Seminars in Hematology
The January 1997 issue of Seminars in Hematology was the last official publication of the Polycythemia Vera Study Group (PVSG). Summaries of these articles are available below as reviewed by a non-health care professional member. An article on interferon treatment for polycythemia vera was also part of that issue, but this review is included on a separate link available at the main webpage.
Prologue & Epilogue - closing of the Wasserman-PVSG era; lessons from the study of PV
Experience of the PVSG with essential thrombocythemia
PVSG report on hydroxyurea in patients with PV
Management of PV with hydroxyurea
Erythropoietin receptor mutations (and a note on familial myeloproliferative disease)
Long-term evolution of PV in patients treated by phlebotomy & 32P
Anagrelide
FISH - fluorescence in situ hybridization (chromosome testing)
In vitro (test tube) erythropoiesis in PV and other myeloproliferative disorders
SEMINARS IN HEAMTOLOGY Vol. 34, No. 1, January 1997, POLYCYTHEMIA VERA edited by Nathaniel Berlin
"Prologue: Polycythemia Vera: The Closing of the Wasserman-Polycythemia Vera Study Group Era" by Nathaniel Berlin
"Epilogue: Broader Lessons From the Study of Polycythemia Vera" by Paul Berk
I believe it was Charles in our group who asked what had happened to the Polycythemia Vera Study Group (PVSG). Now we know. This fourth and last series of publications officially closes the PVSG. The four series have been published at about ten-year intervals in Seminars in Hematology - 1966, 1975-76, 1986, and the current issue - Jan. 1997. (In addition there have been other individual reports and the book - POLYCYTHEMIA VERA AND THE MYELOPROLIFERATIVE DISEASES edited by Wasserman, Berlin, and Berk; published by Saunders in 1995.) In the prologue Berlin outlines three eras in the history of polycythemia vera (PV). The first he calls the "Vaquez-Osler era" which began in 1892. Venesection (phlebotomy) was introduced, as well as the agent - hydrazine, and x-radiation. The "Lawrence-Donner Laboratory era" is the second - beginning in 1939 when the first patient was treated with radioactive phosphorus (P32). Wasserman worked in the Donner Lab where arterial blood saturation, blood volume levels, and measurement of erythropoiesis (red blood cell production) with radioactive iron were first applied to the diagnosis of PV. (Important, for example, in determining the type of polycythemia - vera, secondary, relative.) The third period began in 1967 with the establishment of the PVSG by Wasserman. Funding was provided by the National Cancer Insitute. Diagnostic criteria were determined and the three randomized arms of treatment - phlebotomy-only, chlorambucil, and P32 were set up. Berlin, and also Berk in the epilogue, bemoan the loss of American funding for long-term trials for PV/MPD. Neither federal funding nor major pharmacutical support is available for "studies of an uncommon disease." As such, it was up to Najean in France to continue following the PVSG protocols - P32 and phleb-only arms (chlorambucil having been closed due to high leukemic conversion). The Israelis have followed the protocol with hydroxyurea. Berlin remarks that the leadership in the PV field rests now, particularly in Europe where a French and an Italian group have been established (instrumental in starting the European Collaboration on Low-Dose Aspirin in PV, for example). Berlin reviews what has come out of the PVSG era - clonal disorder of stem cell origin, erythroid precursors are hypersensitive to erythropoietin (Epo) AND to insulin-like growth factor. Epo receptor seems to be normal (except in familial high-affinity hemoglobin polycythemias - not vera)."PV is a progressive disorder. Those who do not develop a thrombotic or vascular disorder or cancer eventually develop myelofibrosis and some....acute leukemia." (There may be a separate pure, idiopathic, benign erythrocytosis.) The progression to MF is beginning to be better understood - also the role of platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF beta). Hydroxyurea has been effective in reducing thrombotic occurrences, but may be leukemogenic - particularly if used with P32 or alkylating agents. Questions have arisen with phlebotomy-only treatment. Two new treatments - anagrelide and interferon have emerged. Attention is just beginning to focus on Epo-producing tumors causing polycythemias (apparently secondary sources of polycythemia not always recognized in the past). Berlin identifies the new era as that of "molecular-biology" where it is hoped that research at the cellular level will provide clues and answers leading to better treatment (cure??).
Berk in the epiloque covers some more subtle or subjective issues that have risen through the PVSG era. He reminds the reader of far-reaching medical advances made through PV research - clonal origin of malignancy, levels of cell involvement in hematopoiesis, stimulating hormones like Epo, cytokines - to name just a few of the discoveries that have had an effect beyond PV. Berk also lists a modified algorithm for the evaluation of polycythemia (vera, relative, secondary). Berk includes the improvements made in life-expectancy, but also states that research for better treatment (and cure) needs to continue. He notes that, at least at the clinical level, the leadership in PV study has passed from American to European hands.
A VERY quick overview of the various articles' contents (we'll do a more complete review of each in the coming weeks):
"The very long-term evolution of PV: an analysis of 318 patients initially treated by phlebotomy or P32 between 1969 and 1981" by Najean and Rain. The result of these two PVSG arms is recorded. Good results as far as P32 in regard to disease remission and lack of resistance (ceasing to work after a time), though leukemogenicity is a factor. Phleb-only arm can't really be evaluated statistically. More later.
"From efficacy to safety: a PVSG report on hydroxyurea in patients with PV" by Fruchtman, et al. Looks at long-term survival with HU and phleb-only. More on this later.
"Management of PV with hydroxyurea" by Tatersky and Sharon. Israeli results with HU are generally favourable - used alone. Risk of leukemia needing to be weighed with risk of thrombotic events.
"Experience of the PVSG with essential thrombocythemia: a final report on diagnostic criteria, survival, and leukemic transition by treatment" by Murphy, et al. Quite a lengthy report including the difficulty of diagnosis - possible subsets, leukemia conversion (rare in untreated patient), long-term outcome. In general the statistics given are for an older group of patients. It is noted that a younger, predominately female group of ET patients might not fit the conclusions drawn.
"Interferon alfa:effects of long-term treatment for PV" by Silver. We have a review of this article online. If you missed it and would like it, please let me know.
"Anagrelide for control of thrombocythemia in polycythemia and other MP disorders" by Petitt, et al. A brief overview includes dosage information and response; along with possible adverse reactions. Attractive for young patients because it does not appear to produce secondary malignancies. HOWEVER, a warning is given that it should NOT be used in pregnancy - the small size of the molecule could cross the placenta.
"FISHing among myeloproliferative disorders" by Najfeld. Fluorescence in situ hydridization is unique in its use of analysis of genes and chromosomes because it can work on a single cell level. This means, for one thing, a better(and easier) detection of chromosome abnormalities. More later (I hope - it's pretty technical!!!)
"In vitro erythropoiesis in PV and other MP disorders" by Weinberg. This in vitro (test tube or dish) stuff is quite interesting. Details on what exactly is found in those EEC tests on PV patients and what the significance might be. How does Epo and the various other colony growth factors affect PV blood in a different way than non-PV blood? PERHAPS OF THE MOST SIGNIFICANCE - PV blood is VERY sensitive to insulin-like growth factor (IGF-1). (Previously thought that a sensitivity to Epo was significant, but the sensitivity to IGF appears to be much stronger.) More later.
"Erythropoietin receptor mutations and human disease" by Gregg and Prchal. Layman's terms - receptors are the "doormen" of each cell. The Epo-receptor "takes" the Epo from the outside of the cell, through the membrane, and into the cell where it can perform its particular function. Receptors appear to be normal in PV (although whether they can begin to function abnormally as an affect of the disease - acquired - still needs to be studied). However, in familial polycythemia (secondary, not vera) such as high-affinity hemoglobin abnormalities - the Epo-receptor DOES appear to be abnormal.
