National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for

Use of Tumor Markers in Testicular, Prostate, Colorectal, Breast and Ovarian Cancers

Supplemental Data Comments Received Table: Independent Comments received

Testicular Cancer – Comments received.

From Professor Barry Hancock, Cancer Research Centre, Weston Park Hospital, Sheffield,

UK.

I thought this was very good. I was going to make a couple of comments regarding hCG assays,

then thought not because the controversies in gestational trophoblastic neoplasia are not relevant

to testicular cancer. I asked Rob Coleman to look at the document and will forward his comments

also. Thank you for inviting our views.

Thank you for your comments.

From Dr Grahame Howard, Department of Clinical Oncology, Western General Hospital,

Edinburgh, UK.

This is an excellent summary .I cannot comment on the laboratory aspects but the clinical section

reflects our practice. The guidance on frequency of follow up and marker tests is different to our

own practice and reflects USA practice. I am not sure if this is relevant to you and I could give

you our follow-up protocols if you wish. [Requested]

Thank you for your comments and for the copy of the protocol.

From Professor Michael Seckl, Department of Medical Oncology, Charing Cross Hospital,

London, UK.

1/ Page 2

Regarding a trial of antibiotic treatment for a testicular mass prior to performing an ultrasound, I

think this would be a rather low key approach especially for a painless mass as described in the

lines above. Probably should delete this and suggest urgent referral for ultrasound. Of course if

the patient has a hot red swollen testis that would be different and a trial of antibiotics might be

reasonable.

2/ Page 2

The authors describe performing a testicular biopsy in suspected testicular cancer. This would

seem a bit unusual and contrary to good practice in the UK and US. One would worry about

seeding of tumour along the biopsy track leading to secondary spread to groin nodes etc.

3/ Page 3 line 5. The authors say that surveillance for stage 1 seminoma is not recommended in

the US. (Well perhaps not by everybody, but a number of key opinion leaders such as Craig

Nichols now support it and it is the number 1 choice on the NCI webpage for the treatment of

testicular cancer!)

1

4/ Page 10 Genetic aberrations

The authors talk about A gain in 12p without introducing what it is. It would be nice to have it

explained/introduced. Similarly in the middle of the page after ref 74 they talk about.....due to

epigenetic changes.... again it would be nice to have a bit more information, such as due to

epigenetic changes such as ...methylation ? silencing ??.....

5/ Page 12 Staging

In this section the authors seem quite happy with the use of surveillance for stage I seminoma (at

odds with earlier in the text)

6/ Page 13 I am a bit confused as to which stage patients are getting RPLND or chemotherapy

here. I think this needs to be made clear in the text.

7/ Page 14 'Surveillance'

I think this section needs clarifying. Does primary therapy mean orchiectomy or does it mean

chemo or RT? The paragraph talks about patients with good risk disease treated with surgery

alone. This is a bit confusing as good risk sounds like good prognosis, where the patients must be

treated with chemotherapy.

Otherwise with a bit of tidying up and being reviewed from the clinical point of view then this is

an interesting and useful set of recommendations.

Thank you for your comments. Suggested changes have been made to the manuscript.

From Professor Rob Coleman, Cancer Research Centre, Weston Park Hospital, Sheffield,

UK.

This is an excellent review and a very useful resource. My only comment is that for the adjuvant

treatment of seminoma, short (single) course carboplatin should be mentioned as an alternative to

radiotherapy (p3). The reference for this is:

Oliver RTD., Mason MD., Mead GM., van der Maase H., Rustin GJS., Joffe JK., de Wit R.,

Aass N., Graham JD., Coleman R., Kirk SJ., Stenning SP for the MRC TE19 collaborators

and the EORTC 30982 collaborators. Radiotherapy versus single-dose carboplatin in adjuvant

treatment of stage I seminoma: a randomised trial. The Lancet 366: (July 23) 293-300, 2005.

Thank you for your comments. This reference has been incorporated [Ref 10].

PROSTATE CANCER

From Mr Prasad Bollina, Department of Urology, Western General Hospital, Edinburgh,

UK.

Thank you for giving me a copy of the manuscript of NACB Guidelines for the use of tumour

markers. This is very interesting and informative and I am grateful for giving me the opportunity

to look at this manuscript. I hope it is in order for me to make one or two observations and

comments.

2

PSA - Age Specific Reference Ranges:

I note with interest that the NACB recommends against the use of age specific reference levels

and has changed its previous recommendation. As you know this would be of immediate

relevance to us with our group proposing to implement the age specific reference levels across

[specified geographical region]. The manuscript does highlight the evidence of a significant

proportion (20-25%) of cancers being picked up in the PSA range of 3-4ug/L. I think this is pretty

strong evidence and as you know clinical practice is strongly moving towards this.

The guidelines in the manuscript quite rightly do not endorse a general recommendation on mass

screening but advise PSA testing based on informed decision making by the individual. If this is

the case then a single cut off level of 4ug/L may have the risk of the individual not able to make

valid informed decision making and may be unfair to ignore the fact that a PSA level of 3-4 may

be significant for the younger age group.

Use of % Free and Free PSA

The use of % free and free PSA is being recommended and as you know it's use is in clinical

practice, although somewhat patchy. I must say we have not looked very closely at this issue and

I now wonder whether we should consider this as part of our protocol here in [specified

geographical region].

No response required

From Professor Fritz Schröder, Chairman, Department of Urology, Erasmus Medical

Centre, Rotterdam, the Netherlands.

Thank you for your letter that includes the draft report on prostate markers intended for

publication in Clinical Chemistry. I have gone through the manuscript and I must say without

reading every single word. Let me comment on those issues that came to my mind.

The PSA cut-off point of 4 which is referred to on page 3, second paragraph and which is

discussed on page 4 in the section on age specific reference ranges.

I believe that PSA cut-off values in 2007 cannot be discussed without making reference to the

findings obtained within the control arm of the Prostate Cancer Prevention Trial (PCPT by

Thomson et al NEJM 2003 and 2004 as well as in JAMA 2005. This latter paper contains

information on the sensitivity and specificity distribution and the sensitivity of detecting

potentially aggressive cancer in the lower PSA ranges. There is at this moment no easy way to

resolve this problem. I would feel that it makes a lot of sense to include other predictors such as

the ones described in a paper by Roobol et al in The Prostate 2006. Clearly, prior use of PSA for

early detection, a previous biopsy, and prostatic volume should be mentioned as potential

predictors of biopsy outcome and they should also be put into context with the problem of PSA

cut-offs in the sense that they can be used to improve specificity of testing in the low PSA ranges,

including PSA 4-10.

3

Results from the PCPT have been incorporated.

Your tables 6 and 7 take over recommendations from various agencies to utilise PSA velocity as

a diagnostic criterion. I could not find the text that relates to this issue. Let me just say that the

reference of Carter relates to clinical prostate cancer in situations where the patient later dies of

the disease. The paper by Berger is severely biased by case selection and attribution bias. The

paper should distance itself from these recommendations.

PSA velocity is discussed on p. 35, last paragraph.

Otherwise I don’t really have comments. Once again, let me stress that due to a limited time I

have not read the paper completely. The references to our own work seem to be adequate.

From Professor Hein von Poppel, Chairman, Dept of Urology, Director of the European

School of Urology, University Hospital of KULeuven, Leuven, Belgium.

Thank you for sending me the paper on PSA for review. It is a very interesting overview and I

just have a couple of minor remarks.

One of the very important things is that prostate cancer cure rates after radical prostatectomy (and

radiotherapy) are only about guaranteed in patients with very low PSAs. Catalona has published

10 and 15 years disease free survival rates of 95 % in patients with PSA lower than 2 and 90 % in

patients with PSA lower than 4, etc. This means that still 10 % of patients with prostate cancer

detected with a PSA below 4 die of the disease. If a screening program is set up in order to

decrease mortality it is exactly the cancers with a low PSA that have the best chance of

benefiting. So it is my belief that a cut-off level of 4 µg/ml cannot be used.

Again, following the data of Catalona, a 50-year old male should have a PSA below 0.9, so I am

not sure that on page 3 you can mention that men ≤ 50 years would have a PSA-level of about 1.5

µg/L.

Data are from Lilja and colleagues, Reference 126.

I think it is very important to stress that blood samples that are just stored in the waiting room of

general practitioners to be picked up and analysed 24 hours later without being refrigerated are

probably useless. I am not sure what this means for the free PSA, but you mention that this is

even more susceptible to decay.

Now addressed on p. 38, which references a relevant systematic review.

Fritz Schröder has recently been reporting that the screening can be done without digital rectal

examination. I believe that for your paper it is extremely important to ask Fritz to review it and I

am pretty sure that he will love to do that. He is responsible for the European Screening Trial and

he just came up recently with new data that can surely improve your review in a substantial way.

Professor Schröder kindly reviewed this paper.

