Identifying and Over Coming Immune Barriers at the Level of the Tumor Microenvironment..Melanoma .Jim Breitfeller

Eleventh International Conference on Progress in Vaccination Against Cancer

Dr. Thomas Gajewski
University of Chicago,Chicago,IL,USA

"Immunotherapeutic approaches for the treatment of melanoma, such as tumor antigen-based vaccines, can frequently boost immune responses. However, clinical responses as measured by tumor shrinkage are seen in only a minority of patients. This observation has prompted careful analysis of the tumor microenvironment for biologic correlates to clinical response and also to identify mechanisms of tumor resistance. Patients with advanced melanoma treated with antigen-specific vaccines had pre-treatment tumor biopsies analyzed by gene expression profiling. Supervised hierarchical clustering was performed based on clinical outcome. An expanded bank of tumors was analyzed to increase the sample size and better understand gene patterns.
Two major categories of melanoma metastases have been observed.

One subgroup of patient has an inflamed phenotype that includes expression of chemokines, T-cell markers, and other immunoregulatory factors. Clinical responders to melanoma vaccines appear to fall within this subset. This group also contains the highest expression of negative regulatory factors, including PD-L1, IDO, and FoxP3, suggesting that these immunosuppressive mechanisms may dominantly inhibit anti-tumor –cell function in those patients. In addition, absence of B7 expression supports classical T-cell anergy. Preclinical experiments have confirmed a critical role for these mechanisms in limiting anti-tumor T–cell efficacy in vivo, giving candidate treatment strategies for translation back into the clinic.

A second subset of patients is represented by tumors which are non-inflamed and lack chemokines for T cell recruitment. Therefore, a major barrier in these cases appears to be failed T –cell migration into tumor sites. Experimental strategies to augment T-cell migration can have important anti-tumor effects in preclinical models. The presence of the "inflamed" gene signature was associated with a type I IFN transcriptional profile, and murine experimental models have confirmed a critical role for type I IFN signaling in promoting adaptive immunity."


So,In the first subset, tumors had a suppresive nature that may be over riddden by Anti-CTLA-4 (Yervoy) and or Anti-PD-1 Therapy

The second subset was missing the "danger signal" inflammatory cytokines and chemoattractants most likely due to STAT3 signaling from the Tumor.

Stimulation of Toll-like receptor 4 (TLR-4) activates macrophages and results in the release of TNF-alpha. It is hypothesized that melanoma inhibits macrophage activation by suppressing TLR-4 signaling.








Cytokines are small proteins which allow cells of the immune system to communicate with one another via cytokine receptors expressed at the cell surface.
Activated macrophages defend against tumors by secreting cytokines to recruit secondary immune cells, presenting antigen to T cells, and by direct tumor cytotoxicity. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to melanoma cells, and produce less superoxide, nitric oxide, and tumor necrosis factor-alpha (TNF-alpha) than those from nontumor-bearing mice. Similar impairment of macrophage activation occurs in vitro using media harvested from cultured melanoma cells.

Activated Macrophages secrete the following cytokines under different conditions:
IL-1,IL-12,IL-6, IFN-gamma/alpha/beta and TNF-alpha


IL-6


Interleukin 6 is a pro-inflammatory cytokine and is produced in response to infection and tissue injury. IL-6 exerts its effects on multiple cell types and can act systemically.

IL-6 stimulates liver secretion of acute phase proteins
IL-6 stimulates B-lymphocytes to produce antibodies
IL-6 in concert with IL-1b causes T-cell activation
IL-6 induces STAT 3 Signaling
IL-6 Plus TGF-b induces the Th17 cell phenotype


IL-1 beta

Interleukin-1b is a pro-inflammatory cytokine which is secreted by macrophages activated by a number of stimuli including TNF-alpha, bacterial endotoxin and IL-1b itself.
IL-1b exerts its effects on many different cell types locally at the site of production and systemically (at a distance).


IL-12


Interleukin-12 is a heterodimer consisting of a p35 and a p40 subunit. Both subunits are required for receptor binding and biological activity.
IL-12 stimulates growth of activated Natural Killer (NK) cells, CD8+ and CD4+ T- cells.
IL-12 increases NK and T-cell g-IFN production which shifts T-cell differentiation towards a Th1-type response.
IL-12 increases NK production of TNF-alpha which can act synergistically with IFN-gamma.
IL-12 suppresses IL-4 induced IgE production.


TNF-alpha


Tumor Necrosis Factor alpha is made by many other cells as well as macrophages, which are major sources, especially after priming by Interferon gamma.
TNF-alpha initiates a cascade of cytokines which mediate an inflammatory response. TNF-alpha effects are mediated through two types of receptor, a 75kDa TNFR-a receptor and a 55kDa TNFR-b receptor.
TNF-alpha regulates the expression of many genes in many cell types important for the host response to infection.

IFN-gamma/beta

Macrophages, and many other cells produce these Type I interferons which act as immunomodulatory, as well as antiviral cytokines. Distinct receptor from interferon gamma, mediates overlapping or competing effects on macrophages. Cellular signalling pathways involve Jak/Stats, and other pathways.

So, if Melanoma suppresses Macrophage Activation, then the tumor microenviroment is missing IL-6, IL-1b and other cytokines.

If you look at the above micrographs, you will see that the two patients that had relapsed (10710 and 10737) Had IL-1b and IL-6 missing. The macrophages were not activated!!!! The "Danger Signal" known as inflammation was missing!





The missing combination of IL-1 and IL-6 meant there is no T-cell activation. And no induction of the Th17 phenotype. It is now becoming a lot more clearer based on Dr. Gajewski's findings.


Now might be the time for a critical re-evaluation of our overall approaches to targeting STAT3 and for developing new models for disrupting the protein in order to accomplish the goal of delivering clinically useful direct STAT3 inhibitors as novel anticancer agents in a timely manner.






“It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.”

~Charles Darwin~
Take Care,
Jimmy B

Characteristics of the Innate Immune ResponseMelanoma ..Jim Breitfeller

The immune system protects against pathogens that penetrate the physical barriers of the skin and mucous membranes lining the digestive, respiratory, and reproductive tracts. It is subdivided into the innate and the adaptive immune systems. These two systems work differently, but collaboratively, to provide a powerful defense against microbial invaders. Increasing evidence suggests that the immune system also plays a role in detecting and eliminating tumor cells, and can be manipulated therapeutically against cancer.

Innate Immunity

Characteristics of the Innate Immune Response

The innate immune system provides a rapid but nonspecific response to the most common foreign pathogens.1 This system, in some form, is present in all animals, and some elements of it have existed for more than 500 to 700 million years.2 Cells of the innate immune system have specialized receptors (eg, Toll-like receptors) that recognize molecular structures or patterns that are characteristic of—and often indispensable parts of—common pathogens.3 As such, they recognize these pathogens immediately, even without having encountered them previously, and can react promptly. Disadvantages of the innate immune system are that it can recognize only a limited number of molecules, has limited ability to recognize viruses once they have entered normal cells, and has no "memory" and therefore cannot provide lasting protective immunity against these molecules.
The innate immune system is often sufficient to protect against the small quantities of common pathogens humans come into contact with on a day-to-day basis.2 When additional "help" is needed, the innate immune system activates and modulates the adaptive immune system.2,3
Cells of the Innate Immune Response

Macrophages.
Macrophages are the "sentinels" of the immune system. Present in large quantities under the skin, in the lungs, and in the tissues surrounding the intestines, these cells are in key positions to detect microbes where they first enter the body.2 The name macrophage means "large eater," and its primary responsibility is to rid the body of debris as well as pathogens, largely but not exclusively via phagocytosis.2,3

In their usual resting state, macrophages sample their environment and serve as "housekeepers," scavenging dead cells, cellular debris, oxidized lipoproteins, and other normal cellular by-products.2,3 When exposed to certain cytokines (eg, interferon gamma) released by other immune cells, such as helper T-cells and natural killer cells, macrophages become primed or activated. The activated macrophage engulfs a pathogen, containing it in a phagosome, which then fuses with a lysosome full of antimicrobial enzymes that destroy the pathogen. After digesting the pathogen, macrophages release various chemicals that increase the flow of blood to the area, trigger capillaries to allow extravasation of blood cells into the affected tissue, stimulate pain signals from nerves in the area, and release cytokines that facilitate communication with other cells in the immune system. As will be described in more detail later, activation also causes the macrophage to upregulate major histocompatibility complex (MHC) class II receptors on its cell surface, and protein fragments from the invading pathogen are transported to the MHC receptors and presented there for detection by helper T-cells and natural killer cells.2

Macrophages also have cell surface receptors (eg, the Toll-like receptors mentioned above) that enable them to detect molecules (eg, lipopolysaccharide, mannose) that are not normally found on human cells but are common cell wall components in typical pathogens.2 When a macrophage detects such molecules, it becomes "hyperactivated." The macrophage stops proliferating and becomes a virtual killing machine, growing larger and increasing the number of lysosomes and its rate of phagocytosis. It also actively migrates toward a foreign invader, even extending out "feet" to grab it up.2 In this state, macrophages also secrete tumor necrosis factor (TNF) alpha, interleukin (IL) 1, IL-6, and IL-8. These inflammatory cytokines help kill tumor cells and virus-infected cells and activate and summon other cells in the immune system.2,3

Neutrophils.
Neutrophils are highly phagocytic cells. Produced from myeloid precursors and with a lifespan of only 2 to 5 days, these cells circulate through the bloodstream, where they are within easy reach of all cells in the body until they are summoned.2,3

Cytokines and chemokines released by macrophages and mast cells draw neutrophils to the area of infection.2,3 It takes only about 30 minutes for neutrophils to exit the bloodstream and arrive fully activated at the site of an infection.2 Once there, they not only perform phagocytosis, they secrete cytokines (eg, TNF) to summon other immune cells and release various antimicrobial products from granules into the extracellular space.1-3

Mast cells and eosinophils.

