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Do higher levels of CD20 equal better treatment outcomes with Rituxan?

A research study from Slovenia has set out to determine if Rituxan is of benefit to patients with B-cell lymphomas (including CLL), and if it is what patients benefit most. This study looked at the quantity of the cell surface protein CD20, that is required on B-cells so that monoclonal antibodies like Rituxan [Rituximab (MabThera)] have something to attach to. The amount of CD20 is determined by florescent markers added to the cells and viewed under a microscope. The more CD20 a patient has the brighter the markers show. Generally, CLL patients don't have a lot of CD20 so most are classified as 'dim' others are 'bright' and a few are very 'bright'. In NHL patients the opposite is generally true, and it has been known through observation that CD20 monoclonals work better for NHL patients than they do for CLL patients.

Monoclonals antibodies like Rituxan deplete the quantity of CD20 each time they are used, so the effect and response diminishes with every treatment. There is some return of CD20 levels on B-cells, however this varies between patients.

Only a few studies have addressed the question of whether higher CD20 expression equates with better treatment outcomes. The aim of this study was to assess the relationship between the level of CD20 expression and overall survival (OS), disease-free survival (DFS) along with the overall response rate (ORR) in B-cell lymphoma patients.

One hundred and fourteen patients with different histological types of B-cell lymphomas treated with rituxan and chemotherapy between 2003 and 2007 were enrolled in the study. All patients had CD20 expression assessed prior to the beginning of treatment.

A cut-off value of 25.000 molecules of equivalent soluble fluorochrome (MESF) of CD20 was determined. The study found that 80% of patients with CLL fall below this cut-off level. Patients with CD20 levels that fall above the cut-off point had a significantly longer OS than patients with a CD20 expression level below the cut-off value.

The researchers concluded that the cut-off limit of CD20 expression suggested to have the predictive significance of better outcome was in our series set at 25.000 MESF. This cut-off value should be considered when the decision regarding treatment with Rituxan is taken. However they cautioned that, these results warrant further studies on larger groups of patients.

Source: PMID: 20811695

Abstract: Link Here

More on CD20 antibodies: Anti-CD20 monoclonal antibodies: historical and future perspectives

and here: Link Here

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Sub-cutaneaous, self-administered Campath - tiny study from CLL researchers

A tiny study of 29 CLL patients published in the Journal Cancer has looked at patients self-administering subcutaneous alemtuzumab (Campath) to treat residual disease.

Patients in partial remission (PR), nodular PR (nPR), or complete remission (CR) who had disease in bone marrow established by 2-color flow cytometry analysis were enrolled and received alemtuzumab 30 mg subcutaneously 3 times weekly for 4 weeks, and patients had the option to self-administer alemtuzumab.

23 patients were considered to be 'responders', if those in PR converted to an nPR or a CR, and patients in nPR who converted to a CR, and patients in CR who had no evidence of disease on 2-colour flow cytometry analysis after treatment. (4 of 4 patients achieved a CR, 8 of 9 patients achieved an nPR, and 11 of 16 patients who achieved a PR.) Higher plasma alemtuzumab levels at the end of treatment were correlated with a longer response duration.

It was found there was a trend toward a higher response rate but a shorter response duration with subcutaneous alemtuzumab.

The research concludes that " The current results demonstrated that self-administered, subcutaneous alemtuzumab was safe and active for residual disease and that plasma alemtuzumab levels and real-time minimal residual disease evaluation are important endpoints to monitor in future alemtuzumab consolidation trials."

Abstract: Link Here

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Autoimmune cytopenia in CLL

A study just published in the journal Blood looks at the prevalence, characteristics, clinical correlates, and prognostic significance of autoimmune cytopenia in patients with CLL.

From a total of 960 CLL patients, it was found that:

• 70 patients had autoimmune cytopenia (7%). 19 of these were detected at diagnosis, 48 during the course of their disease and 3 prior to CLL diagnosis

Of these 70 patients:

• 49 patients had autoimmune hemolytic anemia (AIHA)
• 20 patients had immune thrombocytopenic purpura (ITP)
• 1 patient had both(AIHA) and (ITP)

There was a clear association between autoimmune cytopenia and poor prognostic variables (i.e. high blood lymphocyte count, rapid blood lymphocyte doubling time, increased serum beta-2 microglobulin level, and high expression of ZAP-70 and CD38). Nevertheless, the outcome of patients with autoimmune cytopenia as a whole was not significantly different from that of patients without this complication.