You are mentioning that salvage radiation therapy is best administered at the slightest PSA

increase. And you are correct that a PSA rise after radical prostatectomy is just an undetectable

4

PSA that becomes detectable. One of the problems with slowly rising and late recurrent PSAs is

that probably a certain amount of patients just have benign glands that are responsible for the

PSA relapse. It is difficult to recognize them and in order to distinguish a benign from a

malignant relapse you need about 2 or 3 years to see the slope of the PSA rise. Many clinicians

are therefore not willing to wait for 2 years and then give radiation treatment but will deliver

radiation treatment as soon as a 2 or 3 times PSA increase has been documented.

In the guidelines for early detection of prostate cancer the Americans, the French and probably

soon the EAU and Europa Uomo [The European Prostate Cancer Coalition - a patient

organisation] will advocate to start screening at 40 years of age with a repeat PSA at 45 and one

at 50. It will be much more the PSA velocity than the absolute value. Patients that will be

underneath 1µg/L at 50 years of age will then not need annual testing afterwards. Patients with a

high PSA velocity will be submitted to biopsies. Indeed prostate cancer develops in most patients

between 40 and 50 years of age and it is in this category that dangerous cancers can develop that

mostly can kill patients at 60 to 65 years of age.

Issues relating to screening are discussed on p. 33.

In your treatment chapter on page 9 you mention cryosurgery next to radical prostatectomy and

radiation therapy. I believe that high intensity focused ultrasound and cryosurgery should not be

mentioned together with the 3 other classical treatment options. The long-term results of HIFU

and cryosurgery are not yet well enough documented to make these treatment strategies

acceptable for routine use.

“Alternative treatment modalities (e.g. cryosurgery or high intensity focused ultrasound) await

evaluation of their long term results” has been added to p. 35.

“When the disease becomes refractory to …” I think one could mention that after first line

hormone treatment there is also second and third line hormone-therapy with for instance

ketoconazole, aminogluthetimide, estramustine phosphate, corticosteroids, estrogens… Many

patients will come to the end of all these hormonal manipulations before they are considered

candidates for Taxotere.

Incorporated on p. 35.

You describe that radical prostatectomy is an option only for patients with organ confined

disease. We have extensive experience with radical prostatectomy in patients with extra-capsular

disease (T3a) and also the Mayo Clinic has updated their experience in 2005 in a paper in the

British Journal of Urology. More and more American centres these days are reporting on their

results of radical prostatectomy in T3 cases and I think this therefore needs to be adapted in your

draft.

In Table 7: I believe that the statement that PSA for the early detection of prostate cancer should

be used in conjunction with digital rectal examination needs to be reconsidered after Fritz

Schröder’s remarks.

5

No changes were required based on comments from Professor Schröder.

A small last remark: The Tyrol trial has been re-analysed so there is more recent reference.

A recent reference is included. [Reference 177].

Again many thanks for giving me the opportunity to see the paper.

From Dr Györg Sölétormos, Clinical Chemist, Herlev Hospital, Denmark.

I have read with great interest the NACB Guidelines for the Use of Tumor Markers in Prostate

Cancer and have a few comments.

In the Pre-analytical specimen processing and storage section, second paragraph the text reads as

follows: “Taking into account the intra-individual biological variation of PSA in blood, a change

of 20-30% between serially collected specimens is required before the difference can be

considered clinically significant (ref 39, Eastham et al, JAMA 2003; 289: 2695-2700).”

Firstly: The referenced paper was not designed to investigate biological variation of PSA.

Secondly: The paper does not estimate the magnitude of biological variation.

Thirdly: The paper does not report that a change of 20-30% is needed to overcome the biological

variation.

What the paper did was to acknowledge that there is a component of variation besides analytical

variation influencing the result of PSA measurements. However, the paper did never estimate the

magnitude of biological variation. Therefore, it is not correct to use the paper as a reference in

this context.

The European Group on Tumor Markers (EGTM) has recently published a thorough review on

the biological variation PSA: Biological variation of Total Prostate-Specific Antigen: A survey of

Published Estimates and Consequences for Clinical Practice; Clin Chem 2005; 51: 1342-51 by

Söletormos et al. The EGTM reported that a fair estimate of the biological variation of PSA is

20% for men above 50 years within the concentration range of 0.1-20 µg/L. This means that the

95% Confidence Interval (CI) for a singe result is 33% (P < 0.05). For a change in concentrations

during monitoring the CI is 50%, which means that the baseline concentration has to change 50%

to be significant at P < 0.05.

Recommendations based on NACB guidelines should keep a strict focus on using statements

based on evidence.

The concept of biological variation should be addressed correctly.

The impact of biological variation on single measurements, replicated measurements, serial

measurements as well velocity should be addressed as the NACB guidelines are intended to help

users with interpretation of PSA data.

These comments have been incorporated (page 40).

From Dr Gerard Davis, Principal Scientist in Cancer Core R & D, Abbott Diagnostics

Division, Illinois, USA.

6

I reviewed the attachment [i.e. the Prostate Cancer manuscript]. The draft looks refined. I do not

have any suggestions for any changes.

No response required

From Dr Joseph Watine, Clinical Biochemist, Centre Hospitalier Général, Rodez, France.

In the NACB grading system for tumour markers (chapter one), very much emphasis is put on

systematic reviews and meta-analysis. This sounds very good indeed. In contrast, I have had a

look at the prostate cancer chapter, and I saw that none of the five PSA systematic reviews that

have been published in 2005 or 2006 [1-5] are quoted by the authors.

All five of these systematic reviews have now been cited appropriately.

Having read the NACB recommendations, it seems necessary to stress below the conclusions of

at least two of these five systematic reviews which should certainly be taken into account by the

authors of this chapter:

Given that only two randomized controlled trials were included, and the high risk of bias of both

trials, there is insufficient evidence to either support or refute the routine use of mass, selective or

opportunistic screening compared to no screening for reducing prostate cancer mortality.

Currently, no robust evidence from randomized controlled trials is available regarding the impact

of screening on quality of life, harms of screening, or its economic value. Results from two

ongoing large scale multicenter randomized controlled trials that will be available in the next

several years are required to make evidence-based decisions regarding prostate cancer screening

[2].

The overall benefit of monitoring serum PSA after treatment for prostate cancer remains

controversial. Considering the potential dangers associated with incorrectly assuming the efficacy

of new forms of treatment, the toxicity of administering salvage therapies of uncertain efficacy

after BF has been identified and the anxiety associated with tracking post-treatment serum PSA,

additional studies must be done to determine the appropriate use of this marker in properly

treating patients after therapy [5].

The on-going randomized clinical trials are discussed on p. 33. The above views are also

echoed in NACB Prostate Cancer Panel Recommendation 5.

References

1. Evans R, Edwards A, Brett J, Bradburn M, Watson E, Austoker J, Elwyn G. Reduction in

uptake of PSA tests following decision aids: systematic review of current aids and their

evaluations. Patient Educ Couns. 2005 Jul;58(1):13-26.

2. Ilic D, O'Connor D, Green S, Wilt T. Screening for prostate cancer. Cochrane Database Syst

Rev. 2006 Jul 19;3:CD004720.

3. Lee R, Localio AR, Armstrong K, Malkowicz SB, Schwartz JS; Free PSA Study Group. A

meta-analysis of the performance characteristics of the free prostate-specific antigen test.

Urology. 2006 Apr;67(4):762-8. Epub 2006 Apr 5.

7

4. Roddam AW, Duffy MJ, Hamdy FC, Ward AM, Patnick J, Price CP, Rimmer J, Sturgeon C,

White P, Allen NE; On behalf of the NHS Prostate Cancer Risk Management Programme.

Use of Prostate-Specific Antigen (PSA) Isoforms for the Detection of Prostate Cancer in Men

with a PSA Level of 2-10 ng/ml: Systematic Review and Meta-Analysis. Eur Urol. 2005

Sep;48(3):386-99; discussion 398-9. Comment in: Eur Urol. 2005 Dec;48(6):1059-60; author

reply 1060-1.

5. Vicini FA, Vargas C, Abner A, Kestin L, Horwitz E, Martinez A. Limitations in the use of

serum prostate specific antigen levels to monitor patients after treatment for prostate cancer. J

Urol. 2005 May;173(5):1456-62

From Dr David Alter, Pathology and Laboratory Medicine, Spectrum Health – Blodgett, Grand Rapids, MI, USA

I was reviewing fPSA-testing issues with reference to their clinical utility and use of a specific

reference range for reflex testing so I checked the draft guideline. I found what appeared to be a

fair discussion of the issue that correlated with my knowledge base; however, when I did some

literature review in PUBMED, I found a number of references in the last two years that clearly

and specifically supported a reflex range of 2.5 to 10 ng/mL with one reference going against the

need to test below and above the range.

We believe that these issues have now been addressed satisfactorily.

COLORECTAL CANCER

From Professor Robert Bast, Department of Experimental Therapeutics, University of

Texas M.D. Anderson Cancer Center, Houston, Texas, USA.