These cells lie beneath exposed surfaces of the body (ie, the skin and mucosal barriers) and can survive for years. Their best-known function is to provide a defense against parasites. Mast cells are phagocytic and also contain granules of chemicals, most notably histamine. Eosinophils are poor phagocytes but do carry granules. When a mast cell or eosinophil detects a parasite, it "degranulates," that is, it unloads the chemicals.

Contributing Writer: Lauren Cerruto
Contributing Editor: Bernard A. Fox, PhD
Editor-in-Chief: Jeffrey S. Weber, MD, PhD





Take Care,

Jimmy B

Ah haaaaa!!!!!!!!! Road map to Recovery Melanoma..Jim Breitfeller

Follow the Yellow Brick Road!!!



"What we can learn from individual patients is often overlooked in oncology," he said, adding that many of these remarkable cases have led to the development of new treatment strategies for melanoma such as vaccinations against specific antigens and bone marrow transplantation. "From clinical observation, we can learn a lot from these remarkable cases."



Alan Houghton M.D., Chief of Immunology at Memorial Sloan-Kettering Cancer Center, New York





Well here are all the outside pieces to the Melanoma Puzzle. Now this may not work for every one but it did work so far for me. My last hurtle was how did my Immune system have the right antigen to present.



Without that piece of the puzzle, there would be no effective response.



Bobby Luker, this is for you and everyone who is still on the Yellow brick Road.







Take care



Jimmy B

Newswise Medical News | Immunologists Identify Biochemical Signals That Help Immune Cells Remember How to Fight Infection

Newswise Medical News | Immunologists Identify Biochemical Signals That Help Immune Cells Remember How to Fight Infection

Shared via AddThis

Blame it on the Tregs!!!! Melanoma.. Jim Breitfeller

As you Know I have been off line lately. I have been following up on some research that I have been doing and actually fired another letter to Dr. Rosenberg. It all has to do with Tregs (CD4+ CD25+ T-cells). They are Known for surpressing the immune response that we want. In Adoptive cell transfer done at the NCI, Rosenberg and colleagues do Lymphodepleting step before injecting the patient with (TILs) tumor-infiltrating lymphocytes. This is a systemic chemotherapy that transient elimination of host immune system includes:


suppressive cells
regulatory T (Treg) cells,
myeloid derived suppressor (MSC) cells,
Natural killer T cells (NKT) cells
cells competing for cytokines
transiently increased function of antigen presenting cells (APCs)

By doing this It gives the TILs a fighting chance of survival and activation. Il-2 is added for maintance and survival of the TILs.

So I came across a paper "CTLA-4 Blockade Confers Lymphocyte Resistance to Regulatory T-Cells in Advanced Melanoma: Surrogate Marker of Efficacy of Tremelimumab?"

Author by:
Cédric Ménard1,2, François Ghiringhelli1,4,5, Stephan Roux1,2, Nathalie Chaput1,2, Christine Mateus3, Ursula Grohmann6, Sophie Caillat-Zucman7, Laurence Zitvogel1,2 and Caroline Robert1,3

Authors' Affiliations: 1 Center of Clinical Investigations, CBT507, 2 Institut National de la Sante et de la Recherche Medicale U805, and 3 Department of Medicine, Dermatology Unit, Institut Gustave Roussy, Villejuif, France; 4 Department of Medicine, Centre Georges Francois Leclerc, 5 CRI Institut National de la Sante et de la Recherche Medicale 866, Faculté de Médecine, Dijon, France; 6 Department of Experimental Medicine, University of Perugia, Perugia, Italy; and 7 Institut National de la Sante et de la Recherche Medicale U561, Hôpital St. Vincent de Paul, Paris, France

Purpose: Anti–CTL antigen-4 (CTLA-4) monoclonal antibody (mAb) has led to encouraging antitumor activity associated with immune-related adverse events in patients with heavily pretreated melanoma. However, mechanisms of action and surrogate immunologic markers of efficacy have not been reported thus far.

Experimental Design: We monitored the immune responses of 10 melanoma patients included in a phase II clinical trial, which evaluated the efficacy of a second line of therapy of tremelimumab anti–CTLA-4 mAb in patients with metastatic melanoma. The frequency of blood leukocyte populations in association with T cell and regulatory T cell (Treg) functions were evaluated.

Results: Prior to therapy, patients with advanced melanoma presented with a severe CD4+ and CD8+ T cell lymphopenia associated with blunted T-cell proliferative capacities that could be assigned to Treg. Tremelimumab rapidly restored the effector and memory CD4+ and CD8+ T-cell pool and TCR-dependent T-cell proliferation that became entirely resistant to Treg-mediated suppression. Progression-free survival and overall survival was directly correlated with the acquisition of a biological response defined as the resistance of peripheral lymphocytes to Treg-inhibitory effects (obtained in 7 of 10 patients).

Conclusion: CTLA-4 blockade seems to be a valuable strategy to revive reactive memory T cells anergized in the context of stage IV melanoma, and our work suggests that memory T-cell resistance to Treg resulting from anti–CTLA-4 treatment might be a biological activity marker for tremelimumab in patients with melanoma.

I am the process of obtaining this Paper. I believe it holds another piece of the Melanoma puzzle.


Take Care

Jimmy B

PUBLIC MEETING: CYTOKINE THERAPIES: NOVEL APPROACHES FOR CLINICAL INDICATIONS Melanoma Jim Breitfeller

PUBLIC MEETING: CYTOKINE THERAPIES: NOVEL APPROACHES FOR CLINICAL INDICATIONS
DATE: March 26-27, 8:00 a.m.
LOCATION: NYAS Conference Center, 7 World Trade Center, 250 Greenwich St. at Barclay St., New York City, NY

CONTACT: Raymond Donnelly, FDA CDER, 301 827-1776,

Email: Raymond.Donnelly@fda.hhs.gov;

Kristy Kilpin,
NYAS, 212 298-8639, Email: kkilpin@nyas.org.

This event is a public, two-day forum that will focus on the clinical use of cytokines and cytokines antagonists as therapeutic agents for the treatment of human diseases, including cancer and autoimmune disorders such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.

FDA upcoming Meetings



Guess What I did, You got it.

I hope it doesn't fall on deaf ears.

Jimmy B

The Innate Immune System is able to recognize self-targets and initiate Inflammatory Response Melanoma..Jim Breitfller

Recent advance in autoimmunity research reveals that the innate immune system is able to recognize self-targets and initiate inflammatory response in a similar way as with pathogens. This is what anti-CTLA-4 blockage has done. Accordingly, alterations in cell morphology are recognized by the innate immune system resulting in an acute inflammatory response (Carroll and Holers,2005).

"Our immune system is an impressive piece of biological machinery using
specialized cells and soluble mediators to protect us from foreign substances such as bacteria, viruses and cancer cells. The immune system is historically divided into two parts, the innate and the adaptive immune systems. Although working together for effective protection these two systems differ from each other in many respects. The innate immune response has componentsrecognizing characteristic features that are common within groups of microbes and cannot distinguish the fine differences between foreign substances. The principal components of innate immunity are physical and chemical barriers, phagocytic cells, blood proteins and cytokines which all react to repeated infections in essentially the same way in which they first responded. The defining characteristics of adaptive or specific immunity are exquisite specificity and the ability to remember and respond more vigorously
to repeated exposures to the same pathogen. The key components of
the adaptive system are B- and T lymphocytes, which can differentiate to
effector or memory cells after stimulation by foreign molecules known as
antigens (Ags)."

