The Immune System

T-regs Revealed

The Body's Way of Controlling the Mayhem

Key Players in a Delicate Balancing Act

The human immune system performs a delicate balancing act between safeguarding the body's own vital tissues and aggressively destroying pathogens and cancers. In recent years research has revealed how a key role in this act is performed by immune system cells called T-regs. We review the critical role of these important players in an article titled Tregs Revealed. Understanding how these cells function and their relationship to the rest of the immune system is central to designing effective therapies for CLL — and to controlling the unwanted side effect of autoimmune disease.

 

 

Effectors

Natural Killer (NK) Cells

Date: 10/1/03

by Chaya Venkat

In previous articles we discussed how T-cells change from mild-mannered lymphocytes into armed and primed-to-kill CTLs (cytotoxic T lymphocytes). T-cells are the sophisticated arm of our immune system. In order for them to become CTLs, they have to be given the right activating commands, and directed to kill a specific target expressing a specific antigen marker on its surface. Once they are primed, they become extremely effective killing machines, each one a serial killer capable of killing many target cells, one after the other. Their extraordinary efficiency and killing wrath directed at a very specific target is what makes CTLs so powerful, such an important part of the immune system.

Total dependence on CTLs can also be the weak link in our defenses. Imagine a pathogen, a virus or a tumor for example, that is able to hide itself so that no tell-tale antigens signaling "danger" are exhibited on its surface; or perhaps the problem is that there are not sufficient snitches (dendritic cells) to do the job of presenting the enemy's antigen markers to the T-cells. In the absence of appropriate antigen marker on which to fixate, the T-cells will not commit to kill. Many tumors evade killing by CTLs by precisely this route. They either do not display any dangerous looking antigens on their cell surface, or they put out enough conflicting signals that the dendritic cells and T-cells are totally confused. Without a clear mandate, T-cells do not engage, they do not transform to become CTLs.

This is where Natural Killer (NK) cells become important. Under the microscope they look like large lymphocytes, not that dissimilar from T-cells or B-cells. But unlike B-cells and inactive T-cells, NK cells contain the deadly granules of perforin and granzyme. Unlike T-cells, NK cells do not need to be indoctrinated with a specific antigen . They are called "natural" killers because they are born armed and ready at all times, able to swing into action with a lot less fuss and bother. They target tumor cells and protect against a wide variety of infectious microbes. Like CTLs, these natural killer cells are armed with the same granzyme and perforin weapons, carried as inert granules within themselves. The killer cell gets real close to the target cell, then perforin is released to bore a hole and build a tunnel into the target cell. Granzyme is then released to seep through the perforin tunnel into the vulnerable innards of the target. This combination of perforin and granzyme is one of the hardest combinations to evade, few cells survive a perforin and granzyme attack. Here is a link to a past article in CLL Topics:  How T-cells Kill - Serial Killers, Up Close and Personal.

CLL cells are adept at disguising themselves - they are not very "antigenic" (no glaringly obvious and dangerous looking antigen markers on their surface, to trigger CTL attack). They are also able to fool dendritic cells most of the time. These tricks make CLL cells hard to kill by CTLs. But how about NK cells? These natural killers are not fussy about getting the right antigen signal and so on and they are more inclined to kill first and ask questions later. How do CLL cells evade getting killed by NK cells?

In many previous articles we discussed the relationship between cancer and chronic inflammation. Chronic and inappropriate inflammation sometimes provides good background for cancer cell growth. You must have heard the story of the boy who cried wolf too many times. It is a little bit like that. Chronic inflammation, perhaps as a result allergic reactions, gets the immune system used to the constant state of alertness, without any real reason to be all agitated. Pretty soon, the call to arms is ignored as just another false alarm, and the immune response is muted. An immune system that has become lazy in responding to threat signals is prime ground for cancer cells to establish themselves. It should therefore come as no surprise that histamine, one of the classic proteins involved in allergic reactions, is often found in excessive quantities in cancer patients. Histamine is a common link between cancer and inflammation.

NK cell production is stimulated by cytokines such as interleukin-1 beta (IL-1 beta), interleukin-2 (IL-2) and interferon gamma (IFN gamma). NK cell formation is inhibited by histamine, and other products of inflammation such as prostaglandins (PGE2). So that is one of the links between cancer and chronic inflammation. Besides making the immune system generally lazy, the over abundance of histamine and prostaglandins suppresses the formation of natural killer cells, the elite troops of the innate immune system.

What does all this mean, and what earthly use is it for us as CLL patients? Well, knowing how things work is a good point to start in defining what we can do about changing them. As we said above, interleukin-2 (IL-2) is a potent stimulator of NK cells. Well, one of the options to improve current therapy is the use of IL-2 as a way of enhancing the effect of Rituxan. In fact, some of the work done in heavily pretreated NHL patients with Rituxan and IL-2 as an immune system booster have shown good results. Here is a link to an article that describes a paper by Gribben, et. al. Combination Immunotherapy with Rituxan and IL-2.

The first abstract below by Josèe Golay lays the foundation for us. Much of the ADCC cell kill in Rituxan therapy is carried out by NK cells, and these natural killers are even more effective in the presence of IL-2. Short term exposure to IL-2 makes NK cells more lethal, long term exposure to IL-2 just grows more of these lethal killers. 

The second abstract, hot off the presses in the latest issue of "Leukemia" describes in detail how one can grow expanded armies of NK cells using IL-2 outside the body. But more than this article, I found its review (in the same issue) by Dr. Neil Kay quite interesting. Dr. Kay suggests that rather than looking at armies of NK cells grown outside the body, it might make sense to use IL-2 therapy to grow the armies inside your own body. Put that together with the Golay article, and you have the logic for a clinical trial that might be interesting: Rituxan therapy with IL-2 as an immune booster.

In fact, Dr. Kay seems to have been an early convert (or trendsetter!) in this regard. The third abstract is all the way back from 1987, where Dr. Kay proposes IL-2 therapy as a way of dealing with NK-cell defects in CLL patients.

Most of us have heard of "Tagamet", one of the first drugs that were truly effective in reducing acid production in the stomach. "Tagamet" is the brand name, the generic name is "cimetidine". While you may recognize Tagamet and perhaps even cimetidine as heartburn medicine, many of you may not realize that it is also a histamine blocker. It turns out that our friend, histamine, is the culprit in hyperacidity as well. When a histamine molecule attaches itself to a receptor called the H2R in the gut, this act turns on the acid pumps. Cimetidine works by virtue of being able to block histamine, and therefore prevent the acid pumps beting switched on. Tagamet (and its generic versions, since it is off patent) are readily available in the drugstores, and no prescription is needed to get them.

Cimetidine has an enviable toxicity profile, established over more than a decade. It is a relatively cheap, over-the-counter drug. The fourth abstract below asks the provocative question in the title "Does cimetidine improve prospects for cancer patients?" The next two abstracts seem to suggest the answer is "Yes". One of the problems with major surgery is the risk of infections. Patients have weakened immune systems after the trauma of getting cut open, as in colorectal or cardiac surgery. There is clear evidence that NK cell populations are depleted after surgery, and this contributes to the risk of infections. Interestingly enough, administration of cimetidine covered this risk, kept the NK cell populations from taking a dive downwards. This raises the question: can cimetidine, either by itself or in combination with the inhibitors of NF-kB pathway, prolong remission after Rituxan therapy? Another clinical trial, anybody?