EXPERIENCE OF THE POLYCYTHEMIA VERA STUDY GROUP WITH ESSENTIAL THROMBOCYTHEMIA: A FINAL REPORT ON DIAGNOSTIC CRITERIA, SURVIVAL, AND LEUKEMIC TRANSITION BY TREATMENT by Murphy, et al., publ. in Seminars in Hematology, volume 34, no. 1, 1997.
Is there a distinct disease of essential thrombocythemia within the myeloproliferative disorders? Has the criteria for diagnosing this entity changed since the PVSG first set down ET criteria 20 years ago? Is there a subset of ET composed of younger, primarily female patients? Is there a progression of ET to PV to MF to acute leukemia? Are there any indicators of which ET patients progress and when this might occur? These are some of the questions that the article deals with in its final report on ET within the PVSG. (No, not all the questions are answered completely!!) The authors note that, yes, there is a subset of younger, predominately female, patients for whom the conclusions this report finds may NOT apply. Actually they do express concern and caution in the use of hydroxyurea for the young ET patient. (The PVSG patients were an older group of ET'ers - median age in the 60's - and, as such, were all considered at a high risk of thrombotic events and treated with myelosuppressives. There are a number of published articles on ET in younger patients, can thrombotic risks be identified, progression to PV, etc.) Before reviewing the entire article contents, let me "pull out" three interesting points that may be of importance to all ET'ers (and PV'ers). The first two points deal with changes in the PVSG criteria for diagnosing ET. The PVSG criteria are still commonly used today (though additional tests such as endogenous erythroid or megakarycyte colonies, platelet measurements, spleen size, can also be done). ET, according to the article, is still primarily a diagnosis of exclusion. In other words - with a high platelet count (over 600,000) all other possibilities ((PV, CML, MDS, MF, or reactive (secondary) thrombocytosis)) must be ruled out first. The updated diagnostic criteria is listed in the article and discussed somewhat. I won't go into that except for the two points that might be new to some. FIRST - failure to identify the Philadelphia chromosome can not rule out CML; a particular DNA or RNA gene arrangement must also be checked. In the same area - if another particular chromosome abnormality is found (for example, the 5q deletion), it may point toward MDS (myelodyplastic syndrome). SECOND - one of the original PVSG criteria for ET was the presence of stainable iron in the bone marrow. (Its absence being an indicator of PV.) It is now reported that ET may have negative iron marrow stain (even reactive thrombocytosis can be caused by iron-deficiency). They report that if iron deficiency is noted, PV can be excluded if a trial of iron therapy fails to increase RBC mass. BUT, the absence of stainable iron alone is NOT enough to rule out ET. The THIRD point of particular interest to ET (and PV) patients in the article deals with the issue of hydroxyurea (HU) for treatment. As previously mentioned in postings on HU, there seems to be a high percentage that convert to leukemia when HU is used with another, P32 or alkylating, agent. In this PVSG report it was 5 of 7 patients that had been switched from HU that developed leukemia. (Small numbers though some other reports have higher - some enough to be called "statistically significant.") The difference in this report is that they note the HU was given first. (The order is not given any significance in the other reports. In Weinfeld, for example, HU is listed as being given second in some. My thoughts only - HU is more likely to be given first now, then something else if the HU isn't working; earlier it was the HU that was the "new kid on the block" and more likely to be given second.) The report postulates that perhaps the higher incidence of leukemia is because these were patients in a subset more likely to get leukemia since they had poor disease control with HU. (My thoughts again - if this is true, we should see this leukemic conversion also with those who have switched from HU to anagrelide or interferon. This has not been reported so far with 8 years or so of switched treatment. And, I'm not sure that disease control was the issue in the earlier reports of switching to HU.) ......On a personal note, it was these reports that gave Norm and me concern in looking for an alternative if the interferon did not do the job. We had initially thought of HU, but once on HU, Norm would not want to switch to a different alkylating agent if his disease became resistant to HU. (And there are reports of this.) So we began looking more seriously at pipobroman which has been used long-term in Europe (and, according to reports, "ignored" by the PVSG)...... In summarizing the report - 91 patients (platelets above 1 million) were followed and evaluated. However, there were possible questions on the diagnosis of 41 of these patients. Various attempts were made to confirm diagnosis in these patients, BUT since there were CML, PV, or MF questions about these 41, the final leukemia stats need to be evaluated accordingly. It is noted that leukemia in untreated ET patients is rare. In 1986, a review uncovered 15 cases of acute leukemia in ET, but 14 had been treated with P32 or an alkylating agent. A literature review from 1986-1992 found 15 cases of acute leukemia after treatment with HU-only. Of the 91 PVSG patients followed for up to 15 years, there were 41 deaths. Twelve of these were from acute leukemia (others were vascular accidents, tumors, and others causes related to age). Neither hemoglobin, hematocrit, platelet count, nor spleen size was helpful in identifying patients at risk for leukemic conversion. A slightly higher WBC (white blood cell) count WAS present at diagnosis (median 14.3 compared to median 11.2) and was considered jsut barely "statistically significant." Bone marrow biopsies were not particularly helpful either EXCEPT that there was a higher conversion to leukemia (2 of 4) in those patients that had ET/MF questions about their diagnosis. (Spleenomegaly, leukoerythroblastic blood, and mild marrow fibrosis - with this picture one wonders why they had an ET diagnosis in the first place, but this possibly reveals some of the problems with a straight PVSG criteria for diagnosing ET.) There were nine patients that moved to a new classification of PV or MF. Six of these were from the original group of 50 patients for whom a diagnosis question was NOT an issue. Of the 22 patients treated by HU alone, 14 were still alive with a median follow-up of 8.2 years. The highest number of patients to move to leukemia, as mentioned, was in the group switched from HU to another treatment. Conclusions of the report - the median survival for the group was just under ten years. Conversion to leukemia was 20% at 10 years. (These sound like lousy stats to me, but their median age was over 60, and all were treated.) The authors continue to recommend HU for the older patient at risk of thrombotic events. THEY QUESTION THE USE OF HU FOR YOUNG ET PATIENTS. ....This is in contrast with Fruchtman's report ("From efficacy to safety:...") reviewed below. He concludes that HU is PERHAPS (sort of a weak endorsement)the safest for young PV patients since it has been used the longest, etc. Just over 60 percent of PV patients started on the HU arm of the PVSG were alive after 15 years; just under 45% of those started on the phleb-only arm were still alive after 15 years. Also, not yet reviewed, the Israeli report from this journal concludes that HU is the treatment of choice for young PV patients. However, I do not believe their data is very complete
"FROM EFFICACY TO SAFETY: A POLYCYTHEMIA VERA STUDY GROUP REPORT ON HYDROXYUREA IN PATIENTS WITH POLYCYTHEMIA VERA" by Fruchtman, et al. (publ. in Seminars in Hematology, vol. 34, no. 1, 1997.