Source: Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 189

Early Immunostimulatory Effects of IgE- and IgG Antibodies

author FREDRIK HJELM

With this in mind, I had an Innate immune response when the CTLA-4 engage the B7 receptor. The response was an inflamatory response on day 15.

Base on the Innate immune response froma natural antibody. It takes about two week to generate a response.This response I posulate as the danger signal needed for T-cells to begin their frontal attack on the foreign invaders (The Cancer Cells).

I am in contact with Kirkwood and Medarex trying to convince them of my theory. I has been a hard sell.

response:Fine theory but just theory that has never been tested in relevant clinical setting…





Jimmy B

Cutting Edge Technology “Infection-mimicking materials to program dendritic cells in situ”Melanoma .. Jim Breitfeller

Cutting Edge Technology “Infection-mimicking materials to program dendritic cells in situ”

Thanks to Donald Bohlken for bring this to my attention.

“I thought you might be interested in that attached article from the January 25, 2009 issue of the journal "Nature Materials" concerning a Harvard study of a new vaccine methodology which resulted in a 90% survival of mice infected with a melanoma strain which would normally kill them in 25 days. The article speaks of this result as a "cure".

The article synopsis notes: "Cancer vaccines typically depend on cumbersome and expensive manipulation of cells in the laboratory, and subsequent cell transplantation leads to poor lymph-node homing and limited efficacy. We propose that materials mimicking key aspects of bacterial infection may instead be used to directly control immune-cell trafficking and activation in the body. It is demonstrated that polymers can be designed to first release a cytokine to recruit and house host dendritic cells, and subsequently present cancer antigens and danger signals to activate the resident dendritic cells and markedly enhance their homing to lymph nodes. Specific and protective anti-tumour immunity was generated with these materials, as 90% survival was achieved in animals that otherwise die from cancer within 25 days. These materials show promise as cancer vaccines, and more broadly suggest that polymers may be designed to program and control the trafficking of a variety of cell types in the body."
This is Nano and Transdermal Technology at its best.

Implants Mimic Infection To Rally Immune System Against TumorsMain Category:

Melanoma / Skin CancerAlso Included In: Immune System / Vaccines; Medical Devices / Diagnostics; Biology / Biochemistry

Article Date: 25 Jan 2009 - 0:00 PST

“Bioengineers at Harvard University have shown that small plastic disks impregnated with tumor-specific antigens and implanted under the skin can reprogram the mammalian immune system to attack tumors.

The research -- which ridded 90 percent of mice of an aggressive form of melanoma that would usually kill the rodents within 25 days -- represents the most effective demonstration to date of a cancer vaccine.

Harvard's David J. Mooney and colleagues describe the research in the current issue of the journal Nature Materials.

"Our immune systems work by recognizing and attacking foreign invaders, allowing most cancer cells -- which originate inside the body -- to escape detection," says Mooney, Gordon McKay Professor of Bioengineering in Harvard's School of Engineering and Applied Sciences. "This technique, which redirects the immune system from inside the body, appears to be easier and more effective than other approaches to cancer vaccination."

Most previous work on cancer vaccines has focused on removing immune cells from the body and reprogramming them to attack malignant tissues. The altered cells are then reinjected back into the body. While Mooney says ample theoretical work suggests this approach should work, in experiments more than 90 percent of the reinjected cells have died before having any effect.
The implants developed by Mooney and colleagues are slender disks measuring 8.5 millimeters across. Made of an FDA-approved biodegradable polymer, they can be inserted subcutaneously, much like the implantable contraceptives that can be placed in a woman's arm.
The disks are 90 percent air, making them highly permeable to immune cells. They release cytokines, powerful attractants of immune-system messengers called dendritic cells.
These cells enter an implant's pores, where they are exposed to antigens specific to the type of tumor being targeted. The dendritic cells then report to nearby lymph nodes, where they activate the immune system's T cells to hunt down and kill tumor cells throughout the body.”

Source: Http://www.medicalnewstoday.com/articles/136473.php

Implants Mimic Infection To Rally Immune System Against Tumors



Donald Bohlken actually contacted one of the collaborators, Dr. Glenn Dranoff of the Dana Farber Cancer Institute and Harvard Medical School. He indicated: "We are initiating efforts to bring this to clinical testing, but it will take some time to adapt the procedures to patients. A one year time frame is a reasonable guess."

If this crosses over from the mouse model to the human model, we may have a winner on our hands. A 90 % response and if they are complete responses, this technology would surpass any therapy out there to date. The main take away is that they are prompting our immune system to recognize the tumors by activation of the T-cell. Rosenberg and Hwu and other colleagues are doing just that with (ACT) Adoptive Cell Transfer. Kirkwood, Camacho, Webber, Hodi, Maker, O’Day and Wolchok did that with anti-CTLA-4 blockage.

I can see the Light at the end of the tunnel!!!!!!!!!!!!!!!!!!!!!!!!!!!

Thank you Don for advocating for us and your Brother Ron who is a fighting Warrior of Melanoma.

I will post the research paper on Melanoma Missionary for everybody.

Jimmy B

The Orchestration of an Inmmune Response Melanoma ..Jim Breitfeller

The Orchestration of an Inmmune Response

This is the title of my next piece. I don't know all the players (responders)but, it will make you stand up and yell bravo!!!!!. If you have ever seen the Boston Pops or any orchestra play, you would be amazed. All the instuments (Cells) have to be in tune (the right microenviroment). The Sounds (Cytokines) coming and going (sending signals). It is a sight to see. Your Immune system is the Orchestra.And when it is working right, nothing can evade it. This next to last piece is my about my journey and how Dr.Kirkwood pulled off a masterful concert. I will try to back up my theories with fact. I am not a Oncologist, not a biologist, I am THE PATIENT!!!!!!!!



Jimmy B

Tumors Evading Detection!!!!! Melanoma.. Jim Breitfeller

Tumors Evading Detection
Lack of costimulation:

Many Melanoma tumor cells do not have the B7 protein on their surface so this co-stimulatory second signal cannot take place. Theoretically, they should cause an immune response but they do not stimulate an effective anti-tumor immune response. The first signal originates from the binding of the T cell receptor (TCR) to its antigen-MHC, and provides the specificity of the interaction. Without this signal, the cell enters anergic state and can act as a T reg cell. Expression of B7 on the surface of a cell is the costimulatory signal necessary to allow for the cytolytic CD8+ T cell attack on the tumor. B7 display renders tumor cells capable of effective antigen presentation, leading to their eventual eradication.

Secretion of immunosuppressive cytokines:

Another way tumors evade detection is by secretion of certain cytokines. They are low-molecular weight proteins that use their communication ability to regulate the immune response. Cytokines can act upon either the cells secreting them (autocrine) or on neighboring cells (paracrine) to generate activities in the targeted cells. This means they can act as light switches for on and off immune responses. For example, interleukin-2 activates a cell-mediated immune response, while interleukin-10 suppresses cell-mediated responses. Many types of cancer, including Melanoma, take advantage of this ability to down regulate this appropriate immune response to help extend their survival and proliferation. This causes cancer patients to fail in mounting a successful attack on the tumors. Immunosuppressive cytokines secreted by cancer cells include transforming growth factor-beta (TGF-beta), interleukin-10 (IL-10) and vascular endothelial growth factor (VEGF).

TGF-beta is one of the most potent immunosuppressive cytokines characterized to date. It is capable of affecting the proliferation, activation and differentiation of cells participating in both the innate and acquired immune response.TGF-beta inhibits the profilation T-cells, B cells, Natural killer cells (NK), and macrophages.TGF-beta also converts T-cells, which normally attack cancer with an inflammatory (immune) reaction, into regulatory (suppressor) T-cells, which turn off the inflammatory reaction. Another of TGF-beta's affect is on cytotoxic T lymphocytes (CTLs) This is very important for anti-tumor immunity because of their cytotoxic effects. TGF-beta down-regulates many of the processes necessary for CTL activation. Without this activation, there is no assault on the tumor cells from the CTLs. In addition to suppressing proliferation, TGF-beta has been shown to induce apoptosis (cell death) in B and T cells.

Another immunosuppressive cytokine is IL-10. It is capable of inhibiting the prodction of of pro-inflammatory cytokines like IFN-gamma, IL-2, and GM-CSF made by cells such as macrophages and T helper cells. IL-10 also displays potent abilities to suppress the antigen presentation capacity of antigen presenting cells. Secretion of IL-10 in the vicinity of a tumor can render the tumor totally insensitive to CTL-mediated lysis. It is most likely that the tumor’s microenvironment is altered enough to block or turn off the discharge granules that would lyses the tumor cell. However, it is also stimulatory towards certain T cells, mast cells and B cells. It enhances B cell survival, proliferation, and antibody production. As you can see, IL-10 has many rolls to play when it come to the immune system.

