Risk of Ductal Carcinoma In Situ (DCIS) Progressing to Invasive Breast Cancer (IBC) D. Craig Allred, M.D., Professor of Pathology, Breast Center, Baylor College of Medicine, Houston, TX

The goals of this presentation are to challenge the widely held opinions that there are just two subtypes of DCIS (i.e. noncomedo and comedo) and that comedo DCIS is the most likely to progress to IBC.

DCIS is the most common (90%) type of noninvasive breast cancer and is important by giving rise to most IBCs. A practice has developed over the years to simply dichotomize DCIS into noncomedo and comedo subtypes based on the notion that the histological features of this lesion are either well or poorly differentiated, respectively. This is in contrast to the acknowledged histological variation of IBC ranging on a continuum from very low to very high grade morphological abnormalities, and this diversity has been expressed by numeric grading systems scoring features like growth pattern, nuclear grade, and mitotic index. In fact, DCIS shows the same wide-ranging histological diversity as IBC which can also be quantified by similar grading systems. Recent studies have shown a strong correlation between the histological and biological differentiation of DCIS along this continuum. For example, the average rate of tumor cell proliferation gradually increases from 1% in the lowest grade to over 50% in the highest grade lesions. Overexpression/amplification of the erbB2 oncogene and mutation of the p53 tumor suppressor gene also increase in the same direction. In contrast, the expression of estrogen and progesterone receptors decreases gradually from almost 100% to 25% from the best to the worst differentiated.

Another commonly held belief is that so-called comedo DCIS is the most dangerous clinically in the sense of being more likely than noncomedo DCIS to progress to IBC, and this notion is based on several types of evidence. For example, comedo DCIS accounted for up to 75% of newly diagnosed lesions in the pre-mammography era and most were large and palpable. Their largeness was taken to indicate aggressiveness and their commonness as being likely precursors for most IBC. They looked bad under the microscope and had aggressive biological features which correlated with poor prognosis in IBC, so the same was assumed for DCIS. The rate of axillary nodal metastases was higher with comedo than noncomedo disease (2-3% vs. <1%). Many studies suggested that the local recurrence rate of comedo DCIS managed by lumpectomy was 2-3-fold higher than for noncomedo lesions, especially with shorter follow-up, and half of all recurrences were invasive.

While these are compelling reasons to think that comedo DCIS has a particularly bad prognosis, there are reasonable alternative interpretations for much of this evidence and other equally compelling observations support the idea that low-grade noncomedo DCIS is MORE likely to progress to invasive disease. For example, some investigators speculated that comedo DCIS were common historically because they had trouble becoming invasive and thus grew large enough to become palpable. Poorly differentiated histology and aggressive biological features are clinically meaningless when associated with noninvasive disease. Because comedo DCIS are often large, it is possible for pathologists to miss foci of invasion due to routine limited sampling, which could explain a higher rate of nodal metastases. Then there is other evidence. For example, at least 80% of IBC have an associated component of DCIS, and the rate approaches 100% with comprehensive histological sampling. Surprisingly, the majority (up to 75%) of DCIS in a breast with IBC are lower-grade noncomedo lesions. The biological correlate to this is that the 20% rate of erbB2 overexpression in IBC is much closer to the 10-15% rate associated with noncomedo DCIS than the 60-70% rate observed with comedo disease. In addition, some clinical studies of DCIS managed by lumpectomy are beginning to show that, with long follow-up, noncomedo DCIS has a higher rate of recurrence as IBC.

With mammography, the majority of newly discovered DCIS are now noncomedo lesions. They are usually smaller and more confined than comedo lesions, making them easier to manage with conservative surgery. However, they are probably as likely, if not more so, than high-grade lesions to progress to IBC if unrecognized or inadequately treated and, therefore, should be taken very seriously.

Gray-scale and color Doppler US for local recurrences of breast cancer Bruno D. Fornage, M.D., Professor of Radiology & Surgical Oncolory, M. D. Anderson Cancer Center, University of Texas, Houston, TX

Because sonography (US) cannot demonstrate microcalcifications with sufficient reliability, only breast cancer recurrences that present as a mass can be visualized with US.

Gray-scale Sonographic Appearances
The US appearances of recurrent masses are not significantly different from those of primary tumors, but the recurrent lesions are usually smaller at the time of diagnosis. Malignant tumors appear as a focal hypoechoic mass with irregular or spiculated margins and disruption of the normal architecture of the breast. In contrast to fibroadenomas and other benign masses, a recurrent tumor, like a primary carcinoma, may exhibit a “taller-than-wide” shape, with the tumor’s longest diameter being perpendicular to the skin (length-to-anteroposterior-diameter ratio less than 1). This shape is highly characteristic of malignancy. If the mass is sufficiently large, some heterogeneity of the echotexture may be noted. Intratumoral clustered microcalcifications can be visualized as minute bright echoes within the hypoechoic tumor. Acoustic shadowing-once considered an essential diagnostic feature of cancer-may be lacking. Absence of compressibility and adherence of the tumor to the surrounding tissues during dynamic US examination are very important clues suggesting malignancy.