Thank you for the opportunity to review the draft guidelines for tumor markers in colorectal

cancer. Overall, they are accurate, balanced, concise, clearly written and well referenced.

Data with regard to TIMP-1 are certainly of interest. In looking toward the future,

however, it would be important to define where increased sensitivity would be of value

for the management of individual patients. Will greater sensitivity for primary stage I and

II disease actually permit application to early detection? Will there be greater sensitivity

for detecting resectable recurrent disease? Is there complementarity with CEA?

Prognostic data must be linked to decisions regarding adjuvant therapy.

We agree with these points regarding TIMP-1 and state “Although these preliminary findings

with TIMP-1 are promising, the marker cannot be recommended at present either for detecting

early CRC or for evaluating prognosis in patients with this malignancy”. We are certainly not

recommending clinical use of TIMP-1, at this point in time.

8

From Professor Duncan Jodrell, Consultant Medical Oncologist, Western General Hospital,

Edinburgh, UK.

It's an interesting document and seems very reasonable.

In terms of local practice, I'm not sure whether CEA is one of the pre-operative assessments, and

we certainly don't use it for clinical decision-making. It is interesting that the NACB panel

recommends its measurement in the absence of evidence... I guess it is a level C recommendation,

rather than something stronger.

Yes, this is a C strength of recommendation

I was also interested by the statement that "a 30% rise in CEA should be regarded as evidence of

disease progression"...but then qualifies it by saying it should be established whether it is a false

positive. Perhaps it should state "may" rather than "should".

This reviewer is correct. This alteration has been made

Are MSI and MLH1 etc. gene testing offered as a "service" currently ... or simply a by-product of

research? ... I guess that may have implications locally, but seems to make sense that it should be

a service.

We state in Table 10 that these assays are available in specialized labs.

From Professor Callum Fraser, Consultant Clinical Scientist, Ninewells Hospital, Dundee,

UK.

I cannot really comment with any degree of expertise on the tumour markers other than fecal

tests. As you know, I have been involved with bowel screening in Scotland since 1998 so do have

some expertise in fecal analyses.

Thus, what I have done is to copy both the part of the document on fecal markers and the

conclusions and I have added my comments in red [shown here in italics].

I must say that the material is rather dated. However, I do know that the NACB likes

feedback. They published my thoughts on fecal testing in their POCT recommendations.

It could be vastly improved to be honest but maybe I have given too much detail. It

would be good to get the errors of fact sorted, though, and at least the FIT terminology

used [it's 2007 after all!].

Please enjoy the following. Will this suffice? I really do not know much about the other markers

in the article. Let me know if you need more. The Allison/Lawson editorial is also attached - I am

a great fan of Allison's very sensible works and writing.

Comments made shown in italics in following extract from first draft:

9

Fecal markers

The most widely used fecal marker involves testing for occult blood, i.e., the fecal occult

blood test (FOBT). FOBTs can be of 2 main types, i.e., the guaiac test and the immunochemical

test.

It is better now to clearly distinguish between guaiac tests [FOBT] and faecal immunochemical

tests (FIT) – reference 68. Allison JE. Colon Cancer Screening Guidelines 2005: the fecal

occult blood test option has become a better FIT. Gastroenterology 2005;129:745-8, tells the

story very well.

Moreover, this is incorrect – there are not two types – there are three commonly used types. The

omission is the “throw in the bowl” type tests – these involve a paper coated with a chromogenic

dye (TMB) and peroxide. The heme portion of hemoglobin, present in blood, oxidizes the

tetramethylbenzidine that is on the test strip and causes a blue/green color change.

The guaiac test measures the presence of haem in haemoglobin

No, it doesn’t – it measures the “pseudoperoxidase” activity of heme

while the immunochemical test detects human globin. As haem is also present in certain fruits

and vegetables, intake of these foods may give rise to false-positive results in the guaiac test.

No, heme is not present in fruits and vegetables – they may have plant peroxidases. In any case,

this is unimportant – plant peroxidase interference, which gives false positives, can be eliminated

by allowing the feces to dry on the FOBT before adding the developer. See - Sinatra MA, St John

DJ, Young GP. Interference of plant peroxidases with guaiac-based fecal occult blood tests is

avoidable. Clin Chem 1999:45:123-6.

It is more important perhaps to discuss meat intake. However, there is a meta-analysis that shows

that dietary restriction is not required. See - Pignone M, Campbell MK, Carr C, Phillips C. Meta-

analysis of dietary restriction during fecal occult blood testing. Eff Clin Pract 2001;4:150-6.

Certain medicines such as non-steroidal anti-inflammatory drugs can also interfere with the test.

In contrast, the immunochemical test please use FIT here and elsewhere is not affected by these

factors. As well as being subjected to less interference, the immunochemical test exhibits superior

sensitivity and specificity compared to the guaiac test (62).

I think that the authors would do very well to read a very recent review that gives an update on

FOBT and FIT and other options. The authors could update this paper based on the literature

cited therein. Allison JE, Lawson, M. Screening tests for colorectal cancer: A menu of options

remains relevant. Current Oncology Reports 2006; 8:492–98. There is an excellent recent

literature from Israel, Italy, France and Uruguay that shows the role of FIT.

10

The authors do not discuss here the role of quantitative FIT. This is most important. By changing

the analytical detection limit, one can choose the positivity rate and thereby change the sensitivity

and specificity. An excellent example is - Vilkin A, Rozen P, Waked A, et al.: Performance

characteristics and evaluation of an automated-developed and quantitative, immunochemical,

fecal occult blood screening test. Am J Gastroenterol 2005, 100:2519–2525.

In addition, the authors do not mention the very important aspect of fecal testing – the format of

the test. Cole SR, Young GP, Esterman A, et al.: randomized trial of the impact of new faecal

hemoglobin test technologies on population participation in screening for colorectal cancer J

Med Screen 2003, 10:117–122 is an important study showing that, by simplifying sampling and

removing the need for restrictions of diet and drugs, FIT increases participation in screening.

Also FIT are better at lower GI problems – this is very important.

Despite these limitations, a number of large randomized trials have shown that screening with the

guaiac test reduced mortality from CRC (63-67).

There are also other approaches possible, combining FOBT and FIT – see Fraser CG, Matthew

CM, Mowat NAG, Wilson JA, Carey FA, Steele RJC. Immunochemical testing of individuals

positive for guaiac faecal occult blood test in a screening programme for colorectal cancer: an

observational study. Lancet Oncology 2006;7:127-31

Although several guaiac tests exist, only two have been evaluated in large-scale screening,

i.e., the Haemoccult II and the Haemoccult Sensa.

This is completely incorrect. hema-screen [Immunostics Inc, Ocean, New Jersey] has been use in

the UK Colorectal Cancer Screening Pilot. See - UK Colorectal Cancer Screening Pilot Group.

Results of the first round of a demonstration pilot of colorectal cancer screening in the United

Kingdom. Br Med J 2004; 329:133–35. Also see - Steele RJC, Parker R, Patnick J, et al. A

demonstration pilot trial for colorectal cancer screening in the United Kingdom: a new concept

in the introduction of healthcare strategies. J Med Screen 2001; 8:197–202. This is admitted in:

LaStella VP, Allison JE (reply). Fecal occult blood testing options. Gastroenterology

2006;130:285.

The efficacy of the immunochemical test in reducing either the incidence or mortality form

CRC has not yet been investigated in large population-based studies. However, based on

available evidence, it should be at least as accurate if not more accurate than FOBT, in screening

for CRC (68).

Correct - add Allison JE, Lawson, M. Screening tests for colorectal cancer: A menu of options

remains relevant. Current Oncology Reports 2006; 8:492–98 to the reference list as a very nice

discussion of this point.

11

In agreement with other Expert Panels (69-71), the NACB recommends that all subjects 50

years or older should undergo screening for CRC. Multiple screening procedures for CRC exist

however (67-69), and to-date no one procedure has been shown to be significantly superior to the

others. The option chosen may therefore depend on availability, personal preference and risk of

developing CRC (70).

This is very nicely discussed in: Young GP, St John DJ, Winawer SJ, Rozen P, for WHO and

OMED (World Organization for Digestive Endoscopy). Choice of fecal occult blood tests for

colorectal cancer screening: recommendations based on performance characteristics in

population studies: a WHO and OMED report. Am J Gastroenterol 2002; 97: 2499–507.

According to the National Comprehensive Cancer Network (NCCN), FOBT should be

performed on 3 successive stools specimens that are obtained while the patient adheres to a

prescribed diet (69). This organization specifically recommends the Haemoccult SENSA as the

testing method. Both the NCCN and the American Cancer Society recommend against use of

FOBT of a specimen obtained at digital rectal examinations (69, 70).

Bini EJ, et al. Prospective evaluation of the use and outcome of admission stool guaiac testing:

The Digital Rectal Examination on Admission to the Medical Service

(DREAMS) Study. J Clin Gastroenterol 2006;40:821–827 is a nice up to date study with an

extensive bibliography of past studies on this topic.