The cytokine VEGF is produced by most tumors.
Vascular endothelial growth factor (VEGF) is a cytokine that is produced by most tumors. This growth factor enables the tumor to expand vascularly when is in its growth phase. VEGF production can be induced in tumor cells that are not receiving enough oxygen.

Regulatory T-cells (Tregs) (suppressor T cells) are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens.1

Tumor Growth kinetics

The cell cycle has four stages:

1. G1 phase when the cell increases in size and gets ready to replicate its DNA.
2. S phase when the cell synthesizes or copies its chromosomes
3. G2 phase in which the cell prepares to divide
4. M phase when mitosis occurs.

When the various growth inhibitory proteins and checkpoint controls which regulate this cycle become disabled due to mutations characteristic of cancerous cells, the cell cycle is no longer under tight regulation. Tumor cells are capable of proliferating so quickly that the immune response is not fast enough to keep their growth in check. The growth of the tumor cells outpaces the immune response and escape the detection of the immune system. Lack of cell cycle controls leads to excessive proliferation of tumor cells.





I am getting closer!!!!

Take care

Jimmy B

Did the clinical trials on CTLA-4 Therapy Sell us short and we missed the boat??..Melanoma.. Jim Breitfeller

My experience with CTLA-4 Blockage Therapy:


On 9-13-2006 I received my first and only dose of CTLA-4.

9-19-2006 – “The New CTLA-4 trial, has sent me for a loop. I wake up exhausted. I have to push myself to get out of bed. Along with the fatigue, my muscles ache like they have lactic acid in them. Well, no pain no gain.”

9/27/06—“Saw the Physician Assistant, Melissa. It appears that the CTLA-4 has stimulated my immune system. In the pass week, I noticed that there was redness around the area where my tumors are located. Also it is becoming quite tender in that area. This is Great news!!!!! It appears that the treatment my have kick started my immune system. The only way we will know for sure is another CT scan. That is not scheduled until November 29th .

Oct 10, 2006—“I still have fatigue but I am managing it. What really bothers me is my right arm where they removed the lymph nodes. I can't get the swelling down which in turn puts pressure on my nerves. I have to baby it to try to bring down the swelling. Some days it feels like it is caught in a vise. I am going ask if they can drain the excess fluid if possible.”

Oct 12, 2006—“A couple of days ago, Dee noticed two new growths on my back. I was hoping for the best. Anyway, we got confirmation from the Hillman Center that it is 2 new tumors growing. This really stinks. I think it is time to take out the “Weed be Gone”. This is not what I was hoping to hear.” The therapy with CTLA-4 was terminated”.
“So, it is on to the next trial. I am not sure what is going to be, but they mention Interleukin -2.”

“Improved understanding of the functions of CTLA-4 led to the hypothesis that blocking its engagement could result in unopposed CD28 activation of T-cells coupled with suppression or depletion of regulatory cells. In essence, blockade of CTLA-4 leads to "taking the brakes off" the immune system.”1

Antitumor activity has been observed in melanoma after treatment with anti-CTLA-4 antibodies, as well as the potential for autoimmune-related toxicities. The Anti-CTLA-4 antibodies would bind to the B7 receptor. This in turn would keep the T-Cells activated for a prolonged time.

“It has been seen that CTLA-4 antibody blockade alone has had minimal effects in other mouse tumors, however, including the poorly immunogenic B16 melanoma2 and SM1 breast cancer.3 However, the combination of CTLA-4 blockade and vaccination with GVAX (irradiated tumor cells engineered to secrete cytokine granulocyte-macrophage colony-stimulating factor [GM-CSF]) resulted in significant tumor regression in B16 melanoma.2”

The immune system is a powerful weapon, and the body does not use it lightly. Cells in this system must all be activated before they can begin proliferating and carrying out their instructions. For T-cells, two steps are required for activation. This safeguard can be likened to the two keys needed to open safe deposit boxes, in which the box holder has a key unique to that box, and the bank teller has a key that fits all boxes, but both keys are needed to open any particular box.4

This first interaction involves the CD4 or CD8 proteins which form a complex with the CD3 protein to bind to the MHC molecule of the (APC). Antigen-presenting cell This is also called "Signal 1" and its main purpose is T cell activation. IL-2 aids in the “Cell to Cell” communication.

However, this is insufficient for producing a T cell response by itself. In fact, lack of further stimulatory signals sends the T cell into anergy. Anergy is a term in immunobiology that describes a lack of reaction by the body's defense mechanisms to foreign substances.

The Second costimulatory signal necessary to continue the immune response can come from B7-CD28 and CD40-CD40L interactions. The primary role of the B7 proteins is to give a second signal to the T cell. The B7 protein/receptor is present on the Antigen-presenting cell and is able to interact with the CD28 receptor on the T cell surface; this is also known as "Signal 2". There are other activation signals which play a role in immune responses.

On these T cells there is are family receptors whose job is downregulate the T cell activation so the immune system maintains metabolic equilibrium so the immune system doesn’t start an autoimmune response and cause it to attack itself. One of these receptors is Cytotoxic T lymphocyte-associated antigen (CTLA4). So Using the CTLA-4 blockade, the two keys are there to produce the wanted immune response which is to attack the foreign molecule (The Tumor)


Tremelimumab and ipilimumab, two fully human Monoclonal Antibodies (mAbs) that block CTLA-4, have progressed into clinical trials and demonstrated antitumor activity in cancer patients. Most clinical investigations have been performed in patients with advanced melanoma .


In looking at Phase 1 and Phase 2 Clinical trials:

Table 1. Phase I and II Clinical Trials of CTLA-4 Blockade in Metastatic Melanoma5

In the Living Medical Textbook “Oncology” Chapter 4 and looking at the Efficacy Data, I notice that Dr. Maker had the best (CR) complete response 8%.

“Tumor Regression and Autoimmunity in Patients Treated With Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade and Interleukin 2: A Phase I/II Study”

The way I derived the percentage was to take the number of complete responses and divide it by the number of patients in the trial, times 100. I also noticed that most of the complete responses were with a combination / additional therapy. This is SCREAMING OUT that this agent needs to be done in combination with other agents.

When Pfizer did there phase three trial against dacarbizine, the trial was done as a single agent. “Pfizer's randomized phase III of tremelimumab versus the fool's gold standard dacarabzine/DTIC was stopped due to futility.”6

I believe that CTLA4 blockade has clinically significant activity in melanoma and that we need to come back and address how to use this agent in combination with other agents.

I know that Dr. Rosenberg has tried with IL-2 and saw no additive improvement. I believe the trial was flawed in that the dosing was given at the same time.

“We have just not learned how to use these drugs to their maximum effect. Strategies that focus on complementary combinations intended to increase their immunostimulatory effect seem to be the right path. I just hope that we will not abandon CTLA4 blockade based merely on results of protocols that were not fair assessments of their possible activity”6

I am an example of the right Protocol:

“On September 6th, I had another CT scan and MRI for the next trial which was Anti-CTLA-4 Blockage . On September 13 I had my first infusion. On October 8, 2006 My wife noticed two new growths on my back. It was confirmed on October 11th it was new tumors.

Dr. John Kirkwood has decided that IL 2 Interleukin 2 would be next course of action.

I have completed another round of tests (CT scan, Pulmonary Function, and a Nuclear Stress Test). The CT scans shows 40+ nodules in my lungs ranging from 15mm to less than 5mm.

I am slated to be High dosage IL-2 on November 1st 2006.

I am presently washed out an IL-2 clinical trial that started in November 1, 2006. On the fourth cycle I had a heart attack and the doctors determined to abort the IL-2 on February 2, 2007.

On August 23 2007 there was no change to the tumors in my back or lungs but also no growth.

In October 24 2007 I got the word that the tumors and the lung nodules were shrinking.

In April 14, 2008, the 40 + nodules in my lungs decrease to 2.

In July 2008, the nodules in my lungs were undetectable and the ones on my back were all but one gone. Presently, CT and MRI

In November 2008 show no signs cancerous activity.

I did one cycle of CTLA-4, during that time I notice that there was redness around the tumor area and was soar. At the time we (the physician assistant) thought it was an infection. In retrospect, it was the activation on my T cells. Two new tumors appeared and it was decided to go on to interleukin-2 (High dose). Well , I believed that the IL-2 started the cell to cell communication, which was the costimulant to the T cell and there my immune system began the assault on the foreign invaders (the tumors).

I have seen this with another patient but did the IL-2 first with no success. But then went on to the CTLA-4 therapy and the tumors shrank.

I believe we are on to some thing big!!!!!!!!!! We must Investigate this combination therapy.