No differences were observed according to the time at which cytopenia was detected (i.e. at diagnosis, during the course of the disease).

Importantly, patients with advanced (Binet C stage) disease due to an autoimmune mechanism had a significantly better survival than those in advanced stage related to a massive bone marrow infiltration (median survivals: 7.4 years vs. 3.7 years; p=0.02).

The results of this study emphasize the importance of determining the origin of cytopenia in patients with CLL for both treatment and prognostic purposes.

Abstract: Link Here

More on ITP: Link Here

More on Coombs Test: Link Here

More on AIHA: Link Here

More on AIHA and Rituxan treatment: Link Here

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Monoclonal B-cell lymphocytosis (MBL) in Families with CLL

A familial study by National Cancer Institute in the U.S. (NCI), has looked into the relationship of MBL and CLL. According to a report published in the February 12, 2009 issue of the New England Journal of Medicine, monoclonal B-cell lymphocytosis (MBL) precedes CLL in the vast majority of patients.

But is there a strong association between MBL and CLL in familial CLL cases?

Researchers looked at 505 patients with no personal history of lymphoproliferative disease in 140 families. Each family had two cases of CLL in their histories.

Age was the most important determinant where the probability for developing MBL by age 90 years was 61%, and males had a higher risk of MBL than females as is the case with CLL.

The researchers concluded that: "MBL patients had significantly higher mean absolute lymphocyte counts (2.4 x 10(9)/l) and B-cell counts (0.53 x 10(9)/l) than those with a normal B-cell immuno-phenotype. Our findings show that MBL occurs at a very high rate in high risk CLL families."

Both the age and gender distribution of MBL are parallel to CLL, implying a shared inherited risk according, to this study.

Source: British Journal of Haematology, Aug 25th

Abstract: Link Here

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French study looks at Progression Free Survival (PFS) indicators at CLL diagnosis

A French study published in Blood, looks at 4 biological parameters in newly diagnosed, Binet Stage A CLL patients.

Using a group of 339 Binet stage A, newly diagnosed patients they found only 4 prognostic indicators for PFS. They were sTK (serum thymidine kinase), lymphocytosis (is an increase in the number or proportion of lymphocytes in the blood), β2-microglobulin (a protein), and CD38 (from flow cytometry) expression.

Based on these indications they found two distinct groups. The first, were patients with no risk factor or only one risk factor. 85% of this group had no disease progression after a period of 7 years.

The second group, that had two or more risk factors had a PFS of 20 months.

Based on this knowledge the French researchers and the prognostic index they used in the study the researchers "propose an easy, fast, cost-effective strategy for a trustworthy prognostication in (Binet) stage A patients, who currently represent over 80% of the CLL population, allowing physicians to adapt follow-up individually."

Source: Blood First Edition Paper, prepublished online August 25, 2010; DOI 10.1182/blood-2010-06-288274

Abstract: Link Here
Earlier work on sTK: Link to PDF
Earlier work on β2-microglobulin: Link to PDF

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Gene study supports link between vitamin D deficiency and disease, perhaps CLL

University of Oxford researchers have mapped the points at which vitamin D interacts with our DNA – and identified over 200 genes directly influenced by vitamin D. The results are published in the journal Genome Research.

The researchers found 2,776 binding sites for the vitamin D receptor along the length of the genome. These were unusually concentrated near a number of genes associated with susceptibility to autoimmune conditions such as MS, Crohn’s disease, lupus and rheumatoid arthritis, and to cancers such as chronic lymphocytic leukaemia and colorectal cancer.

This new study supports this hypothesis, having found a significant number of vitamin D receptor binding sites in regions of the genome with genetic changes more commonly found in people of European and Asian descent.

The main source of vitamin D in the body comes from exposing the skin to sunlight.

"Vitamin D status is potentially one of the most powerful selective pressures on the genome in relatively recent times," says Professor George Ebers, Action Medical Research Professor of Clinical Neurology at the University of Oxford, and one of the senior authors of the paper. "Our study appears to support this interpretation and it may be we have not had enough time to make all the adaptations we have needed to cope with our northern circumstances."