It seems that this study leaves one with a lot more questions than answers. No doubt the authors are aware of this also, stating, for example,"....formalizing treatment guidelines that universally apply to all patients remains problematic." They also conclude that what is needed, but unlikely to occur given current funding limitations, are very long-term trials of significiant numbers of patients comparing a variety of currently available myelosuppressive therapies, including interferon. Without these trials, the authors conclude that all conclusions and recommendations reached are going to be subject to criticism and controversy. This IS what we have seen and continue to see in treatment options for PV(MPD); this IS why it is so important to be well-informed as a PV patient, AND this IS why our MPD group is so necessary and valuable. That said, the article reviews some of the early conclusions of the PVSG - an increased risk of thrombotic events in the first 3 years of phleb-only patients, that higher-dose aspirin (common at that time) increased hemorrhagic complications (it has been left to the Europeans now to conduct needed trials with low-dose aspirin), and the emergence of hydroxyurea as the most commonly used drug in PV patients given myelosuppressives. Hydroxyurea inhibits an enzyme of DNA synthesis and since this makes it different from previously-used alkylating agents, it was hoped that it would be safe for long-term use. The investigators concluded that HU was effective in controlling thrombocytosis (elevated platelets) and elevated hematocrit, but did allow for continued phlebotomies of up to six/year. They noted that it needed to be used continuously with average daily doses of 500 to 1000 mg. If the drug is discontinued, remissions are extremely short AND REBOUND thrombocytosis commonly occurs in those patients who initially had a high platelet level. Frequent and continuous blood monitoring was required early in therapy because of possible occurrence of cytopenias. However, the long-term mutagenic potential (leukemic conversion) had not been determined. In 1986, Kaplan came out with the report comparing thrombosis and acute leukemia in the Hu/phleb arm with the phleb-only arm of the PVSG. Though the HU group had a greater prior history of thrombotic events, this group had significantly less thrombotic events than the phleb-only. In about a 7-year period there were 5 out of 51 who had events in the HU group(9.8%) while 44 of 134 had thrombotic events in the phleb-only group (32.8%). The current report includes further follow-up (though PVSG was closed, letters were sent to investigators) up to just past 15 years with a medium follow up of 8.6 years. In respect to leukemia conversion, the differences in the two groups are not considered statistically significant - 3 out of 51 Hu patients (5.9%) and 2 of 134 phleb-only patients (1.5%). (Again when they CALL something "statistically insignificant" according to the laws of probability & statistics, I STILL like to see what the stats are!!!) There are a number of tables included and some of the data gets a bit confusing (for me anyway!!!) but let me see if I can summarize it a bit. In respect to spent phase - the differences are not considered statistically significant BUT let me give them to you: 4 out of 51 on the HU (7.8% - this is similar to the spent phase seen in the P32 and chlorambucil arms also in the earlier 1986 report); however, spent phase in the 134 phleb-only arms is 15 to 17 patients (2 switched off phleb-only). This comes out to around 11 to 13%. Now when the spent phase/leukemia is added together and the results in the two arms compared - the results are very similar - the leukemia being higher in HU but the spent phase is lower; vice versa with the phleb-only. (Though there is a slight reflection here possibly of HU when switched to P32 or alkylating agents being MORE likely to convert to leukemia.) The problem I always see when, for example, the 1986 statistics on spent phase for phleb-only are listed is this: if the 44 deaths that occurred in the early years are taken into account (which, as mentioned, is much higher with phleb-only than the other arms) THEN the 16 spent phase (and another 2 or 3 leukemia) is a higher percent of the remaining alive phleb-only. Sixteen (or 19) of 90 is closer to the 18-20% range. (Now we are in the range that Najean finds as well as a 1982 unpublished PVSG report.) Although, the approx. 12% usually listed is still higher than the 8% in the other arms, this is considered "statistically insignificant." ......(It is annoying to me to read reports where these earlier deaths are ignored in forming conclusions. Greenberg's article in the 1995 Seminars in Oncology is a fine article - I have recommended it to many - but he didn't even get the numbers listed correctly. He reported 6 phleb-only had gone to spent phase when the actual number was 16!! Even in this issue of Seminars in Hematology - the Israeli group mentions Messinezy's disagreement with Najean's stats on phleb-only. If one reads the Messinezy reference they give, it is a letter in response to Najean and, to me anyway, seems to try comparing apples with oranges - phleb-only patients who HAVE been treated with other agents!!!)... This current report DOES give an indication of some of these issues by comparing the "overall survival" of the phleb-only and HU/phleb arms. Here, the authors state "a trend for improved survival in patients treated with hydroxyurea is evident." When considering deaths in the two arms (up to 15 years now), there are 54 (remember 43 of these were earlier thrombotic events) of the 134 phleb-only for over 40%. The HU arm is 31%. When THOSE REMOVED from the phleb-only arm to other treatment are taken into account, the deaths are 74 or over 55%. HU is still under 40% with those switched to other agents taken into account......I think this IS very interesting especially considering that the HU arm included from the beginning those that may have been considered to have a more active disease (more thrombotic events) and still allowed for up to 6 phlebotomies a year AND in the second set of stats above included those that were switched to another treatment, generally considered to be because of poor disease control (with perhaps a higher leukemic conversion after the switch).... The authors do make some conclusions. The elderly should be treated with myelosuppressives. They conclude that the spent phase development is the same for HU and phleb-only. (As I commented at length, I believe even their own stats are closer to confirming Najean's conclusions than their own.) However, they do question HU's long-term safety. Concern is expressed over the potential use of HU for treating sickle cell disease. Since the data being used is from the PVSG reports, the authors urge caution. Children have a more actively dividing stem cell pool which could contribute to greater long-term toxicity. They believe interferon may be of benefit, but, as with HU, long-term safety is not yet determined and the cost is a concern.
MANAGEMENT OF POLYCYTHEMIA VERA WITH HYDROXYUREA by Tatarsky and Sharon, > publ. in Seminars in Hematology, vol. 34, no. 1, 1997.
This study follows the PVSG (Polycythemia Vera Study Group) protocol of > HU-only (other than phlebotomy) patients treated at an Israeli clinic. > Although funding for the PVSG study was cut in 1984, the clinic continued > following (and added some) patients through 1995. So a total of 71 > patients were entered (median age 54, median yrs on trial is 7.3) in the > trial period of 1980 - 1995. Nine patients were under the age of 40 (12.4% > - this is younger than the reported median, interesting that Silver also > had a higher percent of younger patients than would be expected by the > standard reported 5% under 40). Sixty-one patients (86%) were of > Jewish-Ashkenazy origin though the ratio in the area population is said to > be under 40%. Phlebotomy-only was the prior treatment of 33 of the 71 > patients and some continued with phlebotomies while on HU. > Twenty-seven patients had died by the end of the study (38%). The study > is not really clear on the cause of death in all of these. In five > patients, a thrombotic event occurred (7%). Only 12 of 25 patients who > started with moderate to marked splenomegaly had, at least, a temporary > improvement. Two had to undergo splenectomy. Four (5.6%) developed acute > leukemia. Six patients discontinued HU (apparently NOT included in the > patients mentioned above) for reasons of toxicity - jaundice, leg ulcers, > hypothryroidism, etc. Four patients (all over 70) developed malignant > tumors. Pruritus improved in most patients. > In respect to myelofibrosis, hydroxyurea did NOT decrease the cellularity > of megakaryocyte content of the marrow. Reticulin content DID increase > slowly over the years. However, the actual incidence of confirmed > conversion to myelofibrosis was only in two patients (2.8%) which makes it > smaller than, for example, the phlebotomy-only arm of the PVSG. The > article does not tell us if these two patients were among those treated > previously with phlebotomies. > The article alludes to other PVSG stats that indicate leukemia may be > higher IN those myelofibrosis patients who ARE treated by HU. (More on > this when we review the new Najean article from this same issue.) The > authors also mention Najean's finding of phlebotomy-only being an increased > risk of MF, but then the authors state that Messinezy disagrees and give a > reference. (As I mentioned earlier, I wonder if they READ the reference > they gave for Messinezy. It is just a letter AND really compares apples > with oranges since Messinezy is not comparing patients who have ONLY been > treated by phlebotomy.) > The article concludes by recommending HU for patients under 60 who need > myelosuppression. This is in contrast to some other articles in this same > issue of Seminars in Hematology. Silver does NOT recommend HU for patients > under 70; Murphy, et al, do NOT recommend HU for young ET patients; and > Fruchtman gives a weak endorsement for HU (leaving an opening for > anagrelide and interferon). Fruchtman also recommends caution and further > study when thinking of HU for sickle cell, for example. Though Fruchtman's > data does show a "trend for improved survival" in those patients treated by > HU compared to those treated by phlebotomy-only. (Remember that just over > 60% of those started in the HU arm were still alive after 15 years, just > under 45% of those started in the phleb-only arm were still alive. > Significant to me especially since the older and previous-thrombotic-event > patients were more likely to be placed in the HU arm. More on this when we > review the Najean article.) > In conclusion, a reminder that this article only details the results of > the clinic's experience with patients treated only with HU or Hu and > phlebotomies. It does not include their data on those patients previously > treated with other myelosuppressive agents and then swtiched to HU.