Jimmy B


References

1. Living Medical Textbook “Oncology” From Projects In Knowledge Contributing Writers: F. Stephen Hodi, MD and Lauren Cerruto http://www.livingmedicaltextbook.org/Activity/index.cfm?showfile=b&jn=1843&sj=1843.01&sc=1843.01.4

2. Van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med. 1999;190:355-366.

3. Hurwitz AA, Yu T F-Y, Leach DR, Allison JP. CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimentral mammary carcinoma. Proc Natl Acad Sci U S A. 1998;95:10067-10071.

4. Sompayrac L. How the Immune System Works. 3rd ed. Malden, MA: Blackwell Publishing; 2008

5. Table 1. Phase I and II Clinical Trials of CTLA-4 Blockade in Metastatic Melanoma5 http://www.livingmedicaltextbook.org/Activity/index.cfm?showfile=b&jn=1843&sj=1843.04&sc=1843.04.2

6. http://www.glgroup.com/News/CTLA4-Blockade-in-Melanoma--Where-Next--23639.html



Jimmy B

Remember I said HOMEWORK..HOMEWORK..HOMEWORK!!!!! ..Melanoma.. Jim Breitfeller

Guess whatI did this weekend!!!!!!



My Homework!!!!!!!


Jimmy B

A Reply back from DR. Markovic, Svetomir N., M.D., Ph.D. ON Emerging Therapies for Melanoma ..Jim Breitfeller

Emerging Therapies for Melanoma

Rajini Katipamula; Svetomir N Markovic
Expert Rev Anticancer Ther. 2008;8(4):553-560.
Posted 07/09/2008




Dear Mr. Breitfeller,
Thank you for your note.

I just got back from a trip and am catching up with e-mail. I must say, I really enjoyed reading your message.

Our thoughts on the matter (as summarized in my brief article you noted) are basically that there are 3 elements to the puzzle that need to be addressed:

(1) the tumor; (2) its vascular supply; and (3) the body's immune system.
Addressing each one of these alone, will only gain little benefits for most, transient benefits for some and awesome results in a lucky few patients. So, why not look at the problem in the context of the patient.

And, that is basically where we are right now. We are crafting therapies that will engage all 3 elements; we measure the impact on all 3 and gain insights into what's working, what's not, and what we need to do next.

The first study we did using this approach was just published in Cancer this month. And, we did reasonably well for the majority of patients (best progression free survival result in a single arm phase II study done by the US cooperative groups in patients with metastatic melanoma).

What was most interesting is that we learned that the drugs we used may have worked in a completely different mechanism... The combination of chemotherapy and an inhibitor of angiogenesis may have yielded the good clinical outcomes via an immunological mechanism (paper in preparation).

I think that there may be something to this, and we'll keep plugging on this path. Hopefully we will be able to lend some insights that may be helpful to our patients and colleagues.

Take care,
Svetomir



--------------------------------------------------------------------------------
From: dbreitfe [mailto:dbreitfe@rochester.rr.com]
Sent: Monday, January 05, 2009 8:28 AM
To: Markovic, Svetomir N., M.D., Ph.D.
Subject: permission to post on Carepages "Emerging Therapies for Melanoma"


Svetomir, I am a cancer patient under the care of John M Kirkwood at the Hillman Cancer Center. I think you paper is very fascinating. Let me give you a little back ground of my dealings with Melanoma:



Last July (2005)I was riding my bicycle to work at the Eastman Kodak Research Labs about 3 miles from home. I was wearing a knapsack to carry my things to and from the labs. I started noticing an ache on my back. I asked my wife to take a look at it. It was just a mole. So I decide to go to the dermatologist. To make the log story short, it was cancer. It took about a month and a half to get it biopsied . (Sept. 26 2005) I contacted a surgical oncologist and on October 27 2005 I had a wide incision to remove the tumor off my back. I also had a PET scan and a sentinel lymph node biopsy done and it showed no cancer except the localized one on my back. I knew from my research that I would be needing adjuvant therapy.

So I started communicating with Sloan Kettering, University of Pittsburgh Cancer Center, and a couple of others including the Wilmot Cancer Center at Strong. They all recommended to start with Interferon treatment. In the meantime, my back got infected and it took until Feb. 14 2006 to heal. On March 6 2006, I started the High Dose Interferon treatment that was to last 4 weeks with daily infusions. The treatment lasted only 2 ½ weeks because my blood sugar went completely out of control. I also have type 2 diabetes.

About 2 weeks after the interferon treatments, I noticed my right lymph node growing to golf ball size and they were aching. I had them biopsied (April 4 2006) and 9 out of 11 nodes were cancerous. On April 24th , I had my lump nodes removed under my right arm. This had move me from a stage 3 to a stage 4. The cancer was spreading quite rapidly. Strong did not have any specialists in the field of malignant melanoma. So, I was in contact with Dr. John Kirkwood in Pittsburgh. So with my oncologist approval, my wife and I went down to Pittsburgh and had a consultation with Dr. Kirkwood (June 12, 2006) Kirkwood wanted another PET scan (June 16 2006) to see if the cancer was contained to my lymph nodes. The results showed 4 tumors on my back, one under my right arm, and two spots, one in each lobe of my lungs. It wasn’t good.

So, On July 10, 2006, I started my clinical trial of Dicarbazine and Patrin cycle 1. Each cycle is 21 days. On day 3, I am injected with dicarbazine at the Hillman Cancer Center in Pittsburgh. On the 11th day I go back to have a check up. Twice a month I traveled to Pittsburgh at least with this trial. I started my 2nd cycle on July 31 2006. On August 17 I had a CT scan and the report showed new tumors in my lungs so they stopped the Dicarbazine trial.

On September 6th, I had another CT scan and MRI for the next trial which was Anti-CTLA-4. On September 13 I had my first infusion. On October 8, 2006 My wife noticed two new growths on my back. It was confirmed on October 11th it was new tumors.
Dr. John Kirkwood has decided that IL 2 Interlukin 2 would be next course of action. I have completed another round of tests (CT scan, Pulmonary Function, and a Nuclear Stress Test). The CT scans shows 40+ nodules in my lungs ranging from 15mm to less than 5mm.

I am slated to be High dosage IL-2 on November 1st 2006.

I am presently washed out an IL-2 clinical trial that started in November 1, 2006. On the fourth cycle I had a heart attack and the doctors determined to abort the IL-2 on February 2, 2007. On August 23 2007 there was no change to the tumors in my back or lungs but also no growth. In October 24 2007 I got the word that the tumors and the lung nodules were shrinking. In April 14, 2008, the 40 + nodules in my lungs decrease to 2. In July 2008, the nodules in my lungs were undetectable and the ones on my back were all but one gone. Presently, CT and MRI
In November 2008 show no signs cancerous activity. I am stabilized for the time being.

As you can see I did the CTLA-4, one dose and then went on to IL-2 until I had a heart attack. Base on your 5 year View, in my case, my overall survival rate has doubled so far.

“Five-year View”

“Several advances are being made in the field of immunological and targeted therapies. In our opinion, the agents of most interest in the next 5 years would be the anti-CTLA-4 antibodies and angiogenesis inhibitors combined with cytotoxic therapy. While most of these novel agents may not have significant single-agent activity, combining them with systemic chemotherapy might be the key to clinical success in melanoma. Ultimately, rational combination therapeutics of multimodal targeting agents (immunotherapeutics, cytotoxic therapy, angiogenesis inhibitors) will likely lead to substantive clinical benefit in this patient population. Our hope is that this massive clinical research endeavor will soon yield the long sought after improvement in the overall survival of patients with advanced melanoma.”

Being a researcher myself, I came up with a theory.

11/06/08 This is my Theory
Posted Nov 6, 2008 9:59am

11/06/08 This is my Theory
The Interlukin-2
IL-2, which works by stimulating killer T-cells to attack melanoma. In some cases with the right body chemistry, helps communicate that message to the killer T-cells and the body begins to fight off the Beast.

In other cases, your body chemistry is different than mine and may lack some sort protein/? or what ever. So in this case, the communication is lost like a drop signal from your cell phone. What you need is another Cell Tower to transmit that signal. That is where CTLA-4 coming in to play. It builds the tower to help with the communication. I may be over simplifying the biochemistry but I am not in the lab to do the right experiments. So, I have to take an educated guess.

So, I did a little online research


As you can see, Dr. Kirkwood and I wanted to induce tumor regression by using my own immune system. If we could get my immune system to recognize the tumors as foreign, then we might have a fighting chance.

So we decide to try the CTLA-4 Therapy, Antitumor response with prolonged time to progression has been seen in patients with melanoma who have received either of the CTLA-4 antibodies and durable antitumor responses have been observed with ipilimumab in patients with melanoma ovarian cancer, prostate cancer, and renal cell carcinoma It has been seen, antitumor responses may be characterized by short-term progression followed by delayed regression.

An important, possibly unique, clinical characteristic of anti-CTLA-4 antibodies is that the duration of clinical response -- and even stable disease -- is often quite prolonged.

This is what I believe is going on in my case but I have no proof.

So lets combine the two clinical trials and that was done by Dr. Rosenberg at the National Cancer Institute. This did not yield the Better outcome.

I think it is because doing the therapy at the same time my be jamming the signals or changing the micro-chemical in the cell and surrounding environment so that it is not inductive to passing on the communication cell to cell.. protein to protein .



But since there was a lag between my two therapies, My cells began to communicate and jump started my immune system. Do I think is a one off, NO!!!!! I have been in touch with another patient that did IL-2 then, CTLA-4 and got the same response. The trick is to get the timing and the dose right!!!!!


So I took it apon myself to to a little research and this is what I came up with. I believe it all makes sense. I still may be over simplifying the actual process but I am not a biochemist. So Here goes:

Dendritic cells (DCs) are immune cells and form part of the our immune system. Their main function is to process antigen material and present it on the surface to other cells of the immune system, thus functioning as Antigen-Presenting Cells (APC).
The dendritic cells are constantly in communication with other cells in the body. This communication can take the form of direct cell-to-cell contact based on the interaction of cell-surface proteins. An example of this includes the interaction of the receptor B7 of the dendritic cell with CD28 present on the lymphocyte. However, the cell-cell interaction can also take place at a distance via cytokines. These components of the immune system communicate with one another by exchanging chemical messengers. These proteins are secreted by cells and act on other cells to coordinate an appropriate immune response.

Cytokines include a diverse assortment of interleukins, interferons, and growth factors.One cytokine, interleukin 2 (IL-2), triggers the immune system to produce T cells. IL-2’s immunity-boosting properties have traditionally made it a promising treatment for several illnesses which include Hepatitis C and Melanoma.

There are several steps to activation of the immune system against a foreign molecule. The T cell receptor must first interact with the MHC molecule. The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is, in general, responsible for recognizing antigens bound to Major Histocompatibility Complex (MHC) molecules. MHC the most gene-dense region of the Human genome and plays an important role in the immune system, autoimmunity.

This first interaction involves the CD4 or CD8 proteins which form a complex with the CD3 protein to bind to the MHC molecule of the (APC). Antigen-presenting cell This is also called "Signal 1" and its main purpose is T cell activation.

However, this is insufficient for producing a T cell response by itself. In fact, lack of further stimulatory signals sends the T cell into anergy. Anergy is a term in immunobiology that describes a lack of reaction by the body's defense mechanisms to foreign substances.

The Second costimulatory signal necessary to continue the immune response can come from B7-CD28 and CD40-CD40L interactions. The primary role of the B7 proteins is to give a second signal to the T cell. The B7 protein/receptor is present on the Antigen-presenting cell and is able to interact with the CD28 receptor on the T cell surface; this is also known as "Signal 2". There are other activation signals which play a role in immune responses.

On these T cells there is are family receptors whose job is downregulate the T cell activation so the immune system maintains metabolic equilibrium so the immune system doesn’t start an autoimmune response and cause it to attack itself. One of these receptors is Cytotoxic T lymphocyte-associated antigen (CTLA4).

It was hypothesized back in the 1980’s that if you replaced the CTLA4 with Anti-CTLA4 that it might block the B7 receptor causing an enhancement of the T-cell activation, leading to a more robust antitumor immune response.

It was shown in mice with a disrupted CTLA-4 genes that their immune response ran unabated causing autoimmunity which was fatal.

It was also shown that the anti-CTLA-4 antibodies had a greater affinity to CTLA-4 than the B7 receptor. So by doing the CTLA-4 Therapy, it allowed signal 1 to become active.

So in the presents of the CTLA-4 antibody Therapy, I my case, we may have extended the antitumor response of the T-cells. This left Signal 1 active.

We then, hit the immune system with High dose of IL-2. This must have stimulated the cell to cell communication (Signal 2) causing the immune response to kick in against the foreign molecule (The Tumor)

AS YOU GUESSED IT, I MUST HAVE JUMP STARTED MY IMMUNE SYSTEM!!!!!!

Just don't know how long it will last.

AS you can see , I am passionate about finding a cure. My Life depends on it. So if I can pass along information that may help others, I do. And that is why I am asking for your Permission on post this information.

I hope to here from you soon

Thanks for your time and Keep the good research coming!!!!

Sincerely,



James M. Breitfeller



The information contained is intended for the confidential use of the intended recipient(s) only and may be legally privileged. If it has been received other than by an intended recipient, it may not be reviewed, disseminated, distributed, copied or retained. Therefore, please notify the sender immediately and delete the original message and all copies.



Thank you



James M. Breitfeller

Adjuvant Therapy in Melanoma: New Combination Cytokine and Other Strategies

Introduction

Melanoma is the leading cause of death among all types of skin cancer and is the fifth most common type of cancer in men in the United States. The incidence of melanoma is increasing; in 2008, the incidence of this disease is expected to exceed 60,000, with 8000 deaths.[1] Fortunately, up to 95% of patients will present with either local or regional disease, which is potentially curable.[2] Although the causes of this increased incidence of melanoma are debated, it is thought that increased exposure to ultraviolet radiation interacts in some manner with genetic factors to initiate melanoma.[3]

Histologically, melanoma is characterized by proliferation of transformed melanocytes.[4] These long-lived cells are normally present at the dermo-epidermal junction and impart pigment to the skin; they are typically resistant to DNA damage and apoptosis, a characteristic of melanoma as well. The principal subtypes of cutaneous melanoma are superficial spreading, nodular, lentigo maligna, and acral lentiginous; rarer noncutaneous primary sites include the mucosal membranes and the pigmented epithelium of the eye. Most types of melanoma are more common among whites, although acral lentiginous melanoma occurs with equal frequency in nonwhites.

Several pathologic and clinical factors are predictive of the risk of recurrence and death in persons with melanoma. The clinical factors include age, sex, location, and lymph node or distant organ involvement, whereas pathologically, the tumor depth (Breslow depth), ulceration, mitotic rate, and presence of microsatellites can determine the risk for recurrence or spread.[5] The recent revision of the American Joint Committee on Cancer staging system for melanoma incorporates many of these factors. Some pathologic features, such as involvement of skin layers (Clark levels) and the presence of tumor regression, have not stood up to rigorous multivariate analysis and are no longer used clinically to determine the risk for recurrence or the current staging classification.[6] Over 60% of melanomas have mutations in the B-type RAF-1 kinase (BRAF).[7,8] About 80% of these mutations are found at exon 15, at a single amino acid residue, usually a substitution for valine by glutamic acid, V599E (now referred to as V600E). This mutation causes increased kinase activation and signaling through the mitogen-activated protein (MAP) kinase pathway.[9,10] Surprisingly, this event occurs with high frequency in benign nevi as well as in melanoma.[11] An additional 15% to 20% of melanomas have mutations in N-Ras, which lies upstream of BRAF.[12] Both of these genes code for proteins that are part of the ERK-MAP kinase pathway. A small proportion of melanomas, especially those originating in sun-damaged skin, the palms and soles, and mucous membranes, have mutations or amplifications in the c-kit gene.[13]

Recent reports suggest that integration of molecular profiling with pathologic and clinical features may result in a better prognostic profile and help to more precisely determine an individual's risk for recurrence.[14] It has been suggested, for example, that the melanoma arising from sun-exposed skin in older persons is more likely to have c-kit mutations than the truncal melanoma arising in younger patients, which is more likely to have BRAF mutations.[14]
Surgical excision with wide margins and sentinel lymph node biopsy (for a melanoma whose depth is ≥ 1 mm) is the treatment of choice for primary melanoma. Several large clinical trials have helped to define the width of margins needed to resect melanoma and reduce the risk for recurrence.[15-17]

Currently, melanoma of ≤ 1 mm thickness is resected with surgical margins of 1 cm, whereas melanoma ≥ 2 mm is resected with margins of 2 cm, and intermediate-thickness melanoma is resected with 1- to 2-cm margins. Mapping the draining sentinel lymph node with use of a tracer and careful histologic examination of this node is now a standard of care for melanoma ≥ 1 mm in thickness.[18] High-risk melanoma, defined as stages IIB, IIC, and III, often recurs after excision and is associated with a 40% to 80% chance of death.[2] Given the high risk for recurrence (and subsequent mortality), there is an obvious need for reduction of recurrence risk. This review will address several treatment approaches that have been employed as adjuvant therapy following primary surgical excision.

Interferon Therapy

The interferons (IFNs) can produce antitumor effects through upregulation of the immune system. In humans, IFNs are structurally divided into 2 classes: type I and type II. The type I family, which is used clinically for the treatment of several diseases, includes alpha (alfa), beta, and omega; the type II family has only 1 member, IFN gamma. The various subtypes are further subdivided for pharmaceutical preparations, eg, IFN alfa-2a, IFN gamma-1b.[19] IFNs can stimulate both the innate and adaptive arms of the immune system; for example, they can enhance major histocompatibility complex (MHC) class I antigen presentation, increasing innate immunity and maturation of dendritic cells and leading to enhanced adaptive immunity. They also have antiangiogenic and direct cytotoxic effects on some malignant cells. However, the exact mechanism by which they produce antitumor effects is unclear.
Two large randomized trials have shown that high-dose IFN-alfa-2b significantly reduces the risk for recurrence compared with observation alone in patients with resected cutaneous melanoma.[20,21] A third large randomized trial has shown a significant improvement in overall survival with high-dose IFN-alfa-2b compared with the GM2 ganglioside conjugated to keyhole limpet hemocyanin (KLH) melanoma vaccine in patients with resected stage IIB-III melanoma.[22] In the first trial, ECOG 1684, patients with thick primary melanoma (≥ 4 mm depth) or lymph node-positive disease were randomized to receive IFN-alfa-2b 20 MIU/m2/day intravenously for 4 weeks followed by 10 MIU/m2 subcutaneously 3 times per week for 48 weeks or to observation. Patients who received IFN-alfa-2b had improved relapse-free survival (RFS) (5-year RFS 37% vs 26%; P = .0023) and overall survival (OS) (5-year OS 46% vs 37%; P = .0237) compared with the observation arm. This led to the approval by the US Food and Drug Administration of IFN-alfa-2b for the treatment of high-risk melanoma after surgical resection. Because this high dose appeared to be effective, the next ECOG trial was conducted to determine whether a lower dose of IFN might be as effective as that studied in ECOG 1684. Therefore, ECOG 1690 randomized patients with thick or node-positive melanoma to 1 of 3 arms: (1) high-dose IFN-alfa-2b (20 MIU/m2/day intravenously for 4 weeks followed by 10 MIU/m2 subcutaneously 3 times per week for 48 weeks) for 1 year; (2) low-dose IFN-alfa-2b (3 MIU/m2 3 times per week subcutaneously for 2 years); and (3) observation. Five-year RFS in the 3 arms was 44%, 40%, and 35%, respectively. The difference between high-dose IFN and observation was statistically significant (P = .05), but the difference between low-dose IFN and observation was not. Of note, no improvement in overall survival was noted between either of the treatment arms and observation.
The GM2 ganglioside vaccine had shown promising results in small phase 1 and 2 trials and was brought forward for further clinical testing.[23,24] The gangliosides are complex carbohydrates found on the outer cell membrane that can be as immunogenic as protein antigens. The resulting trial, ECOG 1694,[22] was different from the 2 prior studies because it compared high-dose IFN to GM2-KLH/21 vaccine in patients with resected stage IIB and III melanoma. High-dose IFN improved both RFS (hazard ratio [HR] 1.49; P = .00045) and OS (HR 1.38; P = .023) compared with GM2 vaccine, and this trial was halted early by the data safety monitoring committee. The benefit attributed to IFN was seen in patients with node-positive disease as well as those with node-negative disease.

Because the findings of these trials and others conducted by European and American investigators were inconsistent, several meta-analyses have been performed to assess the data globally. An authoritative meta-analysis performed by Wheatley and colleagues[25] examined 12 randomized controlled trials and found that RFS was highly significantly improved with IFN-alfa by 17% (HR 0.83; P = .000003). The benefit on OS associated with IFN-alfa trended toward a 7% improvement but was not statistically significant (HR 0.93; confidence interval [CI], 0.85-1.02; P = .1). The investigators subsequently have updated their meta-analysis and shown a slightly better outcome for IFN-alfa in terms of OS.
Inasmuch as IFN-alfa has a short half-life necessitating frequent injection and resulting in side effects from rapid variation in blood levels, it has been modified to reduce clearance and increase its half-life.[19] One such modification is the addition of a 12,000-dalton polyethylene glycol chain, which results in pegylated IFN (PEG-IFN). In the treatment of viral hepatitis, PEG-IFN appears to be as efficacious as IFN with reduced toxicity.[26] To determine whether PEG-IFN was effective in melanoma, the European Organization for Research and Treatment of Cancer (EORTC) conducted a large trial, EORTC 18991, which randomized more than 1300 patients with node-positive melanoma postoperatively to either PEG-IFN for a maximum of 5 years or observation. The 4-year RFS was 45.6% and 35.9% (P = .01) in the PEG-IFN and observation groups, respectively. No difference in OS was noted.[27] The greatest benefit appeared to be in patients with microscopic nodal disease. Overall, patients appeared to tolerate this treatment relatively well.
The major issue limiting the use of IFN in patients with melanoma has been its toxicity. IFN is associated with a characteristic and sometimes debilitating constellation of signs and symptoms that can make its use challenging.

Commonly, patients experience fatigue, myalgia, anorexia, altered taste sensation, fevers, chills, loss of concentration and short-term memory, and other neuropsychiatric abnormalities. Significant depression has been reported in some patients. Laboratory abnormalities associated with use of IFN include elevations in transaminase levels, neutropenia and thrombocytopenia, increased creatinine levels, and anemia. Commonly, patients are dehydrated and need to be encouraged to increase their fluid intake and maintain their activity levels. Given the modest benefit of IFN in terms of reducing the risk for recurrence and death from melanoma, its significant toxicity profile has made it an option for only a select subpopulation of patients at high risk for melanoma recurrence.[19]
Granulocyte-Macrophage - Colony-Stimulating Factor

Although granulocyte-macrophage - colony-stimulating factor (GM-CSF; sargramostim) was initially identified as a colony-stimulating factor for myeloid cells in the bone marrow, it was also found to induce differentiation of dendritic cells (DCs).[28] The DCs, sometimes referred to as antigen-presenting cells, display tumor antigens to the immune system in the appropriate context and are increasingly recognized as a vital afferent arm of the immune system. Both quantitative and functional defects in DCs have been described in patients with cancer. These defects may contribute to "tumor escape" of immune surveillance. Specifically, GM-CSF increases the mobilization, differentiation, and function of DCs[29,30] and hence enhanced antigen presentation to CD4 and CD8 T cells.
GM-CSF appeared to be effective in increasing tumor-specific immunity when used as an adjuvant for vaccines.[30] In the metastatic disease setting, GM-CSF has been injected intralesionally and intrahepatically (with use of a hepatic artery catheter) and administered as an inhalational treatment. A GM-CSF-producing adenovirus appears to induce regression of metastatic melanoma when injected into lesions.[31] GM-CSF inhaled as an aerosol resulted in regression of pulmonary metastasis from melanoma in a phase 1 study.[32] In a phase 1 study of patients with metastatic melanoma, a 32% response rate was noted in patients with liver metastases following hepatic arterial infusion of large doses of GM-CSF.[33]
On the basis of these data, Spitler and colleagues[34] evaluated the role of GM-CSF in patients with high-risk (stage IIIB, IIIC, or IV) resected melanoma. Patients received GM-CSF 125 µg/m2 subcutaneously daily for 2 weeks every month for 1 year. The median survival of patients in this study was 37.5 months and far exceeded the median survival of 12.2 months in historical controls. The limitations of the study included the lack of a placebo arm and heterogeneity between the historical control and study groups. Overall, GM-CSF was well tolerated.
To confirm the findings of this trial and to examine the effect of GM-CSF on DCs, Daud and coworkers conducted a study with essentially the same dose and schedule in a similar patient population.[35,36] OS and RFS were 65 months and 5.6 months, respectively. GM-CSF treatment caused an increase in mature DCs, first identified after 2 weeks of treatment and normalizing by 4 weeks. Patients with decreased DCs at baseline had significant increases in DC number and function compared with those with "normal" parameters at baseline (Figure 1).[36] No change was observed in the number of myeloid-derived suppressor cells (MDSCs). Early recurrence (< 90 days) correlated with a decreased effect of GM-CSF on host DCs, compared with late or no (evidence of) recurrence (Figure 2).[36] Therefore, greater increase of DCs was associated with remission or delayed recurrence. Although this study lacked a control group, the survival data for the GM-CSF-treated patients does appear impressive. The benefits of GM-CSF appear to be greater in those with reduced DCs, but inasmuch as these analyses were exploratory, further investigation is warranted.
GM-CSF is currently being actively investigated in combination with other immunologic agents and chemotherapy. GM-CSF with the anti-CTLA-4 antibody ipilumimab has been explored in a phase 1 trial[37] (L. Fong, personal communication) and has shown some promising activity in prostate cancer. In addition, it has been explored as a component of combination therapy[38-40] and also as a maintenance or consolidation regimen in patients with metastatic melanoma who have benefited to some extent from chemotherapy.[41] There is also an ongoing trial with high-dose IL-2 in combination with GM-CSF (J. Lutzky, personal communication).