Abstracts:

Haematologica. 2024 Sep;88(9):1002-12

Riituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2.

Golay J, Manganini M, Facchinetti V, Gramigna R, Broady R, Borleri G, Rambaldi A, Introna M.

Istituto Ricerche Farmacologiche Mario Negri, via Eritrea 62, 20247 Milan, Italy. 

BACKGROUND AND OBJECTIVES: We analyzed the sensitivity of freshly isolated neoplastic B cells to rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC), using different effector cells.

DESIGN AND METHODS: ADCC was performed by 51Cr release assays in vitro, using peripheral blood mononuclear cells, IL-2-activated or expanded NK cells, neutrophils or macrophages as effector cells. B lymphoma lines and freshly isolated leukemic samples were used as targets. 

RESULTS: NK cells, but PMN or macrophages mediated rituximab dependent cellular cytotoxicity against two B lymphoma lines. Purified NK cells (95% CD56+/CD16+) reached 70% lysis at the highest E:T ratio. By contrast, all freshly isolated B leukemia or lymphoma cases, including 5 chronic lymphocytic leukemia, 1 B-prolymphocytic leukemia, 1 mantle cell lymphoma, 2 marginal zone lymhomas and 2 follicular lymphomas were poorly lysed by ADCC in the same conditions and regardless of CD20 expression levels, reaching a mean of 4% and 27% maximal lysis with PBMC or purified NK cells, respectively. Interestingly, short term IL-2 cultured PBMC, containing 10 % activated NK cells, as well as long-term expanded NK cells, containing 80-95% activated NK cells, became strong ADCC effector cells with rituximab and lysed all leukemic samples to a mean of 57% and 67% at the highest E:T ratio, respectively. 

INTERPRETATION AND CONCLUSIONS: Primary leukemic cells are more resistant than cell lines to rituximab- and NK cell-mediated ADCC but short-term exposure to IL-2 or long-term expansion of NK cells in vitro may provide effective tools to improve the therapeutic activity of rituximab.

PMID: 12969808
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Leukemia. 2024 Oct;17(10):1973-80.

Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy.

Guven H, Gilljam M, Chambers BJ, Ljunggren HG, Christensson B, Kimby E, Dilber MS.

Department of Medicine, Division of Hematology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. It is currently an incurable disease, making new treatment options such as immunotherapy desirable. Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option. Administration of cytotoxic cells such as natural killer (NK) and natural killer-like T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs. A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Here, we report the feasibility of expanding populations of the human killer cells, CD3-CD56+ NK and CD3+CD56+ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients. The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture. The 21-day cultures from both B-cell- and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype. Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly. In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment. Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates. Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B. From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.Leukemia (2003) 17, 1973-1980. doi:10.1038/sj.leu.2403083

PMID: 14513047
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Am J Hematol. 1987 Feb;24(2):161-7.

Restoration of impaired natural killer cell activity of B-chronic lymphocytic leukemia patients by recombinant interleukin-2.

Kay NE, Zarling J.

Natural killer (NK) function in the majority of B-cell chronic lymphocytic leukemia patients is markedly deficient. This study was undertaken to determine if the biological response modifier interleukin-2 (IL-2), which is a potent augmenter of normal individuals' NK activity, could augment the low NK activity in these patients. Peripheral blood lymphocytes (PBL), depleted of B-cells, from most B-CLL patients exhibited low natural killer activity against NK-sensitive K562 cells and against herpes simplex virus (HSV)-infected lymphoblastoid cell lines (LCL). Incubation of patients' B-cell-depleted PBL with recombinant IL-2 resulted in augmentation of their NK activity against both K562 cells and HSV-infected cells. Furthermore, whereas large granular lymphocytes (LGL) isolated from CLL patients are deficient in cytoplasmic granules, which are thought to play a role in NK-cell-mediated lysis, treatment of patients' LGL resulted in increased granulation by 4 hr after treatment with IL-2 and showed a concomitant increase in lytic activity comparable to that of normal individuals.

PMID: 3028132
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Digestion. 1999 Sep-Oct;60(5):415-21.

Does cimetidine improve prospects for cancer patients?. A reappraisal of the evidence to date.

Siegers CP, Andresen S, Keogh JP.

Department of Experimental and Clinical Pharmacology and Toxicology, Medical University of Lubeck, Germany.

BACKGROUND: Evidence first appeared in 1988 that cimetidine as an adjuvant may improve the survival of severely ill gastro-intestinal cancer patients when given peri- or postoperatively. Since then, several studies have appeared which suggest an anticancer activity for cimetidine, although few attempts have been made to corroborate their findings in large, placebo-controlled, double-blind studies. 

METHOD: We reviewed the literature concerning cimetidine's potential anticancer activity, particularly with regard to gastro-intestinal cancers. 

RESULTS: Most studies suggest that cimetidine may improve the outcome in cancer patients by a three-pronged mechanism involving (1) inhibition of cancer cell proliferation; (2) stimulation of the lymphocyte activity by inhibition of T cell suppressor function, and (3) inhibition of histamine's activity as a growth factor in tumours. 

CONCLUSION: Bearing in mind the experimental evidence, as well as the potential and excellent safety profile of cimetidine, more studies are required and justified to clarify cimetidine's protherapeutic activity.

PMID: 10473965
____________

J Tongji Med Univ. 1999;19(4):300-3.

Perioperative cimetidine application modulates natural killer cells in patients with colorectal cancer: a randomized clinical study.

Bai D, Yang G, Yuan H, Li Y, Wang K, Shao H.

Department of Oncology, Second Affiliated Hospital, Hubei Medical University, Wuhan 430071.

Thirty-eight colorectal cancer patients were randomly assigned to treatment group, which took cimetidine in the perioperative period, and control group to which no drug was given. Twenty healthy volunteers served as normal controls. NK cells were measured by immunocytochemical technique. The results showed that NK percentages before treatment in both groups of patients were significantly lower than those in normal controls (P < 0.05). NK cell percentages at admission, before operation, on the 2nd and the 10th postoperative days were 14.84 +/- 4.41, 15.74 +/- 3.75, 17.21 +/- 3.69, 21.05 +/- 4.54, respectively, for the treatment group, and 15.00 +/- 2.77, 13.05 +/- 2.46, 14.21 +/- 2.19, 15.58 +/- 1.68, respectively, for control group. The difference was statistically significant (P < 0.01), suggesting that the perioperative administration of cimetidine could help restore NK cells in colorectal cancer patients.

PMID: 12938523
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J Thorac Cardiovasc Surg. 1998 Aug;116(2):312-8.

Cimetidine reduces impairment of cellular immunity after cardiac operations with cardiopulmonary bypass.

Katoh J, Tsuchiya K, Osawa H, Sato W, Matsumura G, Iida Y, Suzuki S, Hosaka S, Yoshii S, Tada  Y.

Second Department of Surgery, Yamanashi Medical University, Japan.

OBJECTIVE: Depressive effects of cardiopulmonary bypass on cell-mediated immune responses may lead to postoperative infectious complications. We previously reported that cimetidine reduced postbypass depression of the cytotoxic activity of natural killer cells. This study evaluated cimetidine as an agent to preserve cellular immunity after cardiac operations. 