Because of the lack of sensitivity and specificity of FOBT for adenomas and early CRC, a

considerable amount of research in recent years has focused on other fecal markers, especially on

the genes that undergo mutation during CRC carcinogenesis. Amongst the most widely

investigated DNA markers are mutant ras, mutant p53, mutant APC, specific methylated genes,

MSI and long DNA (3, 72-75).

Again please see the Allison and Lawson paper for a very well documented comprehensive

update on this. What about fecal test other than DNA – such as pyruvate kinase M2 (tumor M2-

PK)

A fecal DNA panel was recently investigated as a screening test for CRC in a large

asymptomatic population. Of the 31 invasive CRCs detected, the DNA panel diagnosed sixteen,

whereas FOBT detected only four (p = 0.003). Of the 71 invasive cancers and adenomas with

high-grade dysplasia, the DNA panel diagnosed 29, while FOBT detected only ten (p < 0.001).

Although the DNA panel displayed a higher sensitivity than FOBT, clearly neither test detected

12

the majority of advanced adenomas or carcinomas (76). However, as the DNA-based test was

superior to FOBT, it might be expected to be at least as good as the latter in reducing mortality

from CRC.

This is unbalanced – there are other data that suggest DNA tests are not very good.

I would strongly urge the authors to read the article by Allison and Lawson and assess the many

papers in 2005-2007 that address the use of quantitative FIT. In the world of asymptomatic

population bowel screening, this is definitely seen as the way ahead – not these other

suggestions!

The authors do not mention the use of FOBT or FIT except in asymptomatic population

screening. This is in fact beginning to be very widespread. Germany, Austria, Australia, Scotland,

England, Finland, France, Italy, Japan, and elsewhere are all working with or on national

programmes.

What the authors do not mention is the use of fecal test in primary care in patients with or

without symptoms. A recent article – Smellie, WSA. Best practice in primary care pathology:

review *. J Clin Pathol, e-published on 15 December 2006 – gives some views. These are not

widely held, to be honest. Probably the paper should have a sentence stating: There are no

objective studies on the use of FOBT or FIT in primary care in patients with symptoms of

colorectal cancer.

Moreover, the authors do not mention the potential use of fecal test in surveillance of people who

have had cancer or polyps removed. A fascinating early study can be found at: Bampton PA, et

al. Interval faecal occult blood testing in a colonoscopy based screening programme detects

additional pathology. Gut 2005 ;54:803-6.

This reviewer makes many good points. Most of his suggestions have been taken on board

including the listing of publications by Allison and Lawson.

We do not believe that sufficient work has been carried on pyruvate kinase M2 at this stage in

order to recommend it as a screening test for CRC. We do however, list it in the context of

several other emerging fecal protein-based tests

We have not discussed the use of FOB testing in primary care or postoperative surveillance in

this manuscript, as our aim was to discuss this test only in the context of screening asymptomatic

subjects.

From Dr Barry Berger, Vice President for Laboratory Medicine, EXACT Sciences

Corporation and Senior Visiting Pathologist, Brigham and Women’s Hospital / Harvard

Medical School, Boston, Massachusetts, USA.

13

Thank you for the opportunity to comment on the NACB Guidelines for the use of Tumor

Markers in Colorectal Cancer. We have recently compiled all of the evidence supporting the use

of stool DNA testing for routine CRC screening and I have provided an excerpt of those materials

for your review. These materials were also provided to the ongoing American Cancer Society -

Multi Society Task Force CRC Screening Guidelines review process.

We appreciate the NACB Guideline group's enthusiasm for the potential of fecal DNA test for

CRC screening. We would like to further support its enthusiasm by commenting on the issue of

mortality end-point studies.

Specifically, I would like to respond to the following quotations from the paper:

I. Page 8, paragraph 3, last sentence: As there are no studies to date showing the screening with

fecal DNA markers reduces mortality from CRC, this test cannot be recommended at present, to

screen for CRC.

II. Page 9, last sentence: Its (fecal DNA) potential to reduce mortality from CRC should be

evaluated in a large prospective randomized trial. Guaiac based FOBT (gFOBT) is the only

colorectal cancer screening test for which there are large prospective studies with a mortality

endpoint. Selby's case controlled study of rigid sigmoidoscopy provides some evidence for a

mortality benefit in the sigmoid colon. There are no such studies for colonoscopy, flexible

sigmoidoscopy, double contrast barium enema, or immunologically based FOBT. These tests are

all recommended and used for CRC screening without a mortality study. We feel, as do others,

that given that stool DNA was shown to be superior to gFOBT, logical inference would support

that the mortality benefit would be at least as great as that of gFOBT and a mortality study to

demonstrate that would not be needed (Winawer SJ, et al. Colorectal Cancer Screening: Clinical

Guidelines and Rationale. Gastroenterology 1997;112:594-642). In the current environment, it is

unlikely that mortality endpoint studies will be performed or funded for any CRC screening

modalities now or in the future. We believe that failure to have such a study should not, therefore,

be the basis for failing to recommend this life saving technology. We respectively ask that the

NACB authors group reconsider this position.

Fully 50% of subjects who have used stool DNA screening in daily clinical practice have never

been previously screened for CRC. Despite the current variety of CRC screening tests, these

subjects failed to be screened until the stool DNA process became available. This observation is

true across both sexes and all ages (Berger BM, et al. Colorectal Cancer Screening Using Stool

DNA Analysis in Clinical Practice: Early Clinical Experience with Respect to Patient Acceptance

and Colonoscopic Follow-up of Abnormal Tests. Clinical Colorectal Cancer 2006;5(5):338-343).

Finally, I have a few comments on the in the LOE data (Table 1) starting on page 10. The LOE

for fecal DNA is given as level III. The Imperiale study, which is abstracted in the chart itself, I

believe is Level I based on the Hayes et al paper that is cited ( Ref 11). The actual fecal DNA

14

citations listed may be misordered as they are listed as numbers "72-77". The Imperiale study is

actually reference # 79.

We would ask you to also consider the following references, which we feel are relevant and

which support the clinical use of fecal DNA:

1. Tagore KS, et al. Sensitivity and Specificity of a Stool DNA Multitarget Assay Panel for the

Detection of Advanced Colorectal Neoplasia. Clinical Colorectal Cancer 2003;3(1):47-53.

2. Calistri D, et al. Fecal Multiple Molecular Tests to Detect Colorectal Cancer in Stool.

Clinical Gastroenterology and Hepatology 2003;1: 377-383.

3. Brand RE, et al. Reproducibility of a Multitarget Stool-Based DNA Assay for Colorectal

Cancer Detection. American Journal of Gastroenterology 2004;99:1338-1341.

4. Syngal S, et al. Detection of Stool DNA Mutations Before and After Treatment of Colorectal

Neoplasia. Cancer 2005;6(2):277-283.

We appreciate the time and effort your group has expended in compiling this very useful

document and we would like to assist with any additional information that you feel would

increase its value further.

This reviewer makes a strong case for the use of a DNA panel in screening for CRC. However,

not all experts agree with his view, e.g., see comment of Professor Fraser above. Furthermore, as

far as we are aware, no expert group has to date recommended the use of DNA markers in

screening for CRC.

In the main text, we state the following “it should be pointed out that compared to FOBTs,

measurements of fecal DNA markers is more expensive and technically demanding. Furthermore,

it is not clear which combination of DNA markers provides the optimum balance of sensitivity

and specificity”. Under the heading “Key Points”, we state the following “One of the most

promising fecal CRC screening tests is the fecal DNA panel. This test should be simplified, made

available at reduced costs and subjected to further investigations. Overall, we cannot recommend

that DNA markers be used in screening for CRC, at this point in time. See our paragraph on the

cost-effectiveness of this test.

As regards the specific references suggested, most of these are included in the recent reviews

that we cite.

As regards level of evidence for the Imperiale et al study, this is possibly a Level I evidence

study and this is now indicated. Most studies with DNA panels however, are Level III/IV

From Dr Joseph Watine, Clinical Biochemist, Centre Hospitalier Général, Rodez, France.

2) Chapter on colorectal cancer (see general comments too):

ASCO recommendations have been updated in J Clin Oncol. 2006 Oct 23; [Epub ahead of print]

ASCO 2006 Update of Recommendations for the Use of Tumor Markers in Gastrointestinal

15

Cancer. Locker GY, Hamilton S, Harris J, Jessup JM, Kemeny N, Macdonald JS, Somerfield

MR, Hayes DF, Bast RC Jr. American Society of Clinical Oncology Tumor Markers Expert

Panel, American Society of Clinical Oncology, Alexandria, VA.

It says: “for colorectal cancer, it is recommended that carcinoembryonic antigen (CEA) be

ordered preoperatively, if it would assist in staging and surgical planning. Postoperative CEA

levels should be performed every 3 months for stage II and III disease for at least 3 years if the

patient is a potential candidate for surgery or chemotherapy of metastatic disease. CEA is the

marker of choice for monitoring the response of metastatic disease to systemic therapy”.

Incidentally, it also says: “for pancreatic cancer, CA 19-9 can be measured every 1 to 3 months

for patients with locally advanced or metastatic disease receiving active therapy. Elevations in

serial CA 19-9 determinations suggest progressive disease but confirmation with other studies

should be sought”.

Section “CEA in determining prognosis” (shouldn’t it be “pre-operative CEA in determining

prognosis”?):

- it is written: “There is however, no evidence at present for a beneficial effect of adjuvant

chemotherapy …. CEA concentrations”. Why in this case do the NACB panel

recommend to measure preoperative CEA? If preoperative CEA is of independent

prognostic value, this is a good reason to recommend preoperative CEA measurements in

trials, but if no clinical decision can be based on these measurements, what is their use in

routine practice?

We recommend the assay of preoperative CEA, as it may be of use in surgical planning and

not for deciding for or against the use of adjuvant chemotherapy. Similar recommendations have

been made by ASCO, 2006.

- It is written: “A further reason for measuring preoperative CEA is that it provides a

baseline concentration for subsequent patient management”. It seems to me that this

reason would in fact be the only one (despite its low levels of evidence) and therefore the

word “further” should be deleted or replaced by the word “only”. This might eventually

not change anything in the final strength of recommendation, but this way of presenting

things would sound rather more objective.

No comment needed here, see above.

- Some other tests, e.g. alkaline phosphatase (ALP) are not mentioned [Watine et al 2004

and 2001].

We do not believe that the data relating to ALP is sufficiently strong to include it in our

recommendations.

16

Section “CEA in post-operative surveillance”:

As mentioned in the general comments, liver function tests, ALP in particular are not mentioned,

whereas according to the meta-analysis quoted at the beginning of this section, it might be useful

to associate these tests with CEA.

Section “CEA in monitoring therapy in advanced disease”:

ASCO recommendation is “… if no other simple test is available”. Why do this end of sentence

disappear in the final NACB recommendations? Other tests, e.g. ALP could be mentioned here

too [Watine et al 2004 and 2001].

The 2006 ASCO guidelines clearly state “CEA is the marker of choice for monitoring systemic

therapy in patients with CRC”.

See our comment above, relating to ALP.

Section “Fecal markers”:

See general comments below. [From same reviewer]

BREAST CANCER

From Professor Dorte Nielsen, Consultant Oncologist, Herlev Hospital, Herlev, Denmark.

I believe the review article is comprehensive, thorough and clearly expressed. Further, the topic is

very relevant and of clinical importance.

Unfortunately, as a clinician I am not able to evaluate the sections concerning recommended

assays.

I have the following minor suggestions/recommendations to the present manuscript version:

1. Acceptance of a novel tumor marker requires a thorough validation before being

implemented into clinical use. I suggest this topic to be discussed more in detail. As a reference I

suggest: Werner M, Faser C Silverberg M. Clinical utility and validation of emerging

biochemical markers for mammary adenocarcinoma. Clin Chem 1993; 39: 2386-96.

We have considered this point but feel that it is not directly relevant to our manuscript.

2. Topoisomerase II is a potentially new marker and it should be included into the text.

We agree that topoisomerase II is a potential new marker but we also believe that it is not yet

ready for clinical application

3. Several references are available i.e. Fritz P et al. c-erbB2 and topoisomerase IIα protein

expression independently predict poor survival in primary human breast cancer: a

retrospective study. Breast Cancer Res 2005; 7: R374-84 or Di Leo A et al. HER-2 and

topoisomerase IIα as predictive markers in a population of node-positive breast cancer

patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide. Ann

Oncol 2001; 12: 1081-9.

17

In the legend to Table 12, we state that the enhanced benefit of adjuvant anthracyclines found

in HER2-positive patients may be due to amplification of topoisomerase IIa. We also list a

number of references to support this view.

4. Tamoxifen is biotransformed into the potent anti-estrogen, endoxifen, by cytochrome P450

2D6. Genetic variation in CYP2D6 may predict the tamoxifen resistance. The authors should

in this respect seriously consider mentioning: Goetz MP et al. The impact of cytochrome

P450 2D6 metabolism in women receiving adjuvant tamoxifen. Breast Cancer Res Treat

2007; 101: 113-21.

As with topoisomerase IIa, CYP2D6 is a potential new marker for determining tamoxifen

pharmacokinetics but again is not ready for clinical use.

From Professor John Smyth, Department of Clinical Oncology, University of Edinburgh,

Edinburgh, UK.

Many thanks for asking me to have a look at the draft recommendations for the use of tumour

markers in breast cancer that you are proposing to submit to Clinical Chemistry.

I read this with great interest and think that this is a very comprehensive summary of the present

situation. It is also very well referenced and I have no new markers to suggest or criticisms of the

conclusions.

This is an important and topical area and I hope that Clinical Chemistry like it.

No response required.

From Professor M Tuxen, Consultant Oncologist, Herlev Hospital, Herlev, Denmark.

Table 12 – you can also describe CYP2D6 genotype, which is an independent predictor of breast

cancer outcome in post-menopausal women receiving adjuvant tamoxifen (Goetz MP. The impact

of cytochrome P450 2D6 metabolism in women receiving adjuvant tamoxifen. Breast Cancer Res

Treat 2007, 101: 113-21).

See our response to CYP2D6 above.

Table 12 – tumor cells in axillary nodes is in clinical use. The prognostic value of lymph node,

especially macrometastasis is well known, I am not sure that I understand what you mean with

“magnitude of effect appears relatively weak”. Ref.109 is very old.

To clarify our meaning here, we are stating that the prognostic impact of tumor cells in lymph

nodes is relatively weak. Yes, we agree that ref. 109 is old. We have now replaced it with a 2007

reference.

From Dr Patrick Terry, Genomic Health, Inc.

18

I would like to request that perhaps during the next revision cycle for the National Academy of

Clinical Biochemistry Guidelines for the Use of Tumor Markers in Breast Cancer you could

consider including the follow update for inclusion in Table 12:

Cancer Marker: Oncotype DXä

Proposed Use/Uses: For predicting response to adjuvant tamoxifen in lymph node-negative breast cancer.

This point is now addressed.

Phase of Development: Validated in a prospectively designed study, assay can be carried out on paraffin-embedded tissue. In clinical use

This point is also now addressed.

LOE: I/II 41.

Please include the current published peer-reviewed publication references:

1. Laurel A Habel, et al. A population-based study of tumor gene expression and risk of breast

cancer death among lymph node-negative patients. Breast Cancer Research May 2006,

8:R25.

2. S Paik, G Tang, S Shak, C Kim, J Baker, W Kim, M Cronin, et al. Gene Expression and

Benefit of Chemotherapy in Women with Node-Negative, Estrogen Receptor-Positive Breast

Cancer. J Clin Oncol. Aug 10 2006: 3726-3734

3. Luca Gianni, Milvia Zambetti, Kim Clark, Joffre Baker, Maureen Cronin, et al. Gene

Expression Profiles in Paraffin-Embedded Core Biopsy Tissue Predict Response to

Chemotherapy in Women with Locally Advanced Breast Cancer. J Clin Oncol. 2005; 23(29):

7265-7277

4. Esteva, et.al., Prognostic role of a multigene reverse transcriptase-PCR assay in patients with

node-negative breast cancer not receiving adjuvant systemic therapy. Clin Cancer Res. 2005

May 1;11(9):3315-9.

5. Cobleigh MA, Tabesh B, Bitterman P, Baker J, Cronin M, et al. Tumor Gene Expression and

Prognosis in Breast Cancer Patients with 10 or More Positive Lymph Nodes. Clin Cancer

Res. 2005; 11(24 Pt 1):8623-8631.

6. Cronin M, et al. Measurement of Gene Expression in Archival Paraffin-Embedded Tissues.

Am J Path. 2004; 164(1):35-42.

19

Rather than listing all the above references, we now include a recent comprehensive article on

the Oncotype DX test, written by Paik et al, 2007.

If you expand the genomics review content of the Practice Guidelines For Use Of Tumor Markers

In The Clinic; then may I humbly suggest an important article and reference for your

consideration:

Richard Simon. Roadmap for Developing and Validating Therapeutically Relevant Genomic

Classifiers. J Clin Oncol, 2005; 23(29):7332-7341. URL:

http://www.jco.org/cgi/content/abstract/23/29/7332.

We do not feel that the above reference is directly relevant to our manuscript.

From Dr Jeff Terryberry, Antibody Development, Anogen (Division of YES Biotech), Mississauga, ON, Canada

I found the NACB guidelines to be particularly well organized and useful. However my main

focus at this time is the clinical utility of prognostic markers for treatment, such as your section

on the use of HER2/neu (ErbB-2) for breast cancer treatment describes.

Accordingly, there should be some mention of the use of epidermal growth factor receptor

(EGFR) detection in colorectal cancer for proper intention to treat colorectal cancer with Erbitux

and Vectibix (FDA-approved inhibitors of EGFR tyrosine kinase).

However, Dako's pharmDx "companion diagnostics" kit (IHC methodology) for EGFR is not yet

FDA-approved. Moreover, Tarceva - another EGFR inhibitor - has been FDA-approved for use in

lung cancer (advanced NSCLC).

In addition, VEGFR tyrosine kinase drugs are now approved. Avastin is indicated in the

treatment of metastatic colon cancer, and it is foreseeable that VEGFR companion diagnostics

will follow suit.

In the same vein, I am trying to ascertain how close to clinical reference laboratory use are the

phosphorylation state-specific antibodies (PSSAs) for cancer prognosis. Particularly useful here -

according to the literature - are:

Phospho-Akt (predictor of prostate cancer outcome and breast cancer treatment outcome)

Phospho-HER2/neu (predictor of breast cancer outcome)

I realize this email contains a lot of suggestive material, but I would like your expert opinion as a

representative of the NACB on the status of these markers; and in the case of EGFR possible

addition to your guidelines is already warranted, even though it has yet to gain mainstream

acceptance in clinical practice.

We have tried to address some of the above issues in the current manuscript but would suggest

that they should be considered in more detail when these guidelines are next reviewed.

20

OVARIAN CANCER

From Professor Gordon Rustin, Mount Vernon Cancer Centre, Northwood, Middlesex

HA62RN, United Kingdom

Thanks for asking me to review this manuscript. It is generally very good. My main issue is that

there is a section on post-op use and another on prognosis, which would be far better split into

detection of recurrence. If they want to keep the prognosis bit they should quote Fayers PM,

Rustin G, Wood R, Nelstrop A, Leonard RCF, Wilkinson P, Cruickshank D, McAllister J,

Redman CWE, Parker D, Scott IV, Slevin ML. The prognostic value of serum CA l25 in patients

with advanced ovarian carcinoma: An analysis of 573 patients by the Medical Research Council

Working Party on Gynaecological Cancer. Int J Gynecol Cancer l993, 3, 285-292.

Incorporated [Reference 422].

Although CA 125 has a prognostic place it is not accurate enough to alter management. In

practise clinicians change therapy if clear-cut progression so the part on progression needs

expanding. It should include the original GCIG definition: Vergote I, Rustin GJ, Eisenhauer A,

Kristensen GB, Pujade-Lauraine E, Parmar MK, Friedlander M, Jakobsen A, Vermorken JB. Re:

New guidelines to evaluate the response to treatment in solid tumours (ovarian cancer). JNCI,

Vol 92, No 18, 2000, 1534-5 as well as the web address of the complete definition at

http://ctep.cancer.gov/resources/gcig/respdef.html

Incorporated on p.86 (more recent GCIG paper).

So you should try and get prognosis deleted as a recommended indication. Although other

guidelines have included it unless there are suggestions as to how it should be used I can't see its

value.

Discussed on p.87.

There could be mention of the upper limit of normal being lower than 35 and there are several

papers showing a rise within the normal range indicates recurrence. See Santillan A, Garg R,

Zahurak ML, Gardner GJ, Giuntoli RL 2nd, Armstrong DK, Bristow RE. Risk of epithelial

ovarian cancer recurrence in patients with rising serum CA-125 levels within the normal range. J

Clin Oncol. 2005 Dec 20;23(36):9338-43.

Incorporated on p.87. Reference 418.

Hope this helps and does not make your task more difficult

From Professor Bengt Tholander, Oncology Unit, University Hospital, Stockholm, Sweden.

I have reviewed the manuscript " Tumor markers in ovarian cancer " of Dr DW Chan et al. I find

it excellent, and have no suggestions for changes in the text. I agree with the conclusions of the

authors.

21

I have one comment: Maybe it can be appropriate also to mention a few words of the tumor

marker inhibin, which has been shown to be of value not only in granulosa cell tumors, but also

in mucinous epithelial ovarian cancer. An Australian group has published data. I can look up the

references.

Inhibin now discussed on p.93.

Concerning my own references, they seem rather old, from the beginning of the nineties, but may

be of some relevance for discrimination between malignant and benign disease. I will come back

with another e-mail in this weekend including these references. I leave it to you and the authors to

decide whether they should be included or not.

Incorporated on p.84. Reference 402.]

From Professor M Tuxen, Consultant Oncologist, Herlev Hospital, Herlev,

Denmark.

Thank you again for sending me the draft recommendations for the use of tumor markers. I read

the ovarian cancer with interest and would like to make same comments on it:

1. I think that the section ”Post-operative use” is somehow confusing. The first part describes

CA 125 and second-look operation, the second one the value of CA 125 during follow-up. I

suggest to divide this section into two: section one ”CA 125 and second-look operation” and

from the sentence ”Elevated, rising, or doubling CA 125 …” section to ”Detection of

recurrence”. I suggest to place the sections in the following order: Screening/ early detection,

Discrimination of pelvic masses, Monitoring treatment, CA 125 and second-look operation,

Detection of recurrence, Prognosis.

Sections re-ordered as suggested.

2. I suggest to supplement the text with more references, some important information should be

completed by reference number e.g.

- page 4, line 24, sentence “The cut-off of 35 kU/L ….”

- page 4, line 25, sentence “Values tend to ….”

- page 4, line 28, sentence “Menstrual cycle variations …”

- page 6, line 5, sentence “Sensitivities of 71-78% ..”

- page 6, line 14, sentence “Elevations of CA 125 greater than …”

- page 7, line 19, sentence “In addition to monitoring initial chemotherapeutic

regiments …..”

- page 8, line 9, sentence “Improved survival …”

References added, as suggested above.

3. Page 2, line 7 – it is better to say “The overall mortality of ovarian cancer is still poor ..” as

the overall mortality changes if the 5-years survival improves.

Wording changed as suggested.

22

4. Page 2, line 18 – I am not sure if the metastatic tumors should be described here as a

separately category of ovarian cancer as e.g. germ cell tumors and epithelial tumor can be

metastatic.

“Metastatic tumors” deleted as suggested to avoid confusion.

5. Page 5, line 6 – “The recommendations of the current NACB panel …” can be removed as it

is described in the section “Tumor markers in ovarian cancer: NACB recommendations”.

Not changed as inclusion consistent with other sections. Will await view of journal editors.

6. Page 5, line 9 – 90% in stage II seems to be a very high number, which is not confirmed by

other reviews (e.g. your ref. 30, 70). Ref. 17 is also a very old review, I will suggest using a

newer one.

Additional reference incorporated. No contradictory evidence in original Ref 30.

7. Page 7, line 3-4 – which recommendation will you suggest in this situation? Should CA 125

be controlled at follow-up visit or not?

Now addressed on p.85 (Recommendation 4) and in reference to the OV05 trial (Reference

415).

8. Table 16 – I miss systematizing of the table, what about listing tumor markers in order to year

of discovery?

This has been done.

9. Table 17 – I will suggest moving “Monitoring therapy” before “Detection of recurrence”.

This has been done.

From Professor Joe Duffy, Clinical Scientist, St Vincent’s Hospital, Dublin, Ireland.

I have read over these draft guidelines and must congratulate you and your co-authors for

doing an excellent job. I have one comment/suggestion. In Table 16, I suggest that you

refer to CA 125 under multiple headings (as in Table 17) rather than just "monitoring".

A footnote to Table 16 now directs the reader to Table 17.

I am also attaching a copy of the 2005 EGTM guidelines on CA 125 in ovarian cancer.

These were published a few months ago in Int J Gynecol Cancer.

This reference is now cited in the text (Ref 561; p.50) and in Table 17.

Indirect comments – posted on the UK Association of Clinical Biochemists (ACB) Mail base – June 2006

First posting (Commenter 1)Misinterpretation of CA125 can lead to a clinical fascinoma (sic), see this BMJ lesson of the

week:

http://bmj.bmjjournals.com/cgi/content/full/325/7370/946

and also rapid responses from some list members:

23

http://bmj.bmjjournals.com/cgi/eletters/325/7370/946

The literature is full of pitfalls in the interpretation of elevated CA125 in benign conditions, a

documented proof that clinicians are unwary of its proper clinical utility.

We hope that guidelines such as those presented in this series of papers will help to remind

users of the clinical pitfalls that can be encountered when tumor markers are used inappropriately.

All best practice guidelines so far (ASCO, NACB, SIGN, EGTM) do not recommend CA125

usage for diagnosis of epithelial ovarian carcinoma and limit its usage for monitoring therapy and

prognosis. Clinicians, however, are free to use it off license, and fill the literature with lessons of

"pericardial effusion mimicking ovarian carcinoma" and the like.

As far as we are aware there are no ASCO guidelines for tumor markers in ovarian cancer. We

believe ASCO has as yet developed recommendations for use of serum tumor markers in breast

and colorectal cancers only.

Second posting (Commenter 2)

Contrary to what you wrote before the weekend:

Nowhere in EGTM guideline, have I read that CA125 is not recommended for the diagnosis

of ovarian cancer. EGTM guideline is not clear at all regarding this point.