ERYTHROPOIETIN RECEPTOR MUTATIONS AND HUMAN DISEASE by Gregg and Prchal, publ. in Seminars in Hematology, vol. 34, no. 1, 1997.
(Also a note on FAMILIAL MYELOPROLIFERATIVE DISEASE.)
Erythropoietin is the principal growth factor involved in the production of red blood cells (erythropoiesis). ((We could also call it the "hormone" involved in red cell production, but the typical (out-dated?) definition of "hormone" as an agent produced in one organ of the body, moving to another area of the body for its major "work" is not an accurate one. Erythropoietin is apparently mainly produced (?) in the kidney, but it is now known that it can be released from many different areas of the body (tumors, for example), and it can stimulate red cell production in other areas than the marrow (spleen, for example).)) Anyway, the cells that are going to become red blood cells (erythroid progenitor cells) from the original stem cells (called pluripotent because they could become progenitor white cells or platelets also) ---these erythroid progenitor cells have receptors on them to receive the erythropoietin (Epo). The receptors are called erythropoietin receptors (Epo-R). (Strictly in my layman's words - the Epo-R is a "door" that will "open" (activate) to allow the Epo through the cell membrane into the inner cell. There are up to 1000 or more of these "doors" - Epo-receptors on each erythroid progenitor cell.)
The Epo-R gene has been located on chromosome 19. It would seem logical that mutations of this gene would affect erythropoiesis and, in fact, this has been observed. In vitro (test tube) mutations of Epo-R have resulted in proliferation of erythroid cells; in some mutations the Epo-R is activated with only a little Epo, in other mutations - there is proliferation with no Epo. (Mutations of genes can be of various types - I'm not really up on this stuff, folks!! - but there can be deletions, transfers to wrong segments, all in varying degrees, etc.)
Murine (mouse) studies have also shown a particular virus (no, nothing we've heard of) have affected a protein which binds to Epo-R causing the receptor to activate with proliferation of red cells (all without any Epo).
Before we get excited about Epo-R defects being the cause of polycythemia vera, the authors point out that Epo-R defects have not been noted in PV patients other than in some particular, familial congenital polycythemias - usually secondary polycythemias.
........A note before discussing the article's familial polycythemias. Messinezy (in the 1996 article previously reviewed from MOLECULAR ASPECTS OF MEDICINE) notes that even if Epo-R gene alterations that cause activation do NOT play a role in PV, it is still possible that an abnormality between the "signal pathways" of cells could be affected by Epo and Epo-R. This sounds somewhat like Spivak's note to us that an "intracellular signalling defect" appears to be unique in PV and idiopathic myelofibrosis. HOWEVER, Spivak was speaking of a platelet dysfunction. The Epo and Epo-R abnormalities would not affect platelets or granulocytes (white blood cell), says Messinezy, but only erythrocytes. Bilgrami & Greenberg, on the other hand (1995 - PV in Seminars in Oncology), tell us that there are two types of Epo-R on each cell. Most are "low affinity" to Epo; others are "high-affinity." These authors state that PV patients are lacking in the "high-affinity" Epo-R, but note that it does not seem to affect the total rate of Epo into the cells.......
Back to the article and familial, congenital polycythemias. Epo-R gene abnormalities have been found in some of these families; the authors go into detail about the types of mutations that have been detected. There have been 7 different reported mutations of the Epo-R gene and, in some cases, NON-diseased family members had the same mutations. So, in these cases, the Epo-R gene abnormalities could not by themselves have caused the disease, but must interact with other, unknown factors. On top of that, in some cases family members who DO have the disease, do NOT share the abnormalities of other diseased family members. (Why do I get the impression, that's also the way it is with all myelproliferative syndromes? Of course, in the case of "acquired" abnormalities, it is not unusual that different abnormalities which show up with a conal stem cell origin of the disease; I think.) In addition, though, some of these congenital polycythemia families do NOT show any abnormalities of the Epo-R gene.
The familial polycythemia noted in the article includes the high-oxygen affinity hemoglobin (a secondary polycythemia, not VERA), but also some other polycythemias that show a true elevated red cell mass without the other lines (platelet, white cells) being hyperproliferative. (This is along the line of what we mentioned in the review of the Najean/Rain article - a possible benign erythrocytosis.) The authors in THIS article note that reports of premature cardiovascular disease have recently been noted in families with this Epo-R mutation in spite of a controlled hematocrit.
........Perhaps this would be a good place to ask about a congenital tendancy in the myeloproliferative diseases (not just a "benign" erythrocytosis affecting only the red blood cell line, but affecting ALL three lines as is found with MPD). In Dr. Harriet Gilbert's chapter entitled "Familial Myeloproliferative Disease" in the book POLYCYTHEMIA AND THE MYELOPROLIFERATIVE DISORDERS, 1995; data on 12 kindreds (26 patients) with MPD is described. Berlin, in his chapter of the same book, states that Dr. Gilbert's chapter is on the "rare polycythemias." The impression is that Dr. Gilbert's data is about this unusual congenital erythrocytosis without meeting the other criteria of myeloproliferative disease. In fact, this is not what Dr. Gilbert describes at all. The 26 patients from 12 families appear to have very definite myeloproliferative disorders (PV,ET,MM,MF, leukemia) with a high rate of progression to MM/leukemia in the PV patients. As Dr. Gilbert notes, other literature and documentation exist to indicate that this tendancy of familial PV (MPD) is not as rare as usually thought. (We see this in our own experiences here on the list, as well as Medline searches.) Dr. Gilbert reasons that patients and doctors have not been familiar enough with myeloproliferative disease and its symptoms, often failing to look into family history. (A case in point - in our home we are well aware of Norm's family's history of autoimmune disease, but we only recently learned that his maternal grandmother had died in her early 40's of a stroke. Who knows?)......
Returning to the article! The authors conclude with two more identified diseases that might possibly have Epo-R abnormalities. Diamond-Blackfan anemia (congenital pure red cell aplasia), though known to have a failure of erythroid progenitors responding to Epo (and other growth factors), did not show an Epo-R abnormality in tests. In erythroleukemia, studies continue as conflicting results exist on what and whether Epo-R abnormalities are involved.