METHODS: In a prospective randomized study, 20 patients were divided into two groups of equal size. Cimetidine-group patients received 400 mg of cimetidine intravenously before bypass and a 33 mg/hr intravenous infusion of cimetidine after the operation, continuing until the fifth postoperative day. Control-group patients received conventional perioperative therapy. Lymphocyte subsets, natural killer cell activity, percentage of CD56+CD16+ (percentage of natural killer cells), and percentage of CD11b+CD8+ (percentage of suppressor T lymphocytes) were measured perioperatively. 

RESULTS: Although temporary postoperative reductions in percentages of CD3+, CD4+, and CD56+CD16+ cells were observed in both groups, CD8+ percentages on postoperative day 1 and CD11b+CD8+ percentages on postoperative days 1 and 3 in the cimetidine group were significantly lower compared with those in the control group (p = 0.01,p = 0.004, andp = 0.02, respectively). Temporary postoperative reduction of natural killer cell activity was also observed in both groups, but the natural killer cell activity on postoperative day 1 in the cimetidine group (17.1%) was significantly higher (p = 0.02) than that in the control group (8.20%). 

CONCLUSIONS: Cimetidine counteracts depressive effects of cardiopulmonary bypass on cell-mediated immunity and may possibly reduce postoperative susceptibility to infection.

PMID: 9699585
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Yardsticks and Definitions

Is a PCR Negative Remission a Cure?

Date: 9/9/03

by Chaya Venkat

The word cure is not all that hard to understand as a simple word in English. Ask any cancer patient.  But you would be surprised how hard it is to define "cure" in clinical trials.  It is hard enough to understand the term "Complete Response (CR)", let alone "cure".

You see, it all depends on the yardstick one uses to measure 'cure'. Not long ago, during the early heyday of Chlorambucil as the lead drug for CLL, if your overt symptoms subsided, and the lymph nodes subsided to the point where you no longer looked like a chipmunk, after a physical exam you were declared to be in "remission" by your doctors. There were no automated machines to do dirt cheap CBC's and I doubt anyone kept track of the blood counts except when something was clearly wrong. Later, the definition of a complete response  was upgraded to mean "normalized" CBC, no palpable lymph nodes or spleen, etc., but it was OK to have a few nodules in the bone marrow. The advent of more detailed bone marrow biopsies raised the bar: you could have a "nodal" PR, which meant that everything looked good except for a few pesky nodes in the bone marrow that looked suspicious - and that kept you from being declared a "CR". Flow cytometry was the next step, where peripheral blood and/or bone marrow aspirate could be examined for the tell-tale markers of CLL. This was particularly effective when the flow cytometry  looked for CLL cells defined by more than just one marker, say for example the classic CD5 and CD19 and CD20 markers of CLL cells. 

Now we are getting to the stage where we are looking for the proverbial needle in a haystack to judge the level of CLL cells left behind after therapy. It is hard to find one needle in a large haystack. But if the needle was somehow encouraged to duplicate itself, so that there are several hundred or several thousand copies made of each needle originally present in the haystack, it would be an easy matter to count all the needles, plus or minus a few, and these omissions are too few to change the count by much. That is precisely what PCR (polymerase chain reaction) does.

Unlike other cancers with a specific gene identified with that cancer, CLL patients are a unique bunch. It does not work to look at a particular genetic marker as the "needle". So researchers have found a way to get a sample of your particular brand of CLL cells, prior to therapy. A snippet of protein in the Ig (immunoglobulin) of your CLL cells is chosen as the marker. Then, after you have finished your therapy, a sample of your blood or bone marrow aspirate is used to study the level of residual disease. Prior to PCR technology, our detection limits were no better than, say, 1 in a 1,000. In other words, if the sample examined had 10,000 cells, we could detect the CLL only if there were more than 10 CLL cells present (10 out of 10,000 is the same as 1 out of 1,000, right?). If you happened to have only 5 CLL cells in the 10,000 cell sample being examined, you would get a clean report, no CLL cells detected. Aha! The report does not say there were no CLL cells, just that none were detected. 

With PCR technology, the particular snippet of protein chosen to represent your CLL cells is copied over and over again in the sample, amplifying the number of units of this marker in the sample. You can see this makes it much easier to measure the level of residual disease. In fact, with PCR technology, our detection limits are now about 1 in 100,000 cells. Compared to the previous example, PCR technology has improved our detection ability by a hundred fold. People who got a clean blood report from prior technology may or may not get a pass, using the more sensitive PCR method. Bottom line, a PCR negative report says that you have less than 1 CLL cell in  100,000 normal cells.

As always, there are lots of details and the details are important: such as, what particular snippet of DNA is chosen to represent your CLL, is a blood specimen used or bone marrow aspirate used for testing, the actual methodology of the PCR amplification etc.  The debate continues on whether to use blood or bone marrow aspirate as the sample on which to base residual disease evaluation.  Blood samples are much easier to get.  But some researchers feel that in the case of CLL, the last traces of the disease are more likely to linger and hide in the bone marrow, and that is where one should look for it.  Makes sense.  On the other hand, bone marrow samples are subject to sample error.  Blood through out your body is pretty similar in composition (even that is an approximation, but let us not hassle that one just now), but clearly bone marrow obtained from your left hip region may be different from that taken from your right hip.  In fact, the marrow may be different from a spot just a few inches away! 

Even if one were to over look these little caveats, the fundamental issue to remember is that a PCR negative response does not guarantee that there are no CLL cells in your body, none, zip, nada.  It just means that if there are any at all, they are too few to be measured by even our most sensitive testing procedure.  A new technique may be invented next year, one that ups the anti on PCR technology.  Then the new standard of remission will be changed once more. 

The table below from the reference cited below illustrates the point nicely.  The same set of patients were judged by historical standards, the original NCI criteria and then the revised NCI criteria.  The percentage of CR patients went from 70% to 66% and then nose-dived all the way to 35%, as the standards for CR became stricter.  The last line in the Table represents today's definition of "molecular remission": it is not only required that the physical exam be normal, nothing flagged on the CBC because the bone marrow is functioning just fine, there are less than 30% lymphocytes in the bone marrow aspirate, bone marrow biopsy indicates no suspicious nodules, flow cytometry of bone marrow aspirate shows less than 10% of the cells are CD5, CD19 and CD20 positive, and the last clincher, the bone marrow aspirate is PCR negative for the chosen DNA snippet chosen as marker.  Phew!!

Reference:  Improving the Complete Remission Rate by Michael J. Keating.
ASH Education Book, 1999. This article is no longer available online at the ASH site.

Now that we have some understanding of what PCR technology is, and how it can be used to define ever more precise measurement of residual disease, the obvious question to ask is this:  Why is it important?

Clinical significance of minimum residual disease (MRD):

Long ago, well before you had ever heard of the acronym CLL, there was one single cell in your body that developed, entirely by chance mutation, a particular phenotype that gave it survival advantages.  It lived a little bit longer, made daughter copies of itself a bit more frequently, than its comrades.  Over time, since the cell's descendents multiplied faster than they were killed, their absolute numbers grew.  For a long time, their numbers were below our detection limits, they grew quietly below the radar screen, so to speak.  It is only when their numbers grew to the point that perhaps a routine CBC had a few numbers flagged, and an alert GP was suspicious enough to ask for additional tests, that you were diagnosed with CLL.  Did you know that roughly 3.5% of adults in the general population have clonal cells detectable  by PCR technology?  Another way of saying it, roughly 3.5% of the adult population is "PCR positive"!!