Scars are difficult to evaluate sonographically because of the significant shadow associated with them. It is critical to examine the region of a scar dynamically by increasing the amount of pressure applied with the probe. This usually clears the shadow that was cast by a scar, whereas the shadow created by a true recurrent mass would remained unchanged. Also, scars are retractile and their lateral edges should be concave, whereas any bulging of the margin of a scar should be viewed with suspicion. Short-term follow-up is often needed to confirm the stability of the imaging findings, and US-guided biopsy may be necessary. In that case, extensive sampling through the area of shadowing is required.

Invasive lobular carcinomas are difficult to identify on US, as they are on mammography. This is true for recurrences as well. The significant distortion and fibrosis seen on mammograms may appear on sonograms as areas of marked shadowing without a well-defined mass. Mucinous and medullary carcinomas are relatively well circumscribed. Medullary cancer may be markedly hypoechoic with significant sound through-transmission and may mimic a cyst.

Color (Power) Doppler Imaging Findings
Abnormal Doppler signals reflecting hypervascularity have been reported in the majority of malignant tumors but also in a significant number of benign masses. In malignant tumors, however, neovessels are typically tortuous and disorganized and penetrate the tumor at a 90( angle).

With new US techniques like pulse-inversion harmonic imaging and the use of US contrast agents, detailed mapping of the internal vascularity of solid masses is becoming possible, which should allow more reliable differentiation between benign and malignant lesions. In any case, any new solid mass developing after breast conservation surgery—especially if hypervascular on color Doppler US—should be considered suspicious for recurrence until proven otherwise.

Recurrences in Nodal Basins
US can readily visualize recurrences in the nodal basins that are not amenable to palpation and mammography, such as in the internal mammary chains and the infraclavicular region.

The confirmation of recurrence, whether in the breast or in the nodal basins is obtained within minutes through US-guided fine-needle aspiration.

The Impact of Local Recurrence After Breast Conserving Therapy on Distant Metastases and Death Frank A. Vicini, M.D., Clinical Assoc. Professor, Director of Radiation Oncology, William Beaumont Hospital, Royal Oak, MI

Introduction: The impact of local recurrence (LR) on survival in patients with early stage breast cancer treated with breast conserving therapy (BCT) remains controversial. Although it has consistently been demonstrated that patients who experience a LR after BCT have an increased risk of developing distant metastases (DM), it is uncertain whether a LR signals a more biologically aggressive tumor or is the nidus for future dissemination(1;2). For many clinicians, a LR after BCT is presumed to have no detrimental effect on survival due to the belief that breast cancer is a systemic disease at inception (3). As a result, a LR is considered a marker for DM rather than a cause. In contrast, others believe that preventing a LR may improve survival by avoiding a “secondary” dissemination of cancer cells directly from the LR(4). This hypothesis is corroborated by recent data from three large prospective randomized trials of post-mastectomy loco-regional radiotherapy (RT) and several published meta-analyses on the impact of adjuvant RT on survival (5-10).

The purpose of the current analysis was to evaluate the impact of local recurrence (LR) on the development of distant metastases (DM), overall survival (OS) and cancer specific survival (CSS) in patients with early stage breast cancer treated with conservative surgery (CS) and postoperative radiotherapy (RT) at our institution and to review published data on this critical issue.

Methods & Materials
Between 1980 and 1995, 1169 patients were treated with CS and RT at William Beaumont Hospital, Royal Oak, Michigan. All patients had follow-up greater than one year and (4 nodes involved with cancer. The median duration of follow-up was 7.7 years. A Cox proportional hazards model was performed to evaluate the effect of LR on the development of DM and CSS. A matched pair analysis (MPA) (1:2) was also performed comparing outcome in patients with and without LR controlling for multiple prognostic factors.

Results
Local recurrence was 11% at 12 years. For the entire population, LR led to a poorer OS and CSS at 12 years than local control (LC) (71% vs 81%, p=0.001 and 69% vs 88%, p<0.001, respectively). In a Cox multiple regression model, LR was a significant predictor of cancer specific mortality. The hazard ratio (HR) associated with LR was 2.69 for mortality and 2.67 for DM (p<0.001 and p<0.001, respectively). The median time from surgery to distant metastases was 3.8 years for patients without LR vs 4.7 years for patients with LR. Patients with LR also had two peaks in the rate of DM (at 2.5 and 6.5 years) as opposed to only one (1.5 years) for those without LR. The impact of LR on DM was still evident in patients with small (( 2.0 cm) tumors (p<0.001), negative lymph nodes (p=0.004) or both (p<0.001). Recurrences developing outside of the surgical bed region had no negative effect on survival. In the matched-pair analysis (controlling for age, tumor size, grade, number of positive nodes, and estrogen receptor status), LR was still the most significant predictor of mortality (HR 5.86 for mortality and 6.43 for DM).

Conclusions
Our results suggest that LR is responsible for an increase in DM and cancer-specific mortality in patients treated with CS and RT. This is reinforced by a distinct difference in the time distribution of DM after LR and by the fact that recurrences originating outside of the surgical bed did not affect overall survival. These data reinforce the necessity to insure optimal LC in patients treated with BCT and support the conclusions of recent randomized trials and meta-analyses specifically addressing this issue.

Reference List

1. Fisher ER, Anderson S, Tan-Chiu E, Fisher B, