com/releasedetail.cfm?ReleaseID=1014817 ) illustrate the nuance of a biological constructs. One minor change and what works turns into what harms (5 deaths in the JCAR015 trial against adult Acute Lymphocytic Leukemia patients). Kite's construct however showed favorable and somewhat striking data of a 36% Complete Response among heavily pretreated and resistant DLCBL patients (n=77) and 71% Complete Remissions in PMBCL patients (n=24). The sticking point remains the Cytokine Storm in 13% of cases that can be deadly if not handled appropriately and in Juno's developed CAR-T therapy; Cerebral edema. Whether that was due to their CAR construct or the patient age still remains a debatable point.

What is very encouraging is the ability to manipulate the killer Immune T-Cells into doing the work of eliminating the rogue cells, rather than using Cell-Cycle arresting mechanisms of chemotherapy and direct kill via external beams of radiation. The 36% Complete Response is of significance and an important issue to remember.

Not all cancer cells express a large cohort of the targeted antigens to address. This weakness in the cancer (64% of the 77 patients that did not respond with CR) also suggests the cancer cell's strength in becoming chameleons by avoiding CAR-T therapy assault in not expressing the antigen.

Progress in Lymphoma as in all other cancers is one of a slow march to the finish line with many a pot-holes on the way. But march, we must to reach that finish line!

REFERENCES:

Lukes and Collins

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2149563/pdf/brjcancersuppl00060-0217.pdf

REAL Classification

https://training.seer.cancer.gov/lymphoma/abstract-code-stage/morphology/real.html

WHO Classification

http://www.bloodjournal.org/content/127/20/2375?sso-checked=true

Jegalian AG, Eberle FC, Pack SD, et al. Follicular lymphoma in situ: clinical implications and comparisons with partial involvement by follicular lymphoma. Blood 2011;118(11):2976-2984.

Pillai RK, Surti U, Swerdlow SH. Follicular lymphoma-like B cells of uncertain significance (in situ follicular lymphoma) may infrequently progress, but precedes follicular lymphoma, is associated with other overt lymphomas and mimics follicular lymphoma in flow cytometric studies. Haematologica 2013;98(10):1571-1580

Kritharis A, Coyle M, Sharma J, Evens AM. Lenalidomide in non-Hodgkin lymphoma: biological perspectives and therapeutic opportunities. Blood. 2015;125(16):2471-2476

Thieblemont C, Delfau-Larue MH, Coiffier B. Lenalidomide in diffuse large B-cell lymphoma. Adv Hematol. 2012;2012:861060.

Mamessier E, Song JY, Eberle FC, et al. Early lesions of follicular lymphoma: a genetic perspective. Haematologica 2014;99(3):481-488.

Frick M, Dörken B, Lenz G. The molecular biology of diffuse large B-cell lymphoma. Therapeutic Advances in Hematology. 2011;2(6):369-379.

Love C, Sun Z, Jima D, et al. The genetic landscape of mutations in Burkitt lymphoma. Nat Genet 2012;44(12):1321-1325

Miranda RN, Aladily TN, Prince HM, et al. Breast implant-associated anaplastic large-cell lymphoma: long-term follow-up of 60 patients. J Clin Oncol 2014;32(2):114-120

http://www.cancer.org/cancer/non-hodgkin-lymphoma/about/types-of-non-hodgkin-lymphoma.html

Karube K, Campo E. MYC alterations in diffuse large B-cell lymphomas. Semin Hematol 2015;52(2):97-106.

Lenz G, Wright GW, Emre NC, et al. Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Proc Natl Acad Sci USA 2008;105(36):13520-13525

Béguelin W, Popovic R, Teater M, et al. EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell 2013;23(5):677-692

Miller TP, LeBlanc M, Spier C, Chase E, Fisher RI. CHOP alone compared to CHOP plus radiotherapy for early stage aggressive non-Hodgkin's lymphomas: Update of the Southwest Oncology Group (SWOG) randomized trial. Blood 2001;98:724a-725a

Sehn, L. H., & Gascoyne, R. D. (2015). Diffuse large B-cell lymphoma: optimizing outcome in the context of clinical and biologic heterogeneity. Blood, 125(1), 22-32

Jares P, Colomer D, Campo E. Molecular pathogenesis of mantle cell lymphoma. J Clin Invest 2012;122(10):3416-3423.

Brudno JN, Kochenderfer JN. Toxicities of chimeric antigen receptor T cells: recognition and management. Blood.2016;127:3321-3330.