Should all these people be considered to be CLL patients?  The answer is a clear "no".  In the case of the majority of these people, the same PCR test done a few months later will show them to be PCR negative.  What happened?  Well, in the case of these people, their immune systems did the job they are supposed to do, take care of the little clonal population, kill the defective cells, destroy the "cancer" before it had a chance to become a full fledged cancer.

Several of you who have been through the FRC trials at M. D. Anderson  have had your response measured by this latest and most sensitive technology.  Once in a while we see jubilant posts on the discussion groups, by a member who has just been told he/she is PCR negative.  In some cases, the happy post is followed by another post a few months later, depressed because the latest test showed the patient had become PCR positive.  Is it the end of the remission?  Is it now just a matter of time before this patient starts to show all the signs of the dreaded CLL coming back?  No, it is by no means certain that remission is over, and the cancer is back.  Just as the clonal population went from undetectable levels (PCR negative) to PCR positive (above the detection limits), it may well dip back down into the blessed PCR negative state.  In fact, patients may go back and forth, following a curve that dips down and then pokes it head up above the detection limit once ever so often.  Again, an indication that your immune system plus any medications you are on are able to knock the clonal population back down, as soon as it becomes a sufficient nuisance.

Of course, in some instances, the conversion of PCR negative state to PCR positive state does indicate the beginning of the end of the remission.  The diagram shown here may make it easier to understand these concepts.

How does all this add up?  Who cares about PCR negative or positive or whatever, what does this mean in terms of survival?  I just answered an email from a visitor to Topics website who wanted to know if I could point him to a reference that had survival statistics for FRC therapy.  I told him the reason he was not able to find any was because there are none to be had.  Some of the newer therapy combinations, like the FRC or the RF, as well as the newer technologies like PCR have not been around long enough, and survival statistics are particularly hard to find in an "indolent" cancer such as CLL. Do people with PCR negative response immediately after FRC therapy live longer than those that did not get this desired response?  Most probably.  Does this mean people with PCR negative response are "cured"?  That depends on what you mean by the word.  If they get disease free and trouble free remission that last 10 years, is that a cure?  If ten years is too short, how about 15 years?  If the patient dies of a second malignancy 15 or 20 years later, perhaps a second malignancy that was caused by the heavy chemotherapy that gave the PCR negative response in the first place, would you chalk that up as a cure or as one of the failures?  You see my point, beauty and definition of cure is very much in the eye of the beholder.

Shown alongside are a couple of graphs that give a glimpse into the future.  Again, the time line is just under 6 years, far too soon to come to solid conclusions about survival.  But there seems to be little doubt that out of the 35 patients who obtained a PCR negative response after an auto BMT, 90% of them are still disease free, and none of them are dead, six years later.  But each and every one of the 9 patients who were PCR positive had relapsed by the end of the third year, and 4 of them were dead.  No question about it, people who get stable PCR negative responses after therapy are most likely to have longer disease free remissions, and live longer.  Does this mean they are "cured"?  Only time will tell.

Abstracts:

Full Article: Blood Journal Article

Blood. 2024 Apr 1;97(7):1929-36.

Clonotypic polymerase chain reaction confirms minimal residual disease in CLL nodular PR: results from a sequential treatment CLL protocol.

Noy A, Verma R, Glenn M, Maslak P, Rahman ZU, Keenan JR, Weiss M, Filippa D, Zelenetz AD.

Department of Medicine/Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY

Patient-tumor-specific oligonucleotides were generated for the detection of minimal residual disease (MRD) in a highly specific and sensitive clonotypic polymerase chain reaction (cPCR). The clone-specific region of highest diversity, CDR-III, was PCR amplified and sequenced. Nested CDR-III clonotypic primers were used in a semi-nested cPCR with a sensitivity of at least 1 in 10(5) cells. Patients with protocol-eligible Rai intermediate or high-risk chronic lymphocytic leukemia (CLL) received induction with fludarabine 25 mg/m(2) per day for 5 days every 4 weeks for 6 cycles, followed by consolidative high-dose cyclophosphamide (1.5, 2.25, or 3g/m(2)). cPCR was performed on peripheral blood and bone marrow mononuclear cells. All 5 patients achieving a clinical partial remission (PR) studied by cPCR were positive. Five patients achieved nodular PR (nPR) (residual nodules or suspicious lymphocytic infiltrates in a bone marrow biopsy as the sole suggestion of residual disease). Five of 5 patients with nPR were cPCR positive. In contrast, flow cytometry for CD5-CD19 dual staining and kappa--lambda clonal excess detected MRD in only 3 of the same 5 nPR patients, all of whom were cPCR positive, and immunohistochemistry detected MRD in only 1 of 4 assessable patients. Three of 7 CR patients evaluable by cPCR had MRD. Only 1 CR patient had MRD by flow cytometry; that patient was also cPCR positive. These data support the conclusions that nodular PR in CLL represents MRD and that clonotypic PCR detects MRD in CLL more frequently than flow cytometry or immunohistochemistry. (Blood. 2024;97:1929-1936)

PMID: 11264154
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Blood. 1996 Sep 15;88(6):2228-35.  Links

Eradication of polymerase chain reaction-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation.

Provan D, Bartlett-Pandite L, Zwicky C, Neuberg D, Maddocks A, Corradini P, Soiffer R, Ritz J, Nadler LM, Gribben JG.

Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA 02146, USA.

In chronic lymphocytic leukemia (CLL), clonal rearrangement of the immunoglobulin heavy chain locus (IgH) provides a useful marker for the detection of minimal residual disease (MRD) after treatment. At the time of initial presentation, DNA from patients with CLL was polymerase chain reaction (PCR)-amplified using consensus Variable (VH) and Joining (JH) region primers using complementarity determining region III consensus region primers or a panel of VH family-specific framework region 1 (FR1) primers. The clonal product was directly sequenced and patient-specific probes constructed using N region nucleotide sequences. We amplified and sequenced the CDRIII region and designed patient specific oligonucleotide probes for the detection of MRD in 55 of 66 patients (84%, 90% Confidence Intervals (CI): 74% to 90%) with poor prognosis CLL referred for autologous and allogeneic bone marrow transplantation (BMT). To determine the clinical utility of this technique, PCR amplification was performed on patient samples at the time of and following autologous (21 patients) and allogeneic (10 patients) BMT in whom serial bone marrow samples obtained after BMT were available for analysis. We show that the persistence of MRD after BMT is associated with increased probability of relapse. In all cases that have relapsed to date, the IgH CDRII region was identical at the time of initial presentation and at relapse suggesting that clonal evolution of the IgH locus is unusual in this disease. The finding that a significant number of patients remain disease free and with no evidence of PCR-detectable MRD after BMT suggests that high-dose therapy may contribute to improved outcome in selected patients with CLL.

PMID: 8822943
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Best Pract Res Clin Haematol. 2024 Mar;15(1):179-95. 

Monitoring disease in lymphoma and CLL patients using molecular techniques.

Gribben JG.