Abstract: Link Here
Article from Oxford University: Link Here
Article WebMD: Link Here

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Bioactive Dietary Polyphenols (EGCG) Decrease Heme Iron Absorption

Researchers at the Department of Nutritional Sciences, Pennsylvania State University have published a study about the use of polyphenols such as EGCG, found in green tea and green tea extracts, and GSE from grape seeds. Both can have a negative effect on the body's ability to absorb iron, a necessary component required to carry oxygen in red blood cells. These cells' cytoplasm is rich in hemoglobin, an iron-containing biomolecule that can bind oxygen and is responsible for the blood's red colour.

By using cell from the intestine where iron absorption takes place it was found that polyphenols bind to iron in the intestinal cells, and this prevents them from being absorbed into the blood stream. As the cells die and are replaced, the non-absorbed iron-polyphenol complex, is passed in the faecal matter.

Researchers advise that the most common cause of anemia is iron deficiency and people at high risk of developing an iron deficiency should be aware of what polypenols they are consuming.

Since some CLL patients take EGCG as a possible means of controlling their cancer, they may want to reconsider this in light of this recent study.

Source: The Journal of Nutrition, doi:10.3945/jn.109.117499 Link Here
Other Article: Link Here

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High serum thymidine kinase 1 level predicts poorer survival

The researchers at MD Anderson (MDA) have been looking at serum thymidine kinase 1 (TK1) in CLL patients. Thymidine kinases (TK1 and TK2) have a key function in the synthesis of DNA and thereby in cell division, as they are part of the unique reaction chain to introduce deoxythymidine into the DNA. TK1 is the cell cycle regulatory gene. Two forms of this protein have been identified in animal cells, one in cytosol and one in mitochondria. Activity of the cytosolic enzyme is high in proliferating cells and peaks during the G1-S phase of the cell cycle; it is very low in resting cells.

The the past TK1 levels have been used to assess NHL using inconvenient radioenzyme assays.

In this study, the researchers used a novel chemiluminescence assay to assess serum TK1 levels in patients with CLL at the time of first examination.

They showed that high serum TK1 levels predict poorer overall survival and correlate with unmutated immunoglobulin variable region genes, CD38 and ZAP-70 expression, and subsequent risk of developing large B-cell lymphoma (Richter syndrome).

Similar findings were observed in a subset of patients treated with current fludarabine-based chemotherapy regimens.

The team at MDA conclude that serum TK1 levels analyzed using this convenient new assay may be useful in the risk assessment of patients with CLL.

Source: American Journal of Clinical Pathology, 2010 Sep;134(3):472-7

Abstract: Link Here

More of TK1: Link Here

More on Chemiluminescence arrays: Link Here

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There's a hole in my bucket dear Liza, dear Liza

The concept of slotting CLL patients into 3 buckets (A,B,C) was introduced in 2003 and has been fairly good in arranging patients into simple risk and disease progression groups. But over the past few years, with new research and diagnostic tools like CGH matrix tests that are in some ways superior to the FISH analysis, it is being realized that CLL is far more complex and patient centric than previously believed. The bucket classification is clearly over simplification.

In 2008, Dr. Shelly Gunn MD, PhD, Medical Director of CombiMatrix Molecular Diagnostics using Hemescan, a CGH array, identified submicroscopic deletions of chromosome 22q11 in over 15% of CLL patients studied. The frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. However, to date, this is not tested by FISH and its meaning is unknown. So, what bucket are patients with 22q11 deletions to be placed in? Further, we know that a deletion of 6q21 occurs in about 7% of CLL patients, also 8q24 and the very rare trisomy 18 or 5q deletion. What about CLL patients with more than one genetic abnormality?

Earlier in 2010, Dr. R Houlston in Britain, announce that he had located a number of genetic deletions that indicated that patients with 13 or more 'risk factors' are 7 times more likely to develop CLL. So, what impact if any, does this have on our buckets? It is simply too early to tell.

Then there is, what is now called, F-CLL (Familial CLL) and V-CLL (Variant CLL). What about SLL patients, should they have their own buckets?

Recently, a study published in journal Blood, has confirmed what Dr. Terry Hamblin has observed for a number of years, which is, that there is a sub-group of 17p deleted patients with mutated IVGh genes, as the study states; "despite the poor prognosis of most patients with 17p deletion, a small subgroup of chronic lymphocytic leukemia (CLL) cases with 17p deletion may be relatively static, not requiring treatment for years with relatively long survival."