NACB guideline even states: "It is possible to make some general recommendations about

interpretation of CA 125 results obtained in a "case-finding" setting. Doubling of CA 125

levels in serum above baseline at any interval should prompt physical examination, TVS, and

CT scan.

Abnormalities detected by any of these indicate the need for need for laparoscopy and

laparotomy".

Thus neither EGTM, nor NACB guidelines tries to prevent us from using CA 125 as a

diagnostic tests for ovarian cancer, on the contrary regarding NACB guideline.

I have not read ASCO, or SIGN guidelines, but I suspect that you were right about them (i.e.

their recommendations probably contradict NACB's recommendations and EGTM 's very

unclear recommendations too.

The NACB, EGTM, and SIGN guidelines (as well as others – e.g. those of the Royal College

of Obstetricians and Gynaecologists in the United Kingdom) recommend use of CA125 in the

discrimination of pelvic masses, not for diagnosis in the general population. [As stated above,

there are as yet no ASCO guidelines for tumor markers in ovarian cancer.]

Third posting (Commenter 1)

It is not recommended for screening/diagnosis by ACBI, AJCC, EGTM, ESMO, NACB

according to: Catharine Sturgeon. Practice Guidelines for Tumor Marker Use in the Clinic;

Clinical Chemistry 2002;48:1151-1159.

Fourth posting (Commenter 2)

In this case, why is it written this below in the NACB guidelines?

24

"It is possible to make some general recommendations about interpretation of CA 125 results

obtained in a "case-finding" setting. Doubling of CA 125 levels in serum above baseline at any

interval should prompt physical examination, TVS, and CT scan. Abnormalities detected by any

of these indicate the need for need for laparoscopy and laparotomy"

See above.

Fifth posting (Commenter 2)

NACB (the National Academy of Clinical Biochemistry) guidelines:

http://www.nacb.org/LMPG/Monograph_TumorMarkers.pdf if you want to check it by yourself...

Sixth posting (Commenter 1)

Thanks XXX for the link, indeed as you say NACB and EGTM recommend:

3.CA125 levels should be determined in women presenting with pelvic masses to distinguish

benign from malignant lesions.

This is correct, and is in accord with recommendations of other organizations, as described

above.

General approach – Comment received.

From Dr Joseph Watine, Clinical Biochemist, Centre Hospitalier Général, Rodez, France.

I understand that the NACB guidelines on tumour markers claim to be evidence-based guidelines.

We have done our best to develop a set of practice guidelines, following a reasonably

homogeneous style, in which the strength and type of evidence underlying each recommendation

is clearly stated, together with an estimate of the confidence with which each recommendation

has been made. Guideline development is an evolving process: learning from what has been

achieved here, we would expect further methodological improvements when these guidelines are

next revised.

Therefore, it might be useful to remind that:

Evidence-based guidelines are based on the systematic identification, critical appraisal, and

synthesis of the evidence, or when evidence is lacking, on sufficient consensus among the

guideline development team,

Systematic reviews being the cornerstone of evidence-based guidelines, it is necessary to keep

permanently in mind that the main purpose of systematic reviewing is to assess, and, if possible,

to quantify, heterogeneity and bias in individual studies in order to evaluate or, if possible, to

measure the probability that the benefits of an intervention (diagnostic or therapeutic) outweighs

downsides (ie harms and costs),

There has been relatively little evaluation of the real impact of practice guidelines on health, but it

is clear that some practice guidelines are less valid than others, and some guidelines can even be

invalid, ie they provide recommendations that, when implemented, are unlikely to improve the

balance between benefits and downsides (ie harms and costs). For example, in order to sell their

25

new products as much as they can, the drug industry may select the results of the randomized

controlled trials that they chose to publish. Likewise industry-supported reviews of drugs are less

transparent, have fewer reservations about methodological limitations of the included trials, and

have more favourable conclusions than reviews not supported by industry. Therapeutic guidelines

based on this kind of evidence run a high risk of being invalid, and similar guidelines are more

than likely to exist in the field of diagnostics (see section “Conflicts of Interest” below).

In my view, the NACB guidelines on tumour markers in their present form, at least the chapters

that I have read, could not really pretend to be evidence-based guidelines (see also all other

comments below).

See comment above.

General Comments:

Conflicts of Interest and Other Prejudices:

There is ample evidence that individuals’ judgments of what is fair are biased in favour of their

self interest, or of their prejudices, and this is probably one of the main reason why invalid

guidelines my be published sometimes. Such conflicts or prejudices can lead one or several

members of a guideline development team to overstate or denigrate research results, selectively

withhold pertinent data or discussion, or exaggerate or minimize the shortcomings of the

research. The motivations for this behaviour can be financial (e.g. drug companies, the

diagnostics industry, granting or sponsoring bodies, health insurance organizations), academic or

administrative (e.g. pressure to publish, pressure to increase or to decrease the work-flow of

departments), political (e.g. pressure from patients advocates), personal (e.g. pressure from

friends and colleagues), cultural or educational (e.g. religion or any other prejudice), or based on

any other human passion (e.g. intellectual passion, will of power, vanity). For example, there is

evidence that practitioners who perform a procedure tend to emphasize the appropriateness of the

procedure compared with non-performing practitioners, or that specialists overwhelmingly

recommend the therapy that they themselves deliver. If users of guidelines wish to be able to

decide for themselves to what extent they can trust a guideline, at least sponsorship, or the lack of

it, together with the names, the activities, the professional addresses, and the precise role of all

contributors to the guideline development process should be made explicit. Even if such

precautions are taken, it is extremely difficult, if not impossible, for a guideline development

team to get rid of all their conflicts of interest and of all their prejudices. Therefore an element of

judgment that can distort the evidence is bound to be present in guidelines. Multidisciplinarity

and formal consensus development methods can reduce such judgments (see

“Multidisciplinarity” section, and specific comments below). The NACB guidelines on tumour

markers, in their present form, should be improved as much as possible in this area.

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Conflicts of interest of those involved in developing these guidelines are documented in an On-

Line Supplement, as required by the National Academy of Clinical Biochemistry and the

American Association of Clinical Chemistry.

Multidisciplinarity

In order to facilitate the implementation of a guideline, it should at least be developed by all

professions involved in the management of the patients described in the guidelines. The

multidisciplinarity of the guideline development team, if associated with formal consensus

development methods, may also be a good way of reducing conflicts of interests, prejudices, and

controversies, while allowing members of the team to learn from each other.

We have done our best to involve individuals from relevant professions.

Formal consensus development methods, such as the nominal group technique, the Delphi survey,

and the RAND/UCLA appropriateness method, which combines elements of both the previous

ones, can minimize the influence of individual opinions, and help writers and users of guidelines

to better understand the reasons for disagreement among experts, and solve disagreements as

much as possible, thus increasing transparency, explicitness, and eventually implementation of

guidelines.

Guidelines on the use of laboratory tests are sometimes developed exclusively, or almost

exclusively by laboratorians. Such guidelines may be of limited value, particularly in terms of

validity. For example, among 11 recently published guidelines providing advice on the use of

laboratory tests in non small cell lung cancer patients, only one guideline, which was very much

laboratory-oriented, clearly recommended the use of tumour markers in routine practice, without

providing any good-quality evidence that such a recommendation would be beneficial for the

patients in terms of clinical outcomes, whereas tumour markers may be associated with

downsides, e.g. unjustified therapies with side-effects, or stress for the patients, and costs. Such

guidelines may, however, have the status of a position statement of the discipline and may serve

as a starting point for further multidisciplinary guideline development. It seems to me that the

NACB guidelines on tumour marker in their present form, at least some chapters, e.g. the lung

cancer chapter, are more position statements than evidence-based guidelines. The problem is that

such chapters are misleadingly entitled “guidelines”.

The NACB lung cancer guidelines are to be included in a subsequent paper.

Question Formulation

It is common sense to say that one cannot find a clear answer to an unclear question. This is why

question formulation is at the heart of systematic reviews and of evidence-based guidelines.

Questions should identify the reason(s) why the test is being used and whether its result will

influence clinical decisions and outcomes. A structured question consists of three or four parts: 1)

the patient [P] with a problem 2) the diagnostic intervention [I], 3) its comparison [C] to another

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test or a reference standard (this third part may be missing), and 4) the outcome [O] (thence the

PICO acronym). Using two examples, we illustrate below two types of questions which can be

formulated differently, depending on whether the question is related to the diagnostic accuracy or

the diagnostic utility of a test. These two questions are formulated according to the four- or three-

part model indicated above:

Type 1 question (regarding the diagnostic accuracy of a test): In (1) patients with high levels of

total PSA, how well does (2) free/total PSA ratio, (4) predict prostate cancer (3) as diagnosed by

biopsy of the prostate?