Department of Medicine, Harvard Medical School, Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Over the past decade considerable advances have been made in the sensitivity of detection of residual lymphoma and leukaemia cells. Assays based on the polymerase chain reaction (PCR) can detect one tumour cell in up to 10(5) to 10(6) normal cells. The identification and cloning of breakpoints associated with specific chromosomal translocations has made possible the application of these techniques to a variety of lymphoid malignancies. In parallel, B cell malignancies exhibit rearrangements of their immunoglobulin genes that are also suitable targets for PCR amplification to identify residual cells. Although these techniques provide a useful adjunct to standard methods of detection and diagnosis, their role in determining disease outcome remains investigational. There is confusion as to whether it is necessary to eradicate PCR-detectable lymphoma cells for cure, so it is not yet possible to determine whether the detection of residual lymphoma cells by PCR is an indication to continue therapy. Copyright 2024 Elsevier Science Ltd.

PMID: 11987923
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Learned Immunity

The Thymus and T-cells

Date:7/19/03

by Chaya Venkat

I am really getting annoyed at how the popular press can take perfectly legitimate medical developments and turn them into three ring circus acts. Not only do they dumb-down the science to the point where the sound-bites make little sense, they also hype up the claims to ridiculous levels. Here is one example. 

Recent press reports of a medical development from "down under" have described in very optimistic terms a new approach to curing many different cancers, viral infections like HIV, auto-immune diseases like Lupus, and just about the "fountain of youth" as far as the immune system is concerned. You can read the whole press release by going to the URL below, I have excerpted just a few quotes from it. Unfortunately, barring the hyped-up press releases, there is no clinical or scientific information out on Dr. Boyd's work in technical journals, one press report suggested he is staying mum on advice of his patent lawyers and business advisors. I do not know if this is true, and it is a sad state of affairs if scientists first talk to lawyers and venture capitalists before they talk to patients. 

Background:

The thymus is a very important part of the immune system. The "T" in the name "T-cells" is in honor of the thymus. It is a complex organ, and plays many crucial roles, some of which are transforming unspecialized blood cells into dendritic cells, helper-inducer T-cells and cytotoxic T-cells. When we are kids, the immune system encounters a large number of unfamiliar antigens, and the thymus is responsible for churning out armies of so-called naïve T-cells, primed to recognize and target anything alien. At this stage in our lives, the size of the thymus is at its largest compared to the body weight. By the time we become teenagers, the immune system has acquired a vast repertoire of very long-lived "memory" T-cells, ready to proliferate into exact copies of themselves by the billions in the event of later infections. The need and the influence of the thymus is correspondingly reduced. The trigger for the demotion of the thymus is the surge of sex hormones during puberty. With the flood of estrogen from the ovaries, or testosterone from the testes, the thymus shrinks and becomes a pale shadow of its former self.

It is well known that many of the dreaded diseases of today are fundamentally a result of inadequate functioning of the immune system, or inappropriate functioning of the immune system. AIDs, for example, results in vastly reduced populations of functioning T-cells. Cancer too is a reflection of an immune system that has failed in detecting and eradicating the malignant cells. Autoimmune diseases like rheumatoid arthritis, lupus, etc., are caused by the immune system going after the wrong targets. 

Dr. Boyd's work: 

According to Dr. Boyd, many of the problems we face as we age are due to this fact, that the flow of naïve T-cells becomes a trickle in adolescence, under the influence of surging sex hormones. Without fewer fresh new T-cells entering the pool, the existing aging memory T-cells become less vigilant and also begin to display abnormal antigens and abnormal behavior patterns. This provides fertile ground for cancers and autoimmune diseases of all sorts. The logic then seems straightforward: get the thymus to start working again at peak performance, the way it used to when we were little kids. Since the substantial shut-down of the thymus function is due to sex hormones, it might be possible to re-start the thymus engine by blocking the sex hormones in question. 

Dr. Boyd's technique involves drugs called GnRH (gonadotrophin-releasing hormone) blockers. These drugs have been used for more than a decade to help treat breast and prostate cancers, and endometriosis. GnRH stimulates the pituitary gland in the brain to transmit a hormonal signal that induces the ovaries to release estrogen, or the testes to release testosterone. By over stimulating the pituitary, the drugs creates a feedback loop that disrupts the signal to the gonads, temporarily blocking the release of sex hormones. In breast cancer therapy, GnRH is sometimes used to reduce the amount of estrogen, which is known to cause rampant growth of the cancer. In the case of prostate cancer, GnRH blocks the production of testosterone. 

Clearly, this is a well established approach, this business of blocking sex-hormones to increase thymus function. You can read the publicity version of the science in the press-release below. 

As I said, there are no technical papers of Dr. Boyd's work in peer-reviewed  scientific journals, at least none that I could find. So I dug up a few literature references to GnRH use, and the abstracts are below for your reference. There is a simpler way of blocking sex hormones, that does not sound quite as attractive, it is castration. In fact, the technical name for treatment with drugs such as GnRH is chemical castration. Certainly, in life threatening situations and serious diseases like uncontrolled cancer it is an option to consider. But I doubt too many people would sign up for this therapy in the absence of such serious situations. Calling it the fountain of youth, as has been done by some tabloids, is a bit of a stretch. Let's see, you still need bifocals, your  skin is still wrinkled and your hair is long gone, and you have no sex drive - and this is the fountain of youth? Not! 

The thymus and the hormonal system that control it are so complex, and influence so many other vital systems at such a profound and basic level, I wonder how one would control the process and get just the effects one wants, and not the other kind. We are just beginning to understand how to grow large armies of T-cells outside the body, as in the T-cell therapy trials underway at UCSD. Would this not be a better way of getting more T-cell function, without messing with the thymus or the hormonal balance? I must admit, I am a little gun-shy of scientists telling us they know what happens when we do thus and thus to the hormone system. Ask any woman who has been on hormone replacement therapy after menopause, because it is supposed to protect you against heart disease. This was the heavily supported position of the whole medical establishment for more than a decade. Now we find that HRT has the opposite effect - it may actually increase the incidence of heart attacks. 

I don't know about you guys, but me, I would like to stay away from messing with hugely complex systems such as the thymus and the hormonal systems, if there was a choice. More limited and no doubt less effective T-cell therapy may also have fewer risks than an across-the-board blockage of sex hormones, even if it is only for a period of time. Chemical castration just does not sound all that attractive.

Article:

World First Breakthrough in Cancer Treatment

24 June 2024

"Victorian scientists have made a world-first breakthrough in the fight against cancer, which could give millions of patients a chance of prolonging their lives or even beating the disease. The breakthrough by Melbourne biotech company Norwood Abbey, announced overnight the BIO2003 conference in Washington, is being hailed as one of the most significant advances in cancer treatment in decades". 

"The treatment involves 'kick-starting' the thymus - the central organ for creating the immune system - to produce new immune cells, or T cells. The treatment also boosts the bone marrow - the producer of stem cells for the thymus and all other blood cells. The new treatment, which should be widely available within just two years, increased a patient's ability to fight cancer and other deadly diseases such as HIV/AIDS". 