THE VERY LONG-TERM EVOLUTION OF POLYCYTHEMIA VERA: AN ANALYSIS OF 318 PATIENTS INITIALLY TREATED BY PHLEBOTOMY OR 32P BETWEEN 1969 AND 1981 by Najean and Rain, publ in Seminars in Hematology, vol. 34, no. 1, 1997.
Since this article (like the other final PVSG reports) deals with lots of numbers and graphs, I'll try to make it more interesting (for me, at least!) by summarizing the important material in four main points.
1. The data, as we are aware from Najean's previous reports, supports the conclusion that "maintenance treatment by phlebotomies....induces an early and high risk of progression to myelofibrosis." Therefore the authors, as Silver also conludes, do NOT recommend phlebotomy-only for patients who satisfy the criteria for polycythemia VERA (more on this at the end of the review). The chart (figure 3) is a "graphic" portrayal of these statistics. (You know - "a picture is worth a 1000 words" - the same might be said of a graph.) .......When reading the charts in this report, remember the solid lines represent those on phlebotomy-only; the dotted lines correspond to all those started on phleb-only, so the phleb-only is still included..... As the authors note, it is impossible to follow-up the 104 patients (median age - 62 years) initially started on phlebotomy-only since by the fifth year - 50% (still alive) were receiving myelosuppressives and by the 10th year - 90% (still alive) were on additional treatment. Reasons for the earliest changes are listed as risk of vascular accidents due to age and/or excessive platelets. Subsequent changes are listed as patient lassitude, splenomegaly and spent phase signs, and myelofibrosis. Seventy-three of the surviving original 104 were changed to hydroxyurea (HU) or 32P. The HU was preferred for the younger patients and/or splenomegaly/MF. The median survival for these patients was approximately 18 years. As the author states, since nearly all were on HU or 32P, this data chiefly concerns those treatments. There appears to be quite a drop between the 18th and 20th year since there are only 26 patients alive after this point (one left on phlebotomy). The graph (fig. 2) seems to show considerably less at the 25 yr point (like 6 ??). The authors call these curves (figure 2) "difficult to interpret." (So you can imagine how I feel!!!) ......The percent on the side of the graph (Fig. 2) does not seem to correspond with the actual surviving numbers as indicated by the numbers in parentheses (actuarial survival - based on % expected to live??). If you have a copy of the article and can clarify this chart for me, please do!!!!......
2. Najean has previously reported that perhaps HU begun at the beginning of spent phase could delay the evolution to MF. The authors' present data supports this theory "that myelosuppression may induce a regression or a stabilization, but only of the early phase of this complication." For example, there were 26 patients that switched from phleb-O due to splenomegaly (22 to HU, 4 to 32P). Seven of these had only moderate splenomegaly, 12 had a huge enlarging spleen, and 7 had overt MF/MM. Of the seven with moderate splenomegaly who switched from phleb-O, only one had evolved to MF at the time of the report. Of the 12 with huge spleens (Najean equates this with "spent phase"), 7 have since developed overt MF. Of the 7 cases who had overt MF at the time of the switch, 6 had died within 6 years. The authors also note that the rate of progression to MF (depending somewhat on classification of "spent phase," etc.) "regularly increases with time, without any plateau, whatever the treatment used."
3. As with previous reports, the authors noted an excess of cases devloping leukemia in patients treated by 32P and HU. (Murphy's article in this same issue felt there might be a significance in HU being given first; this was not the case with the leukemic conversion in this report, nor in previous reports.) Whether there is a greater risk to leukemic conversion of MM/MF patients who are treated by alkylating agents (without considering the HU combo) is suggested by some reports (and disputed by others). At any rate, the authors state that all myeloproliferative syndromes represent, to varying degrees, preleukemic states. And the risk of malignant complications increases with time. Twenty-five percent of patients who die with MF are in a state of leukemic transformation state the authors.
4. The fourth point to cover is one of the main purposes of the article - reporting on the PVSG patients treated by radioactive phosphorus- 32P (called "radiotherapy" by the authors). There were 214 cases included in the present report with a median age of 68. The median survival was 13.5 years. The authors were very positive about these figures considering the older age and increased thrombotic risk of those put in this original group. Complete remission lasting 3 years was obtained in 98% of the patients after a single dose. The risk of leukemia with 32P was similar to that of other alkylating agents - 10% at the 10th yr, 17% at the 15th yr, and 25% at the 20th yr. The risk was significantly related to the dose of 32P. The authors did indicate that remissions become shorter with time, though true acquired resistance (just stopped working) was only reported in 6 cases of those 167 patients living and followed over 10 years.
...A note on polycythemia VERA diagnostic criteria. Najean, and others who recommend myelosuppression, are clear that this should be used on patients for whom a polycythemia VERA diagnosis is clear. Najean speaks of a "pure erythrocytosis" without risk of progression to MF. Berlin says there may be a separate entity - sometimes called "benign" or "idiopathic," as well as "pure." These terms generally speak of an erythrocytosis without a known secondary cause, nor meeting P.VERA criteria other than a high hematocrit (red cell mass or packed cell volume - RCM or PCV) and no apparent decrease in plasma volume causing the high hematocrit (relative polycythemia). Messinezy notes that in many of these patients with "idiopathic erythrocytosis" - a secondary cause or other primary (VERA) criteria may eventually develop. Some familial polycythemias - usually are secondary such as the high oxygen affinity hemoglobins - but these may have an abnormal Epo receptor gene and they may be a primary polycythemia. Berlin also mentions the relationship of various tumors to polycythemias (normally would cause secondary polycythemia). Some of these have been missed in the past. It is known now that not only kidney/liver tumors can cause secondary erythrocytosis, but also tumor sites like the cerebellum, skin, esophagus, adrenal glands, ovaries, even uterine fibroids. (In an interesting case covered in a later review from this issue, removal of a pituitary gland tumor reportedly "cured" a case of actual P.VERA.) And there are numerous causes of "relative polcythemia" (hematocrit is elevated not because of more red cells but because of reduced plasma volume). Hypertension, obesity, sleep apnoea, acute alcohol consumption, smoking (also a secondary cause), etc., or any combination of the above can reduce plasma volume. And plasma volume can be tricky to determine (needs to be done before phlebotomy also to use in diagnosis). It is reported that plasma volume can vary in individuals just according to amound of physical activity, temperature of environment, time of day, etc., not to mention the variations of body height versus body weight/surface area. So, when looking at the recommendations for myelosuppression in polycythemia vera, the authors are not considering cases that might be secondary or relative polycythemias, nor a case of "pure, benign, idiopathic erythrocytosis" with no other criteria for polycythemia VERA. (such as bone marrow biopsy hypercellularity or elevated counts in the other blood lines, etc.)......
ANAGRELIDE FOR CONTROL OF THROMBOCYTHEMIA IN POLYCYTHEMIA AND OTHER MYELOPROLIFERATIVE DISORDERS by Petitt, Silverstein, and Petrone, publ. in Seminars in Hematology, vol. 34, 1997.
This report IS said to be the published continuing summary of 942 patients treated at least four years by anagrelide. The authors state that though several hundred other patients received anagrelide during the past 10 years, these patients are NOT included in this report, BECAUSE the anagrelide was given in "desperate attempts to control late stage chronic myeloproliferative disorders." It is also reported that these patients had been given multiple agents and that data on these patients is incomplete. ......I assume this is a reference to the unpublished data that Pam is trying to obtain for us - though this report does give the data for 942, up from the previous reported 577 patients. Whether this is typical with an unapproved (FDA) drug - giving it out to "desperate" patients and then not including that data - I don't know......