Add to this the just published findings that protein levels of CDK4, p27 and p53 define distinct prognostic subgroups in CLL then the bucket analogy is clearly starting to leak badly...

As our knowledge about CLL is gaining in complexity, perhaps the time has come to develop a new more robust framework for patient classification based on both the old and newer prognostic markers and risk factors, so subsets of CLL patients aren't falling beyond the pail.

Source: Risk Buckets ~ 2003
Source: Whole Genone scanning in CLL
Source: Familial CLL and MBL
Source: V-CLL
Source: Rare 5q deletion
Source: 17p deleted sub grouping
Source: Protein expression analysis of CLL

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The CLL path is different for each patient ~ this may explain why

In CLL genomic aberrations identify prognostic subgroups, pointing towards distinct underlying biological mechanisms that are poorly understood. In particular it remains unclear, whether the prognostic subgroups of CLL are characterized by differential levels of leukemogenic proteins.

A study from Germany, published in Haematologica, 2010 Aug 16, took a look at 23 different proteins that are known to effect cell death (apoptosis), cell proliferation, DNA damage, and cell signalling.

Case were categorized hierarchically into deletion(17p), deletion(11q), trisomy 12, deletion(13q) as sole abnormality or normal karyotype. In addition, the expression levels of CDK4, P27 and P53 were quantified over the clinical course and compared to levels in immuno-purified B-cells of healthy individuals.

The results showed that in subgroups with a good prognosis (13q), differential expression was mainly seen for proteins that regulate apoptosis. In contrast, in cytogenetic subgroups with a worse prognosis (17p- and 11q-), differential expression was mostly detected for proteins that control DNA damage and proliferation.

It was also noted by researchers that expression levels of the proteins CDK4 (cyclin dependent kinase - Cyclin D), p27 (Cyclin-dependent kinase inhibitor protein p27) and p53 (the tumour suppressor protein) were higher compared to B-cells of healthy individuals and significantly correlated with increasing hierarchical risk. In addition, no significant longitudinal changes of expression levels of CDK4, p27 and p53 could be detected.

In conclusion it was felt that differences in expression levels of apoptosis and proliferation controlling proteins define distinct prognostic subgroups of CLL and uncover a correlation of levels of CDK4 , p27 and p53 proteins with higher hierarchical risk.

Source: PubMed: Abstract Link Here

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Resveratrol ~ new study shows some promise in the lab

Resveratrol is a phytoalexin produced naturally by several plants when under attack by pathogens such as bacteria or fungi. It has also been found in the skin of red wine grapes, Japanese knotweed and in lesser amounts in mulberries and blueberries. Resveratrol degrades considerably when heated.

A recent study in ANNALS OF HEMATOLOGY has had a look at it as a possible treatment for CLL when combined with Fludarabine or Cladribine EX VIVO (in the lab NOT in a patient). Researchers for the first time found that both resveratrol + Fludarabine and resveratrol + Cladribine caused a higher rate of apoptosis in comparison to the rate caused by a single drug. The percentage of apoptotic cells induced by resveratrol alone was higher in the group of patients with better prognostic markers than in those with worse prognostic markers.

Of most significance, the study revealed that resveratrol induced apoptosis in CLL cells in a tumour-specific manner but did not affect non-leukemic cells, and apoptosis was associated with a decreased BCL2/BAX ratio.

The researchers concluded that:

"The obtained results indicate that resveratrol might warrant further study as a new therapeutic option for CLL patients. This naturally occurring substance may be used as a single agent, especially in older persons for whom there are some limitations for the use of aggressive treatment. On the other hand, a lower purine analogue dose could potentially be used in combination with resveratrol because of their combined effect."

One of the mechanisms of action of resveratrol is the induction of DNA damage, which ultimately leads to apoptosis."

Full PDF Document: Link Here

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Nurses the backbone of the treatment team ~ new fungal infection knowledge survey, somewhat disquieting

A nationwide survey of 230 U.S. oncology nurses recently published, took a look at treatment and fungal infections. Invasive fungal infections are a leading cause of mortality and morbidity in immunocompromised patients.

While a large proportion of oncology nurses are knowledgeable about the risk for fungal infections in this population and the precautions that need to be taken to prevent and/or minimize that risk, there were important gaps in knowledge.

For example, 66% of nurses believed that hematopoietic stem cell transplantation (HSCT) patients and those who developed graft-vs-host-disease (GVHD) were at high to very high risk.