Among the six hospitals testing the research following the trials in Melbourne are the Dana-Farber Hospital in Boston, the Memorial Sloan-Kettering Cancer Centre in New York and the Royal Free Hospital in London. The treatment has been developed by Monash University's Department of Pathology and Immunology. Associate Professor Richard Boyd, head of immunology at the National Stem Cell Centre in Melbourne, said the thymus, which sits in the chest cavity and is involved in all immune responses, was one of the first of the body's tissues to degenerate with age. "One of the natural inhibitors which leads to that degeneration is sex steroids. We have found by 'turning off' Mother Nature and shutting down the sex steroids, the thymus is able to recharge and produce more immune cells," he said. "That is achieved with a simple implant under the skin which has few side-effects and gives patients a much greater chance of fighting cancer and other diseases which attack the immune system. There is a global need for this treatment and every single patient should have a shot at this - it is not going to cure everyone, but it gives everybody at least a fighting chance. In patients with diseases such as cancer and HIV, autoimmune diseases, even transplant rejection, the possibility of creating a new thymus with its unlimited store of new immune cells provides new hope". 
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Abstracts:

J Endocrinol. 2024 Mar;176(3):293-304.

The hypothalamic-pituitary-gonadal axis: immune function and autoimmunity.

Tanriverdi F, Silveira LF, MacColl GS, Bouloux PM.

Department of Medicine, Neuroendocrine Unit, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK. 

GnRH and sex steroids play an important role in immune system modulation and development. GnRH and the GnRH receptor are produced locally by immune cells, suggesting an autocrine role for GnRH. Experimental studies show a stimulatory action of exogenous GnRH on the immune response. The immune actions of GnRH in vivo are, however, less well established. Oestrogen and androgen receptors are expressed in primary lymphoid organs and peripheral immune cells. Experimental data have established that oestrogens enhance the humoral immune response and may have an activating role in autoimmune disorders. Testosterone enhances suppressor T cell activity. Although there are some clinical studies consistent with these findings, the impact of sex steroids in autoimmune disease pathogenesis and the risk or benefits of their usage in normal and autoimmune-disordered patients remain to be elucidated. There are neither experimental nor clinical data evaluating functional GnRH-sex steroid interactions within the human immune system, and there is a paucity of data relating to GnRH analogues, hormone replacement therapy and oral contraceptive and androgen action in autoimmune diseases. However, a growing body of experimental evidence suggests that an extra-pituitary GnRH immune mechanism plays a role in the programming of the immune system. The implications of these findings in understanding immune function are discussed. 

PMID: 12630914 
____________

Cell Tissue Res. 1990 Sep;261(3):555-64.

Reversal of ageing changes in the thymus of rats by chemical or surgical castration. 

Kendall MD, Fitzpatrick FT, Greenstein BD, Khoylou F, Safieh B, Hamblin A. 

Unit of Cell Biology (Division of Biochemistry), United Medical School, Guy's Hospital, London, United Kingdom. 

Differences in the thymus of young and old male CSE Wistar rats were examined by use of routine histological stains on paraffin-embedded sections. There was a highly significant loss of thymic weight and disruption of architecture with age. Both surgical castration and chemical castration induced by a luteinizing hormone-releasing hormone analogue (Goserelin) caused a significant increase in thymic weight and the reappearance of a well-defined cortex and medulla in ageing rats. Cell surface antigens were detected on cryosections after incubation with a range of monoclonal antibodies. The Pan T cell marker (detected with antibody W3/13) showed fewer positive cells in ageing rats, and an increase after chemical castration. The smaller glands of old rats had fewer positive T cells with CD4 (MRC OX35) and CD8 (MRC OX8) antigens, and more after chemical castration in both young and ageing rats, but the greatest changes were seen in the intensity of Class II major histocompatibility complex (MRC OX6) immunoreactivity. In both young and ageing chemically-castrated rats, the number of cells and the intensity of immunoreactivity were greatly increased in the medulla. 

PMID: 2147125
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Tissue Cell. 1998 Feb;30(1):104-11.

Effects of castration on the lymphocytes of the thymus, spleen and lymph nodes. 

Windmill KF, Lee VW. 

School of Nutrition & Public Health, Deakin University, Geelong, Victoria, Australia. 

It is well known that the thymus plays an important role in the development and maintenance of a competent immune system. The thymus atrophies with age, a process that is accelerated after puberty when there is elevation of serum sex steroid levels. We have used a panel of commercial monoclonal antibodies against various T and B cell surface markers to investigate the post-castration histological alterations in the thymus, spleen and lymph nodes of male Sprague-Dawley rats. Castration of 5-week-old male rats produced a significant increase in thymic weight (P < 0.05) compared to age-matched intact animals. The major observations from the immunohistochemical studies were post-castration elevations in staining for total T cells (MRC OX 19 and W 3/13), CD8 cells (MRC OX 8), B cells (MRC OX 12 and MARK-1) and cells bearing activation markers such as IL-2 receptor (MRC OX 39), transferrin receptor (MRC OX 26) and major histocompatibility class II antigen (MRC OX 6). These data suggest that following castration there is an increase in the ability of lymphocytes to respond to activation. As a result, there are elevated numbers of immature thymocytes within the thymus that undergo differentiation/maturation and consequently produce an increase in peripheral T and B cells. 

PMID: 9569683
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Cell Death

Apoptosis versus Necrosis

Date: 7/3/02

by Chaya Venkat

There are two types of cell death, apoptosis and necrosis. I read somewhere, necrosis can be thought of as murder, where the cell is killed by some external factor. Examples of necrosis are skin cells that are killed, undergo necrosis, when burned beyond repair; or tumor cells that are mashed beyond hope of repair due to an ultrasound burst that is powerful enough to do so.

Apoptosis can be thought of as suicide, where the cell obediently kills itself upon receiving the right signals to do so. All normal cells have a defined role to play, and a time comes in the life of every cell when there is no further role to play. When that time comes, all good cells die obediently on receiving the death signal from the body. In some cases, as in cancer, the cell develops a case of not listening very well, and refuses to die on cue. As in the case of CLL, they then proliferate out of control, not so much because they are multiplying too fast, but because they are not dying fast enough. A little persuasion is needed, to get the message across, and this is where a lot of the chemotherapy drugs come in, getting through the death signal to cells that had somehow evaded responding to the message to commit suicide.

Another big difference between the two pathways of cell death, in necrosis the end is messy, the cell's membrane bursts open spewing forth all sorts of fragments of the contents of the cell that has just been killed. If this cell had been an infected cell, or a tumor cell, these fragments may be interesting enough to trigger an immune response, sometimes giving rise to inflammation. Apoptosis on the other hand is characterized by nice packaging of the cell into little packages that can be easily phagocytized (gobbled up) by other cells. Apoptosis therefore does not have the mess and fuss associated with necrosis, and therefore does not cause an immune response or inflammation. Makes sense, does it not, apoptosis is one of the regular, routine and necessary functions of the body. It would not do to have the immune system getting riled up every time a cell does its appointed task of killing itself on command. The police  force (Immune system) needs to come out only for murder cases (necrosis), not death due to natural causes (apoptosis).

Bottom line, necrosis may trigger an immune response, if the fragments of the murdered cell are appropriately presented to the immune system, by a professional antigen presenting cell (APC) such as a dendritic cell, and the presentation is accompanied by all the necessary co-stimulatory signals. In other words, necrotic tumor cell fragments are a necessary component of vaccines of the heat shock variety we discussed in previous articles. If the tumor cells are dead via apoptosis pathways, there is no necrotic peptide fragments, cell death is nice and tidy, nothing for the immune system to respond to, hence no vaccine effect is possible.