Anagrelide was tested originally as an antiaggregating drug - the thrombocytopenia (low platelets) which occurred with human testing was unexpected. It had not been observed in any of the animal testing, including monkeys. So, in 1985 testing began for control of myeloproliferative thrombocythemia. After initial testing on 20 patients (90% responded), the Anagrelide Study Group began in 1988 with testing on 577 patients. These results were published in 1992 (Amer. J. of Med.). Breakdown of the 942 patients is 546 (58%) with ET, 113 (12%) with PV, 179 (195) with CML, and 108 (11%) with undifferentiated myeloproliferative disease......If you add up the numbers I just gave you - it equals 946, NOT 942, as the number repeated throughout the article. Four patients lost to follow-up, just sloppy proofing, or what???....... Mean age was 58 and mean platelet count was just over 1 million. 86% (810) of patients had received prior drugs - including hydroxyurea, busulfan, interferon, and 32P. Reasons for change included age, failure to respond, or side effects.
So, how does anagrelide work reducing platelet count? The mechanism is not really known. At therapeutic levels given to patients, megakaryocyte colonies and colony size are NOT affected. (Megakaryocytes are the big clumps that the platelets come from - my words, obviously.) However, megakaryocyte maturation, ploidy & cell diameter ARE affected. (Ploidy refers to future chromosome generations - whatever THAT means.) Platelet reduction is NOT universal. The authors list it at somewhere between "84 and 94% of patients studied to date." (HOWEVER, later in article we find that only 79% of the patients included in this study had a partial or complete response. The authors appear to be giving data here from the earlier studies.) The antiaggregating effect IS said to be universal, but only at levels 10 times the concentrations given to induce thrombocytopenia. Since the platelet-reduction effect occurs only in humans, it is theorized that anagrelide must be converted in the human body to "as-yet unidentified derivative which in turn affects megakaryocytes." (Another research article theorizes that "more than a single molecular mechanism may be involved in anagrelide-induced thrombocytopenia.") With discontinuation, platelet counts return to pretreatment levels in 5-7 days. This supports the theory that anagrelide interferes with maturation at a late stage (rather than early-on when stem cells differentiate to megakaryocytes/early forms of platelets).
The article then details the patient criteria used, response rates, and dosage. Patient criteria was platelets over 1 million, or over 600,000 with other criteria of MPD being satisfied. A total response was said to occur in 70% (platelet count below 600,000 or 50% less than treatment levels for 4 weeks) and a partial response in 9% (20 to 50% reduction). Initial doses varied from 0.5 mg 4 times/daily for asymptomatic patients to 1 mg 4 times/daily in patients with extremely high platelet counts. Nearly all responded within 1 week with satisfactory levels achieved by 2 weeks. Time to complete reponse was said to range between 17 and 25 days. When patients were already on another drug, anagrelide was given in gradually increasing doses while decreasing the dose of the other drug. NOTE: The authors state, "The drug is excreted by the kidney, and mild dose reductions have been necessary in a few patients (mostly hypertensives) who have developed significant renal impairment."
Side effects - headache by 37% of patients, palpitations - 26% ......NOTE: and again here I find confusing data. This report lists 26% (252 total patients) experiencing palpitations - but authors state that only 14% of Mayo-treated patients had palpitations. HOWEVER, in the previously published report of 577 patients (1992 report of the 577 patients - supposed to be included with these 942 is my understanding) palpitations/forceful heartbeat/tachycardia were observed in 209 (of the 577) and this was 36%!!!! They have added 365 patients and only come up with 43 more experiencing palpitations. I suppose it has to do with the definition of "palpitations" with the earlier report including "forceful heartbeat" and tachycardia...... Other side effects - diarrhea at 25%, as noted by others here in our group, is said to usually be caused by the lactose capsule - use an intestinal lactose supplement the authors say. Fluid retention - 20% (and again smaller numbers are said to be with Mayo patients - from the 1995 report). This fluid retention may be responsible for the mild anemia observed with anagrelide, the authors state. A few patients are reported to have experienced congestive heart failure shortly after initiating anagrelide treatment. NOTE: the authors warn "anagrelide should be used only with great caution in patients with a history of CHF, or in patients in whom such a situation might be expected to appear." How many patients? This report does not say - in the 1992 report on 577 patients, there are 14 CHF listed. This report does list 50 deaths as occurring while on therapy (includes 15 cardiac and 14 pulmonary). No deaths are said to be directly related to the anagrelide though it is stated that the drug cannot be exonerated in 7 instances. (A previous report on fewer patients notes that 2 sudden deaths might be attributed to the drug.) NOTE: Just a reminder that the authors previously reported that hundreds of "desperate" late-stage MPD'ers who received anagrelide are NOT included in this report. Back to other side-effects which include gastrointestinal and dizziness. (The 1992 report gives a more specific listing of side-effects.) 125 patients (13% withdrew due to side-effects. (I assume this does NOT include the 50 that died.) The authors conclude that 90% of patients had platelet reduction. (Well, I thought they said 79% had a complete or partial response. Maybe they're using stats from the Mayo-only study again. When they give the Mayo-only stats, by the way, this is NOT the 1992 report on 577 patients but a 1995 report.) The authors point out that mitosis (cell division) and nucleic acid metabolism (RNA/DNA stuff) does not appear to be affected so oncogenesis (cancer-causing) has not been associated with anagrelide treatment. In this respect, anagrelide is different from many agents used previously to control thrombocythemia. NOTE: A warning is given that anagrelide should NOT be used in pregnancy. "...the molecule is of such a small size that it could easily cross the placenta with disastrous effects on the fetal platelet count."
THOUGHTS: I do have to comment that, although it may not involve anything of any real significance, the way different stats (from a 1995 Mayo-only study) are incorporated into this study is confusing. And these Mayo-only stats are not even the same stats reported in the 1992 report on the original 577 patients of the Anagrelide Study Group. The authors state that these are included to show the better response rate achieved at the Mayo clinic from those noted by physicians who were not as experienced with the drug. Since most patients are NOT going to be treated at the Mayo clinic - I'm not sure where this fits, but perhaps when computing data for a clinical trial - it does. The only other varied stat that "jumps out" at me would be the matter of heart palpitations which I mentioned previously. Another note - in the 1995 article (a text chapter actually), Silverstein states that he believes anagrelide may have a positive effect on fibrosis by affecting release of PDGF (platelet-derived growth factor). There is no such mention in this later report, though Silverstein is one of its authors. I have no idea whether there is any significance in the fact that it is NOT mentioned in this later report; I just bring it to our attention. (Anecdotically, we have reports of one - possibly two? - cases of fibrosis increasing while on anagrelide.) And, a last note, since no leukemia has been noted in these published reports (some patients followed for over ten years now), it IS hoped that anagrelide is NOT leukemogenic in the long-term. Research indicates that anagrelide does not appear to alter the megakaryocyte progenitor cells or mitotic expansion of developing megakaryocyte precurser cells. This, I understand, is positive in respect to leukemogenicity. Anagrelide does appear to affect platelet production by reducing megakaryocyte size and ploidy, and disrupting full maturation. I am not a chemist (or whatever it takes to understand this stuff), but I do point out that anagrelide DOES affect megakaryocyte ploidy and size - this is reported to be a function of DNA in the cell. (See reports listed below.) Certainly, as the reports state, it appears to be an attractive alternative to other drugs used previously known to be leukemogenic, especially when considering treatment for young patients.
"Analysis of the Mechanism of Anagrelide-Induced Thrombocytopenia in Humans" by Mazur, et al., publ. in BLOOD, vol. 79, 1992.