Looking at the other side of this coin, that means that 34% of the nurses surveyed didn't know this group of patients were at high to very high risk of fugal infections!

In general, oncology nurses are increasing concerned about invasive fungal infections. The incidence has risen sharply during the past 20 years. A primary reason for this increase is the greater number of patients who are undergoing HSCT as a cancer treatment.

Infections caused by Aspergillus species have become increasingly common, especially among patients who have undergone HSCT. According to the report, invasive fungal infections have historically carried a mortality rate ranging from 50% to 90%, and remain a leading cause of infection-related mortality among HSCT patients.

Further the survey found that the cost of treating an invasive fungal infection places a high economic burden on patients and society. Hospitalization for a patient with aspergillosis is approximately 17.5 days, with a resultant cost of $82,425. For invasive candidiasis, the cost of treatment ranges from $34,000 to $44,500 per patient.

Survey Results: Invasive Fungal Infections

Medscape Article

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17p deletion CLL may have a relatively static subgroup, not requiring treatment for years

This study comes from Rossi and his colleagues from ULM University.

They state in journal Blood, "despite the poor prognosis of most patients with 17p deletion, a small subgroup of chronic lymphocytic leukemia (CLL) cases with 17p deletion may be relatively static, not requiring treatment for years with relatively long survival."

Rossi and colleagues were able to show that miRs correlated with 17p status and specifically were down-regulated (miR-34a, miR-181b, miR-497) or up-regulated (miR-15a, miR-21, miR-155) in CLL with 17p deletion. In addition, expression of miR-21 and miR-181b was shown to be associated with overall and progression-free survival, respectively.

Although most 17p deletions target a large region on 17p, the TP53 locus is always affected and most cases with 17p deletion will have a TP53 mutation on the remaining allele. This observation, as well as the observation that CLL with TP53 mutation shows a similar outcome as cases with 17p deletion, suggests that p53 is central to the clinical course of CLL with 17p deletion.

It is therefore intriguing that the model build based on miR-21 expression and 17p deletion also held up in a cohort with a low incidence of 17p. This finding raises the question of how miR-21 is regulated in cases with intact p53 status.

As with all important research there are many questions raised by the work of Rossi and colleagues. It will also be important to characterize the independent impact of miR-21 expression in models including 17p and TP53 mutation. Before translating the results into clinical practice, it will be important to confirm the impact of the deregulated miRs in a uniformly treated cohort; ideally, this should be done with purified CLL samples derived from a prospective trial. This is particularly important as the follow-up is relatively short (20 months) and the cohorts studied diverse.

Although the current work does not attempt to identify the basis for the association between miR-21 expression and outcome, the authors hint at some potential targets. It will be important to understand the function of these miRs and, particularly, how they relate and depend on functional p53. A first step toward this goal will be the detailed characterization of not only 17p status, but also TP53 mutation.

Article Source: Link Here

Article in the same issue of Blood on miR-21: Link Here
Croce & Pekarsky miR-29: Link Here

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Reactivation of hepatitis B virus (HBV) in CLL

Reactivation of hepatitis B virus (HBV) is a frequent complication of chemotherapy in patients with HBsAg carriers. In this prospective study,researchers documented chemotherapy induced HBV reactivation risk in patients with hematological malignancies. HBV reactivation risk is influenced by baseline viral load.

The study divided the population into two groups according to HBV-DNA status. HBV-DNA negative patients (n=18) were treated with nucleoside analogues once HBV reactivation was observed. HBV-DNA positive patients (n=12) commenced lamivudine before the initiation of the chemotherapy.

• In HBV-DNA negative patients HBV reactivation was found in 10 patients (55.5%).


• HBV reactivation was significantly more frequent in chronic lymphocytic leukemia (CLL) patients (P=0.008) and in patients receiving rituximab containing chemotherapy regimens (P=0.06).


• Eight patients (80.0%) responded to antiviral treatment after HBV reactivation.


• Two CLL patients experienced a flare-up after the withdrawal of antiviral therapy.


• In HBV-DNA positive patients, HBV reactivation was observed in four patients (33.3%) during lamivudine treatment and in two patients after lamivudine withdrawal.

This small study demonstrated the increased risk of chemotherapy-induced HBV reactivation in CLL patients, for the first time.

Source: Hematology. 2010 Aug;15(4):240-4.
PubMed: Link Here

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