This is oversimplified, as usual, but it may help get across some of these concepts.

 

 

Memory B-cells

Staph Infections and B-Cell Apoptosis

Date: 6/26/03

by Chaya Venkat

Here is a little puzzle to get your little grey cells working. And there is a nice little "reward" at the end, if you read through this article. 

When a healthy person gets a bacterial infection for the first time, the body takes a little time to respond since the bacteria is a new antigen and the B-cells do not have any memory of it. But after that first time, the immune system retains a memory of that particular bacteria, its specific antigens, in terms of **memory B-cells**. The next time around, when the same bacteria strikes again, the body is able to mobilize its resources quickly. Antigen presenting cells ("APCs") such as dendritic cells carry bits and pieces of the bacteria to the lymph nodes, where they present the antigens to the B-cells. This means the memory B-cells are alerted immediately, and they go into overdrive. The required antibodies to attack that particular bacterial antigen are produced quickly, in large quantities, from plasma cells. Each of these antibodies is a perfect fit for the specific antigens displayed by the bacteria on its surface, and they lock on. Complement system is activated and the bacteria is quickly "opsonized", i.e., covered wit  thousands of fragments of complement proteins. Some of the complement fragments kill directly by punching holes in the skin of the bacteria, others enlist the help of effector cells such as neutrophils, T-cells and macrophages. The net result of all this action is that the invading bacteria is killed off quickly, before it has a chance to establish a foothold in the body, before it has a chance to grow large armies of itself. This is the natural process we emulate in monoclonal therapy, what happens for example, when Rituxan (antibody) locks on to CD20 marker (antigen) on the surface of CLL cells. We have discussed in detail how complement, CDC, and ADCC mechanisms work, once the critical antigen-antibody pair is made. 

As you can see, the whole point of having memory B-cells is so that lessons learned the hard way the first time can help the body respond more quickly and efficiently the second time around. This is the basis of all immunizations and vaccinations. In some cases, the bacteria change their stripes subtly from one generation to the next. Immunizations given for one variety is not sufficient to generate fully effective memory cells for the newer version, and repeat immunizations may be necessary for the new version. This is why people need new flu-shots every year. 

OK. now for the puzzle. If the body is supposed to have this wonderful capacity to remember antigens it has seen before, how come people never have any immunity against staph infections? Staph infections are no joke, they cause major complications, including death, in many hospital patients. They are especially dangerous for immune compromised patients such as HIV patients, transplant patients, or cancer patients undergoing chemotherapy. 

Here is the answer. Just as our immune systems have developed over the millennia, finding better and quicker ways of dealing with attacking bacteria, viruses and the like, the little blighters have had just as long to figure out ways around our defenses. It is a cosmic chess game, played at the cellular level. Move, counter-move, each side trying to find holes in the defenses of the other side. As we mentioned above, some pathogens learn to survive by mutating, so that they are never quite the same from one batch to the next. This makes them hard to pin down, hard for our bodies to develop solid memory defenses against them. 

Staph infections go one better. Since our bodies develop "memory" by using B-cells, staph infections start by attacking the very B-cells. They generate a protein, called SpA, that is a deadly toxin to B-cells. This pre-emptive first strike capability of staph bacteria means that our bodies never get a good chance to develop any memory of this particular invader, our B-cells are killed off before they get a good look at what is killing them. This means we have just as hard a time dealing with staph infections for the umpteenth time, as we did the first time. Clever little blighters, right? Only reason why it does not kill us outright is that we have more than one cell line in our immune system, we have T-cells, neutrophils, macrophages etc which are pretty good at killing bacteria, as well as tools like broad-spectrum antibiotics. All staph bacteria succeed in doing is to make the game a little harder to win, since our bodies do not have the advantage of remembering the last attack, and being prepared for the next time around. 

How about situations where we may actually want all B-cells to die, as in the case of B-CLL patients? This may be one explanation for the rare cases of spontaneous remissions one hears about now and then. Invariably, the story goes like this: the lucky individual had a confirmed diagnosis of CLL, nothing out of the ordinary. Then he got some sort of a garden variety infection, which resolved itself in a week or so. Soon after, lo and behold, it was found he no longer had any traces of CLL, a case of spontaneous remission!! Could it be that the patient had a staph infection or something similar to it, which went after his B-cell population, killing them off with 100% efficiency, and in the process killing off all of his CLL cells as well? 

Remember, that is exactly what we are trying to do with Rituxan monotherapy, killing off all of the B-cells, except we have not yet learned how to do it  with 100% efficiency. CLL patients get a nice remission with Rituxan, but generally speaking the disease returns in 6 months or so, when CLL cells left over after the therapy grow back their clonal population. Now if we can find a way of killing off each and every last one of the mature B-cells, which includes good B-cells but also all the CLL cells, we would have a cure for CLL. Eventually, the stem cells in the bone marrow will grow a nice new set of B-cells, good non-cancerous B-cells. All of the feverish activity in monoclonal antibody research for CLL is geared to this one point, how to improve the efficiency of B-cell kill, without killing any of the other cell line. Right now, the only way we seem to be able to kill off close to 100% of the CLL cells is by using combinations of chemotherapy drugs, such as the latest RFC combo therapy. Even here, I say "close to 100%" of the CLL cells are killed because even in "PCR negative" patients there is no guarantee that every single last CLL cell has been killed. "PCR negative" just says that the CLL cells, if any exist, are at concentrations lower than our ability to detect them. It is not yet certain that PCR negative remissions never relapse. 

It would be nice to be able to get PCR negative status, or even deeper remissions approaching a cure, with relatively non-toxic methods such as Rituxan, without needing to add more toxic Fludarabine and Cyclophosphamide components to the therapy. Perhaps some day we will be able to duplicate what the lowly staph bacteria does so well, kill B-cells with equally ruthless efficiency. Of course, we would like  to do this with some control over the situation, without running into the pesky little problem of getting an actual staph infection. You would be happy to know that this is not a crazy idea thought up by yours truly, below is a press release that says they are actually going into clinical trials to try and do the same trick as staph bacteria, in this case in an effort to treat Lupus. (For those of you who may not know what this awful disease is, it is a type of autoimmune disease, where the culprits are B-cells). 

There seem to be more than one way of skinning the CLL cat. I am truly optimistic that the explosive growth in our understanding of immunology will make CLL a "manageable" if not "curable" disease in our life times. 

Press Release:

Date: 28-Apr-2003

University of California - San Diego; Contact: Sue Pondrom 619-543-6163

Staph infection process leading to B cell suicide described by UCSD researchers 

The method that Staphylococcus aureus (staph) infection uses to inactivate the body's immune response and cause previously healthy B cells to commit suicide, is described for the first time by researchers at the University of California, San Diego (UCSD) School of Medicine in the May 5, 2024 issue of the Journal of Experimental Medicine. 

Normally, B cells mount an early defense against invading bacteria. From this immunologic experience, memory B cells are developed with the ability to quickly recognize these antigens and destroy the bacteria if they return in the future. When staph infections occur, however, this important process for immune defense can be corrupted.  