"Anagrelide: A Review of its Pharmacodynamic and Pharmacokinetic Properties, and Therapeutic Potential in the Treatment of Thrombocythaemia" by Spencer & Brogden, publ. in DRUGS, issue 47, 1994.
FISHING AMONG MYELOPROLIFERATIVE DISORDERS by Najfeld, publ. in Seminars in Hematology, vol. 34, no. 1, 1997.
The article introduction gives us reasons why better chromosome testing is necessary, and why in MPD (other than CML/AML), chromosome testing is not routine at many centres. All those fuzzy little chromosomes (you know, the things that look like black & white caterpillars) are just not that easy to analyze. In spite of technological advances in counting, locating, etc., this entire matter of "cytogenetic analysis" has remained difficult and time-consuming. In addition, with MPD there may be a marrow "dry tap" (no cells to examine) or normal karyotypes (set of chromosomes) may be found in the dividing cells being examined. If single cell nuclei, or cells in steps before division, could be examined - abnormalities might be apparent. Along came FISH - fluorescence in situ hybridization. The technique has been around for awhile in research, but application to the clinical setting is more recent. What does FISH do differently from previous chromosome testing? Well, first of all the fluorescence lights up the chromosomes - now we have bright blue and green caterpillars and, the various areas (DNA segments on the chromosomes where breaks, deletions, rearrangements, etc., have taken place during cell division) can now be marked in multicolors. (The article does NOT detail it like this - strictly my layman's definition.) The article DOES point out that with FISH several target areas (DNA segments) on the chromosomes can now be visualized in the same sample. This IS an important advantage. The article then details three types of DNA probes that have recently been developed to utilize with FISH in detecting segments on the chromosomes. ......DNA probes locate the exact spot desired on the chromosome and stick to it with an antibody. Now the segment is "lit up" by the FISH technology and can be analyzed for an abnormality. (I THINK this is how it works!!)..... Anyway, the three types of DNA probes are: 1) centromere and telomere - these light up the centromere area of the chromosome (sort of the middle of the X before division) and telomere (the ends). 2) sequence-specific (also called, I believe, gene probes) - these are probes that can be used to locate specific genes (their DNA segments) on chromosomes. In this way gene deletions, translocations, amplicfications can be identified. (All of these are chromosome abnormalities that can occur.) How soon these probes are available for all genes depends (the authors remind us) on the speed of the Genome Project (mapping all the human genes). Again, new technology has made it possible to view DNA segments (for example, of single genes) that are too small to be seen with conventional technology. 3) whole chromosome probes (also called "paint probes") allow the entire chromosome (with its entire DNA sequence) to be viewed. This is important, for example, in finding translocations. (An abnormality where a portion of the DNA may have moved to an entirely different location on the new chromosome when the cell divided.)
Interphase FISH is the next section of the article. This may well be the most important aspect of the FISH technology when compared to conventional cytogenetic testing. ......"Interphase" refers to the cell BEFORE mitosis (cell division). With the other conventional methods, only the "metaphase" is examined as metaphase is the stage in mitosis when the chromosomes are most condensed and easily distinguished from one another...... The article lists a number of reasons why interphase FISH is an advantage over metaphase observation including the shorter time period (2 to 4 hours) in which the procedure can be completed. What differences have been found when searching for PD abnormalities comparing interphase FISH with conventional cytogenetics? The authors give the results of 9 published reports comparing methods. Although the two techniques were in agreement most of the time, 8 of 9 reports found some discordant results - 48 of 351 patients. In addition, the authors had 18 of 58 discordant results in their own study. Most of these varying results involved chromosomes 7 and 8. Monosomy 7 (1 copy when it should be 2) did not go into mitosis (cell division) and was therefore undetected by conventional cytogenetics. Trisomy 8 (3 copies instead of 2) was also more often observed with FISH ( and occasionally, vice versa). Another advantage of FISH is then detailed - the observation of genetic material on a single cell basis. This has obvious importance "for detection of clonality, lineage involvement, and events involved in the pathogenesis of MPD." (In other words - at what point did the malignant clone appear, how and which various blood lines did it affect, how does MPD develop in different individuals.) With FISH, not only can the individual cell be observed, but the actual genotype and phenotype can be seen at the same time. (Genotype - all the genes whether dominant or recessive, phenotype - only the genes that are expressed.) The authors detail the first applications of this technology. PV patients' cells with Trisomy 8 were tested wtih EPO-stimulation. Trisomy 8 cells of MPD patients have also been checked on various precurser cells (myeloid, lymph) and then checked again down the blood lines to see if the trisomy 8 showed up in the various myeloid cells (neutrophils, eosinophils, basophils) and lymphocytes. Monosomy 7 has been observed in the same manner. The conclusion for both trisomy 8 and monosomy 7 is that these abnormalities (observed with some MPD patients) are restricted to committed progenitor cells of the myeloid line. The deletion of chromosme 20 (another of the more common MPD abnormalities) found in Epstein-Barr virus has been the subject of some important FISH studies. Del 20q through interphase FISH has been found in bone marrow cells but NOT peripheral blood granulocytes. (The Epstein-Barr 20q del was found in the bone marrow precursers of granular WBC's, monocytes and B cells.) The study showed (as already suspected) that del 20q could be acquired as a result of abnormal granulopoiesis (granular WBC production).
Metaphase FISH is the next topic covered in the article. (Remember that "metaphase" occurs during cell division. In conventional cytogenetics this is the point where chromosomes are broken apart, stained, and examined.) The value of FISH in metaphase studies lies in observation of genes and areas known to be implicated in myeloid disorders and, in locating DNA segments (band regions) that are critical in hematopoietic growth factors, receptors, differentiation to various blood cells, etc. With FISH, regions that were too small with conventional cytogenetics can now be observed and viewed within the entire chromosome. For example, it was previously thought that del 20q was a terminal deletion. It is now known (through FISH) to be an interstitial deletion. Other examples are given in the article - all of this is very important in determining the "multistep pathogenesis of chronic MPD."
The article concludes with some of the possible future applications of FISH - simultaneous, multi-color coding of all 24 individual human chromosomes, is one of the more exciting. The reader is also reminded of the time advantage with FISH. Hybridization that takes 16 hours in most labs can be done in 30 seconds. The question is raised on whether more than the 30% of MPD patients that presently display an abnormal karyotype will be found to have abnormalities under future studies. (An earlier chart in the article documents the percent of abnormalities found in studies on over 1000 MPD patients- excluding CML. Consistent with other studies - MM/MF had the highest percent - 44 to 50% abnormalities. MDS has 34%, PV - 29% and ET - 31%.) The authors state that the current studies have provided definitive evidence that chromosomal rearrangements in MPD form in an early progenitor cell that is capable of differnetiating along the myeloid, erythroid, and sometimes the B-lymphoid line, but not the T-cell line. This is said to be consistent with a multistep pathogenesis. FISH also offers the future possibility of monitoring the cell lines in response to targeted therapy.
IN VITRO ERYTHROPOIESIS IN POLCYYTEHMIA VERA AND OTHER MYELOPROLIFERATIVE DISORDERS by Weinberg, publ. in Seminars in Hematology, vol. 34, no. 1, 1997.
Again, some really interesting research summed up in this article, but, also, quite technical. So, please, keep in mind my obvious limitations in interpreting this stuff! Before going into the entire article summary, let me mention what was, for me, the MOST interesting (and new) research.