Type 2 (related to the value of a test in improving patient outcomes): In (1) non symptomatic

male patients aged 50 years or older, how well does (2) the use of PSA as a screening test for

prostate cancer, (4) decrease prostate cancer incidence, prostate cancer mortality, and all-cause

mortality?

As you know, answers to type 2 questions, which need randomized controlled studies with a hard

outcome measure, ie mortality, provide higher levels of evidence than answers to type 1 questions

which “only” need good quality prospective cohort studies with diagnostic accuracy data. In the

NACB guidelines on tumour marker, the formulation of the questions (and therefore of the

answers) are not always that clear. This could and should be amended (see other comments

below).

We agree that how to formulate questions should have been addressed in more detail in the

recommended structure with which NACB Sub-Committees were provided (Supplemental Data

Table 1). We would strongly recommend that this should be done if and when these Guidelines

are updated.

Grading Levels of Evidence and Strengths of Recommendation

A number of approaches have been used to grade levels of evidence and strengths of

recommendations, all of which have important shortcomings. The GRADE working group has

proposed a grading system, which aims to make it easier for users to assess the judgments behind

recommendations. Maybe the system that is indicated in chapter 1 of these NACB guidelines is a

good one as far as grading the levels of evidence is concerned, but in this case, more explanations

would be necessary in this chapter 1. What is sure is that the strengths of recommendations are

missing. Maybe a quite simple solution would be for the authors of the guidelines to associate

each of their recommendations with a degree of consensus (which might perhaps be a not too bad

substitute for strengths of recommendations). For example, they could use a grade A if all of

them support the use of a marker in a particular situation, or B, or C, or D in decreasing order of

degrees of consensus.

We have included Strengths of Recommendation as well as Levels of Evidence in the NACB

Guidelines, adopting the approach recommended by Atkins et al (BMJ 2004;328:1490) for the

former.

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I have had a look at the 3 latest NACB guidelines published on the NACB website ie maternal-

fetal risk assessment (2006), emergency toxicology (2005), and thyroid disease (2003). As

summarized in the table below, none of these 3 guidelines compared their recommendations with

those of others, and none of these 3 guidelines provided the levels of evidence supporting their

recommendations. These NACB guidelines on tumour markers might therefore be considered as

performing better than these 3 other NACB guidelines, except perhaps for the emergency

toxicology guidelines, which provided degrees of consensus (which is perhaps not that bad a

substitute for strengths of recommendations).

NACB Guidelines Comparisons with

others

Levels of evidence Strength of

recommendations

Maternal-fetal risk

assessment (2006)

None None None

Emergency toxicology

(2005)

None None Degrees of consensus

Thyroid disease (2003) None None None

Tumour markers (draft) Yes Yes None

Tumour Markers versus Other Laboratory Tests

NACB is not the National Academy of Tumour Markers. It is the National Academy of Clinical

Biochemistry.

Clinical Biochemistry includes tumour markers as well as all sorts of other tests. For example, as

far as the prognostic assessment of lung cancer is concerned, it is not yet demonstrated that

tumour markers perform better than LDH, calcium, albumin, blood cell counts or other tests that

are usually measured in routine in lung cancer patients. Some studies may have demonstrated that

tumour markers are better, but other study have demonstrated the opposite, none of these studies

being of a design or of a quality that would allow to know for sure which is right or which is

wrong. Why in this case should the NACB panel recommend tumour markers for the prognostic

assessment of the patients rather than other tests, which are already measured in routine in lung

cancer? This would simply increase costs and other downsides, particularly if there is no evidence

that a prognostic assessment based on these measurements can improve therapeutic decisions and

outcomes.

More generally, there is a tendency in all the chapters that I have read to list exhaustively the

tumour markers that have been evaluated in the primary studies, whereas the other laboratory

tests, some of which might be as useful as, or even more useful than tumour markers, are not

listed as exhaustively. In my view, this is another important shortcoming for such guidelines,

particularly since they are supposed to be endorsed by an organization such as the NACB.

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The remit of this project was to provide information as to the clinical circumstances in which

tumor markers provide added value.

Comparisons with others

All chapters that I have read compare their recommendations with those of others, which is fine.

The problem is the way it is done. For example, regarding lung cancer, NACB recommendations

are only compared with EGTM recommendations. As far as I am aware, the only lung cancer

guidelines that support the use of tumour markers in clinical routine practice are those of the

NACB in their present form, and those of the EGTM. Some authors of the current NACB

guidelines were also authors of the EGTM guidelines; in particular Petra Stieber is the first co-

author of both guidelines. This probably means that in fact no real comparison was done. A real

comparison should include at least the recommendations made by some the most prestigious

clinical societies or governmental organisations worldwide, e.g. ACCP= American College of

Chest Physicians; ASCO= American Society of Clinical Oncology; ATS-ERS= American

Thoracic Society and European Respiratory Society; BTS-SCG= British Thoracic Society and

Society of Cardiothoracic Surgeons of Great Britain and Ireland; SIGN= Scottish Intercollegiate

Guidelines Network, particularly since all these organizations recommend against the use of

tumour markers in clinical routine practice.

It is not clear whether the above paragraph refers only to lung cancer or to other cancer sites. If

the former, the commenter will be aware that none of the lung cancer guidelines produced by the

above organizations considered possible use of tumor markers, i.e. no recommendations are made

either for or against their use. Even for those malignancies where tumor markers are mandatory in

clinical practice (e.g. testicular cancers), reference to them in clinical guidelines is often rather

brief.

More generally, in almost all the chapters that I have read, it seems more or less fallacious to

pretend that comparisons that are made between NACB and EGTM are comparisons between two

really independent sorts of people. Apart from the case of the lung cancer chapter already

mentioned, another striking illustration of this situation seems to be the ovarian cancer

comparisons, where three so-called different groups are compared, ie EGTM 1999, NACB 2005

and EGTM-NACB 2002!

What is more, it seems that the comparisons with others (ie true others) that are made tend to be

chosen according the fact that the “others” agree with the NACB panels. For example, in the lung

cancer chapter as well as in the gammopathies chapter, only one “other(s)” (who was in full

agreement with the NACB panels) was chosen.

See above comment.

Finally, the main purpose of making comparisons with others is to argue when controversies do

exist. For example, regarding mass screening of prostate cancer using PSA, the authors of this

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chapter do not tell us why some organizations recommend for and why some others recommend

against (see related example in the next section, Outcomes).

These complex issues are discussed and references provided.

Outcomes

All relevant outcomes should be considered when guideline recommendations are developed, as

their identification is key to informed decisions. An inappropriate choice of outcome measures

may introduce an outcome bias in guidelines, which can result in the promotion of diagnostic

strategies with unproven benefits (but maybe with proven downsides). For example, regarding

screening for colorectal cancer in the average risk population aged more than 50 years, although

this is not mentioned in the NACB guidelines, there are some organizations in the world that

recommend against such a mass-screening. Regarding faecal occult blood test (FOBT) in

particular, systematic reviews of randomised controlled trials have demonstrated that the use of

FOBT as a screening test for colorectal cancer in the average-risk population results in decreased

colorectal cancer mortality. Based on these systematic reviews, some organizations recommend

mass-screening using FOBT, whereas some other organizations do not recommend mass-

screening using FOBT because (1) this procedure would be too expensive and/or not adapted to

their local organization of care, and/or (2) the balance between benefits and harms is not crystal-

clear from an individual patient perspective [Hewitson et al 2007]. What is more, the impact of

FOBT screening on health is not clear when cause mortality is considered [Moayeddi et al 2006].

Publication of Guidelines

There are no agreed reporting standards for guidelines, and the practices vary between publishing

them on a web site, publishing them in peer review journals, and sometimes doing both.

However, as any other scientific work, evidence-based guidelines and their executive summaries

have to be presented skilfully. This is why people with writing and editing skills should be part of

guideline development teams. Among the many points to be addressed during this phase, the

following ones may need special emphasis when drafting diagnostic recommendations:

In a good diagnostic guideline, recommended interventions should be explained and discussed in

detail, particularly if there are disagreements between the recommended interventions and those

made in current practice.

For the sake of clarity and explicitness, algorithms following the logical framework of diagnostic

interventions should be provided.

Benefits and harms arising from using the recommended tests should be discussed too, in

comparison with alternative tests that could be used in the same context.

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Sometimes, when a test is recommended in a guideline, doctors may be at loss of what to do with

the test result because of lack of information about decision limits, biological variation, or other

information mentioned in table 13.1 in Watine et al 2007. A good diagnostic guideline should

provide this kind of information, as well as patient information, materials supporting

implementation, e.g. reminders, clinical audit data collection forms (see next sections).

The implications for research or any other potential areas of interest to the guidelines users should

also be discussed to enhance transferability.

The general comments above are very much based on: Watine J, Oosterhuis WP, Nagy E,

Bunting P, Horvath AR. Formulating and using evidence-based guidelines. In: Price C,

Christenson R, eds. Evidence Based Laboratory Medicine: From Principles to Outcomes. 2nd

edition, AACC Press, 2007.

We would agree that this is a very useful book.

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