In studies with mice, the researchers found that a staph protein, called SpA, acts like a B cell toxin because it mounts a pre-emptive attack to target a specific region on the antigen receptors of B cells, which ultimately causes their death. Although the B cells begin to respond, they are quickly shut down, as the SpA de-activates these B cell antigen receptors, and there is also loss of other surface molecules such as CD19 and CD21, which are important for amplifying immune responses. Then, within a few hours, the SpA toxin induces B cells to turn on themselves in a programmed suicide process called apoptosis. As a direct consequence, the B cells never get a chance to develop the memory cells necessary to recognize and fight future staph infections. 

"This mechanism may explain why staph infections are so common and why many people get them recurrently," said Gregg Silverman, M.D., UCSD professor of medicine and senior author of the paper. He noted that apoptosis usually takes place on a regular basis in a small proportion of cells to control the size of lymphocyte populations and maintain homeostasis, to keep a balance in the internal equilibrium of body. However, SpA co-opts the normal apoptosis process, in effect killing the B cells too early, before they've had a chance to do their job. 

"Inappropriate mechanisms of induced apoptosis are not unique to many infections," Silverman said. "However, we should be able to use the same process induced by SpA, to treat the disease-causing B cells in autoimmune diseases and cancers like leukemia and lymphoma. These studies may also help us to make protective vaccines against staph."  

In follow up studies, the Silverman team will examine people with staph infections to verify that the same process takes place and that it prevents individuals from defending against staph infections. In addition, the investigators are planning a clinical trial in one to two years to treat patients that have autoimmune diseases such as lupus, that occur due to faulty B cells.

 

 

Remission and Relapse

Interplay of Chemotherapy and the Immune System

Date: 4/20/02

by Chaya Venkat

The Observed Pattern

For most of us at some point, w&w is over and the logical choice is chemotherapy. The drugs have improved greatly over the last few years, as have the protocols for administering them. Oncologists have new weapons in their arsenal, such as Rituxan and Campath.

With all these advances, and longer, better quality remissions, there is still the nagging worry that the immune system is further compromised as a result of chemotherapy. The theory of immune surveillance says that in CLL patients with progressing disease the immune system is carrying out some of its functions but not to the level required to eradicate the cancer, or even to reach a stalemate. The rate at which the tumor burden is growing is an indication of the deficit in the immune surveillance. Fast lymphocyte doubling rates suggest an immune system that is far from optimal. A patient with a really 'indolent' version of CLL whose lymphocyte numbers stay pretty much the same over long period of time probably has a pretty good immune system, almost able to do the job of controlling the cancer. 

So, what can one expect after chemotherapy? A remission, yes, but we all know that until a real cure is found, the cancer will eventually return. When it  does, how will the rate of growth of the CLL cells compare with the rate they were growing before the therapy? The chart shown here plots the the absolute lymphocytes versus time for an unnamed patient who went through two series of chemotherapy: Fludarabine from January 5, 1993 to June 15, 1993. The second round was SWOG protocol from May 1994, through November 1994. If you compare the steepness of the curve before any therapy with the two segments after the two series of chemotherapy, it seems that the CLL grew back at a faster clip after each treatment. The therapy got  complicated after 1995, so I did not include that portion of the data. 

It seems to me that in this patient's case, the immune system became less and less able to hold its own against the CLL clonal tide, after each round of  chemotherapy. Nothing surprising here, I suppose, but it is sobering to see the numbers graph so clearly. I wonder how this type of analysis shakes out for patients who did not have classical chemotherapy, but instead had therapy using monoclonal antibodies (passive immunotherapy) such as Rituxan.

Obviously, we are all unique in how we respond to treatment, so all this can be just so much spinning my wheels, with no solid conclusions to be drawn.  By the way, this information is obtained from data in the public domain, and no patient confidentiality was breached.

How Remissions End in Relapses

The rate of accumulation of CLL cells in the body is exactly equal to the rate at which they are born, minus the rate at which they are killed. This much is simple and clear. It is down hill all the way after that, with W. A. G theories, half-understood concepts and brilliant insights that may some day unveil this whole horrid mess.

The first part of the equation, the rate at which the CLL cells duplicate themselves, depends on many factors, among them: 

1. The intrinsic nature of the original clone, for example at what stage in the development cycle of the b-cell did the clone leave the well-ordained path and became a cancer cell. 
2. The presence of growth factors such as colony stimulating factors and various other cytokines that the body produces, to accelerate the rate of  production of specific cell lines. These are very complex and inter-related feed-back mechanisms, and frankly, I do not believe any one has a full understanding of the whole picture. 
3. The nature of the microenvironment that the clones find themselves: is it a good neighborhood, a good place to grow and flourish, bring up their kids, with plenty of sugar to eat and good potential for angiogenesis to keep the food coming? 
4. Development of additional and different sub-populations of malignant cells, with different inherent rates of duplication. Most of the chemotherapy  drugs in use are mutagenic, and capable of further mutating the CLL clonal population. It is well documented, for example, that one of the mutations to worry about, the Richter's syndrome, happens only in patients that have undergone chemotherapy. Transformation into Richter's syndrome is usually accompanied by a much more aggressive disease.

The second part of my equation deals with how fast the CLL cells are killed. This too is defined by a complex set of variables: 

1. The intrinsic nature of the original CLL clone, how good is it at avoiding receiving the apoptosis death signal, and thereby refuse to die like all good  b-cells. For example, the CD-38 level, the IgVH mutation status, the cell's ability to attract Dr. Kipp's nurse-like-cells to protect it from receiving the apoptosis signal, presence or deletion of important oncogenes such as p53, all of these may be factors that play into the intrinsic ability of the cell to survive. We are just beginning to get glimmers of understanding on these important factors that control prognosis. 
2. The immune competency of the individual, does he have a strong immune system, be it 'natural' or goosed-up due to vaccines or gene therapy, in any case an immune system that can recognize the CLL cells as "non-self" and therefore dangerous, and furthermore, is capable of mounting an effective immune response. 
3. Externally administered cytotoxic drugs targeting the CLL cells.

If there is one thing we can be sure of, it is that this is not the whole picture. There are tremendously more complex issues that are not even guessed at  right now. So, what is a poor confused CLL patient to do?

There are several things that you can do little about, such as your CD38 level, IgVH status, etc. However, it is good to know what the status is, so that you have a better fix on your prognosis, and can therefore make better choices about therapy. 

Try to avoid unnecessary exposure to mutagenic radiation or drugs. You may have no choice but accept the risks, at some point, but it helps to be aware of the risks versus rewards, so you and your doctor can make sensible choices. 

Keep your general physical health as good as possible, and keep an optimistic and positive mental attitude. It is well established that both are important in keeping the immune system working better. Don't feed the cancer, but feed your body good nutritious food, keep up a regular program of exercise. Simple precautions such as washing your hands often, breaking the habit of frequently rubbing your face and eyes, and staying away from crowds during the flu season, all of these will cut back on the number and severity of infections you have to deal with. If your doctor approves, get your flu and  pneumonia shots. Also, good dental health is important, I read somewhere that the bacteria in your mouth are one of the best sources for infection. If you need dental work, check with your dentist whether you should take antibiotics before even routine teeth cleaning.

Read up on chemo-preventive approaches such as anti-angiogenesis, see if they make sense to you, and if you and your doctor feel it is appropriate to incorporate them into your life.

Above all, make each day count. We all have a death sentence on our heads the day we are born: CLL patients have just had the benefit of a reminder. So, live the life you want, this is your only chance to do so.