PV PROGENITOR CELLS ARE MORE THAN 100-FOLD SENSITIVE TO INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) THAN NORMAL PROGENITOR CELLS. AND, IGF-1 RECEPTOR ABNORMALITIES HAVE BEEN FOUND IN PV PATIENTS. (Remember that receptors are the "doors" to the cells. Epo-receptors appear to be normal in PV patients.) ONE PV PATIENT (yes, apparently P.vera, not secondary polycythemia)SHOWED NO SIGNS OF PV AFTER REMOVAL OF A PITUITARY GLAND TUMOR (causing abnormal levels of IGF-1).
More on this later. For now, to summarize the rest of the article. A brief explanation is given of the research determining that the myeloproliferative disorders arise from an abnormal stem cell (called "multipotential" or "pluripotent" because it has the potential to become - erythrocyte, granulocyte, lymphocyte, platelet). There is some variation when it comes to the lymphoid line with this abnormality. (Some PV patients show the abnormal clone in lymphoid line; others don't.) In CML there appears to be lymphoid involvement.
Three different possibilities are listed for why different cell lines are mainly affected in the different myeloproliferative disorders (platelet in ET, erythroid in PV). (Though one characteristic of MP is that ALL three lines are affected - fitting the stem cell origin.) 1) different abnormal clones may actually be involved for the different variations of MPD with a different line being affected as the clone responds differently to normal regulatory factors 2) two defects may be required - one, yes, in the stem cell, but then a second abnormality in the committed progenitor cell (one that has already "decided" whether it will be platelet, RBC, WBC, etc.) may be required which then determines WHICH myeloproliferative disorder one gets 3) it is simply random whether the affected stem cell becomes platelet, erythrocyte, granulocyte, and which ever it DOES become - that becomes the mainly affected line
The article then details the various "colonies" that are formed from the multipotential progenitor cell. (The stem cell doesn't just automatically become the mature cell - various "steps" are involved. At each "step" there are various growth factors that have different effects on its continuation to the next level.) For example, the burst forming unit-erythroid (BFU-E) is the earliest identified cell committed to becoming a red blood cell. However, it comes from a prior colony, and goes to another colony - colony forming unit-erythroid (CFU-E), and from there into an erythroblast. (And then you still have proerythroblasts; early, intermediate, and late erythroblasts, and reticulocytes before you get to the erythrocytes - red blood cells. At what point the erythroblast leaves the CFU-E or whatever, I don't know!) As well, there are CFU-G (granulocytes) and CFU-mega (megakaryocytes). (The CFU-M are macrophages, not megakaryocytes.) Some of these colony forms are found in the marrow and blood; others in normal individuals are found only in the marrow.
Now we get to the endogenous erythroid colonies. The article calls this a "hallmark" of PV and states that this IS a diagnostic tool to distinguish PV from secondary polcythemia. (Explanation: in EEC tests, blood from a PV patient, in the test tube, will produce erythroid colonies WITHOUT adding Epo - erythroipoietin - to the mixture.) Secondary polycythemia patients' blood will NOT produce the colonies without added Epo. The only problem with this diagnostic tool is that, although PV patients bone MARROW will always produce the colonies, SERUM (blood) cultures sometimes do not. The author also mentions (without details) the possibility of some familial polcythemias producing EEC. (We've mentioned this in previous reviews.) What about endogenous colonies in the other myeloproliferative disorders? Is it a diagnostic tool? In CML, patients DO form colonies IF burst promoting activity (BPA) is added. (BPA's are growth factors - interleukin 3 is one.) EEC was not definitive in myeloid metaplasia (MM); however, in ET, some recent studies have indicated (mentioned previously online) that positive EEC tests may be an indicator of latent PV - with these ET patients eventually devloping overt PV. (The author does not mention it, but there are reports now of endogenous megakaryocyte colonies as diagnositic tools in ET.) Positive EEC tests have also been observed in patients with Budd-Chiari syndrome- hepatic(liver) vein thrombosis. These positive test results existed before any other PV symptoms. So, to sum up the EEC information from the article - a positive EEC test from a serum (blood) culture IS indicative of PV. However, in its absence, a negative marrow EEC culture would be necessary to rule out PV.
The author then details the research of erythropoietin (Epo) in PV. Much of this information has already been mentioned before online - some in previous reviews from this issue. Epo-receptors do not appear to be abnormal in PV patients; PV patients seem to lack high-affinity Epo receptors, but this does not appear to affect Epo "uptake" into the cell. Also, PV patients appear to have both very Epo-sensitive progenitor cells and normal Epo progenitor cells. It is believed to be these Epo-sensitive cells that give rise "endogenously" in the serum-containing medium. They are believed to be responding NOT to "no Epo" (since Epo is not added to the medium), but rather are responding to the very minute levels of Epo found even in serum-free medium because of biological materials (albumin) found in the medium. (The albumin containing growth factors.) NOW - a very interesting and new (to me anyway) research development - a culture medium which is truly free of biological materials has recently been developed. (In other words - BEFORE in all these tests with Epo and PV, they could not be sure that other growth factors in "minute" amounts were not affecting the outcome.) Previous culture systems were known to have a growth factor - BPA (burst promoting activity) present. NORMAL AND PV BLOOD FAILED TO PRODUCE COLONIES IN THESE NEW TRULY SERUM-FREE CULTURES, EVEN WHEN EPO WAS ADDED, UNLESS A BPA (such as interleukin 3) WAS ALSO ADDED!!! MORE IMPORTANT: ADDING INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) RESULTED IN THE GROWTH OF ERYTHROID COLONIES (specifically BFU-E) EVEN WITHOUT EPO!!! AND, the normal and PV cells responded identically to Epo (with BPA) in this serum-free culture, BUT the PV progenitor cells were 100-fold more sensitive to IGF-1 than the normal cells. In addition, the IGF-1 receptors, as mentioned earlier, exhibit sensitivity in PV patients.
The author also mentions increased sensitivity of PV patients' cells to granulocyte macrophage colony stimulating factor (GM-CSF) and stem cell factor (SCF).
At this point, the patient with acromegaly (caused by a pituitary gland tumor)and PV is mentioned. He had elevated IGF-1 and growth hormone levels. After removal of the tumor, all abnormalities resolved.( ( I obtained a copy of the article detailing this case. It was NOT a case of secondary polycythemia caused by increased erythropoietin released from a tumor, for example. He fit the clinical diagnostic criteria of PV, as well as the bone marrow and EEC test. He had had a splenectomy about ten years previously due to a splenic hematoma. Although the article states that some of the hematologic abnormalities COULD have been caused by the acromegaly, they ALL resolved following the surgery - including the positive EEC (no longer showed colony growth). The original bone marrow was hypercellular, but no fibrosis nor extraneous cells were noted. He had been followed for four years with no return of PV symptoms.))
To return to the original article, it concludes with a note on the apparent hypersensitivity of PV to a number of growth factors. The author mentions that defective intracellular mechanisms (perhaps involved in communication of these growth factors?) may be involved. This last point of the author's reminds us again of Spivak's mention of an "intracellular signaling defect." On the other hand, the article definitely raises questions about the role of Epo in PV. Spivak reported to us that PV cells differentiate faster and that "polcythemia vera erythroid progenitor cells survive better in the absence of erythropoietin than normal ones. Therefore, PV is not a disorder of over production, it is a disorder of accumulation." Well, the sensitivity of PV cells to Epo has been listed in a number of studies - but this article details other growth factors that have a much greater effect than the Epo (indeed the Epo appears to NOT have that effect without the added growth factors). How does this fit in, if at all, to the proliferation/accumulation theory? I haven't a clue!!!