Anti-CTLA-4 Antibodies

The use of monoclonal antibodies to inhibit cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a novel strategy in melanoma. These antibodies have been found to induce tumor regression and improve long-term survival in tumor-bearing mice.[42-44] Early preclinical studies showed that CTLA-4 serves as a natural braking mechanism for T-cell activation, allowing a return to homeostasis after an immune response. Inhibition of CTLA-4 upregulates several downstream targets, including T helper (Th) 1- and 2- produced cytokines (ie, interleukin 1 and 2) and cell cycle machinery (CDK-4, cyclin D3), leading to a more robust antitumor immune response. Two anti-CTLA-4 antibodies are currently in phase 2 and 3 trials: ipilimumab (also known as MDX-010) and tremelimumab (also known as CP 675,206).
Prolonged, but sometimes delayed, responses have been seen in patients with melanoma who have received either of the anti-CTLA-4 antibodies.[45,46] Similar to the effect seen with IFN therapy, immune-related adverse events correlate with more positive outcomes in patients with metastatic melanoma who receive anti-CTLA-4 therapy, including prolonged time to relapse.[47] Recently, it has been reported that tremelimumab was no better than standard dacarbazine (DTIC) or temozolomide for the up-front treatment of metastatic melanoma.[48] Results of an ongoing trial of ipilimumab and DTIC vs DTIC alone in the setting of metastatic melanoma are expected soon. Because anti-CTLA-4 antibodies appear to be effective in the phase 2 setting in metastatic disease, they are also being considered for use in the adjuvant setting. Currently, an ongoing EORTC study is examining ipilimumab vs observation in patients with high-risk melanoma, and combinations of anti-CTLA-4 antibody with GM-CSF and IFN are promising for evaluation because different parts of the immune system may be stimulated.

Vaccines

Tumor cells can express novel antigens or quantitatively different antigens compared with "normal" or nondividing cells. Harnessing the immune system to destroy malignant cells by recognizing these antigens is a major goal of tumor immunotherapy. Tumor antigens upregulated in melanoma include MART-1 (Melan A), gp100, and tyrosinase; other investigators have focused on the cancer-testis antigens, which are upregulated in tumor cells but present only in human germ cells in the body. Although early studies of therapeutic vaccines against melanoma showed some promise, [49,50] randomized, prospective, placebo-controlled clinical trials have failed to prove a benefit. An allogeneic melanoma vaccine not only failed to improve either DFS or OS, but the study arm actually showed worsened survival.[51] Similarly, although the GM2 ganglioside vaccine showed promise in phase 2 trials,[23] a recent randomized study showed worsened survival in the vaccine population compared with an observation group.[52] Although these signals may be confined to these particular vaccines and not necessarily to peptide or DC vaccines, caution is needed when interpreting vaccine studies given these data.

Chemotherapy

Historically, DTIC has been the standard chemotherapy for patients with high-risk melanoma.[53] The use of chemotherapy in the adjuvant setting in melanoma has been tested in randomized trials and has shown no benefit.[54-56] At this time, chemotherapy should not be administered in the adjuvant setting outside of a clinical trial. Whether chemotherapeutic agents may be effective when used in combination with immune-modulating agents is still under investigation.

Other Novel Therapies

As more details are elucidated about the biology of melanoma, new pharmacologic targets are being identified. Among these are immunologic targets such as programmed death (PD)-1, anti-CD40, and anti-41BB (CD137) antibodies, which are currently in phase 1 and 2 trials.[35] PD-1, a member of the tumor necrosis factor family, has shown promising results in murine trials and is currently in phase 1 investigation. CD40 is a receptor expressed on B cells and DCs; it is bound by CD40 ligand found on activated T cells. Phase 1 trials[57] have demonstrated a modest dose-related benefit (0.3 vs 0.2 mg/kg) that has prompted ongoing phase 2 trials. Also under investigation is adoptive cell transfer after lymphoid depletion (homeostatic lymphoid proliferation).[58] A very interesting proof-of-concept study showed that CD4 T cells directed against NY-ESO-1 antigen were able to produce a response in melanoma.[59]
Targeted agents directed against a growth pathway specific to melanoma are also in development. Sorafenib, a multikinase inhibitor with multiple targets including VEGF, has had modest activity when studied as monotherapy, with a 19% rate of stable disease.[60,61] Src inhibitors such as dasatinib, VEGF kinase antagonists such as sunitinib, and c-kit inhibitors such as imatinib are all under investigation in melanoma.[62].Although these agents are currently not known to be effective in the metastatic setting, they may hold promise for early-stage disease.

Conclusion

Currently, only a few adjuvant therapies show any effectiveness in terms of decreasing the mortality rate in patients with high-risk melanoma. IFN is the only agent that has been proven to reduce recurrences after surgery, although its benefit on survival is likely very modest. However, this modest benefit must be weighed against the toxicities, and hence candidates for adjuvant IFN therapy must be chosen carefully.
Among the newer agents, GM-CSF shows promise: two phase 2 studies have shown that GM-CSF administered subcutaneously may be associated with prolonged survival. Currently a major randomized trial is under way to determine the degree, if any, of this benefit. The combination of GM-CSF with other immune modifiers, such as anti-CTLA-4 antibody or IFN, may be the next step in clinical trials, although no trial data are yet available with these combinations. Vaccines have an extensive history in melanoma, but to date no vaccine preparation has shown a survival or recurrence benefit in melanoma, and recent data even indicate a potential for harm. Other agents are on the horizon, including targeted therapies directed toward the specific mutations present in certain melanomas, which could be used in the adjuvant setting. Some exciting cell-based therapies have been shown to work in proof-of-concept clinical trials; more data are awaited prior to studying these agents in the adjuvant setting.

This activity is supported by an independent educational grant from Bayer HealthCare Pharmaceuticals.

Quantifying cytokine levels to differentiate between healthy and disease states...

Thanks to Larry Demejo an recently Retired Kodak Senior Researcher sent me this Article.

Originally Published IVDT November/December 2008
MOLECULAR DIAGNOSTICS
Adapting life sciences technologies to IVDsTechnological advances in life sciences can feed the early-stage assay development pipeline and can be transitioned into the clinical diagnostics setting.

Sara J. Agee and Quynh Anh Lu

It is nothing like have a great team in place to climb Mount Everest.

Technological advances in life sciences can feed the early-stage assay development pipeline and can be transitioned into the clinical diagnostics setting.

Quantifying cytokine levels to differentiate between healthy and disease states...

Measuring Healthy Cytokine Levels

"The life sciences effort toward developing diagnostics feeds the early-stage assay development pipeline, which can later be transitioned into the clinical setting. Singulex is currently focused on developing assays for cytokines, a complex family of secreted signaling molecules that trigger various responses from the immune system in a combinatorial manner. The level of secretion of specific cytokines can have implications for a variety of disease pathologies, including inflammation, cancer, diabetes, and autoimmune disorders.

However, baseline concentrations from the plasma of healthy human subjects for many cytokines have yet to be defined by prior assay technologies. Establishing such baseline levels of cytokines will provide an important comparison before validating levels across disease states associated with inflammation. This new information will benefit future development of diagnostics that use inflammation as an indicator for such diseases as rheumatoid arthritis, autoinflammatory disease, and asthma.
Some studies have also shown a role for cytokines in staging and/or diagnosing cancer.9 For example, based on the Luminex platform, the Bio-Plex assay by Bio-Rad Laboratories Inc. (Hercules, CA) has been used to implicate elevated IL-6, IL-17, and G-CSF as indicative of breast cancer–positive patients.10 The benefit of this technology is the ability to multiplex, so a large number of cytokines were surveyed. However, this outcome came at the expense of sensitivity, and as a result, this study did not include levels from healthy subjects free of any symptoms. Better, more-sensitive immunoassay technologies could improve the clinical relevance and diagnostic value of these biomarkers in the future."

Quantifying cytokine levels to differentiate between healthy and disease states


What a way to start the New year.

If they Measure the Cytokine levels in Melanoma Patients, and find that they are deficient, they can start CLTA-4 or Li-2 or Adaptive Cell therapy. The Onlogogist has a starting reference point. It will give the oncologist some insight.

Way to go Larry!!!!!!!

Now that is team work!!!!!

Jimmy B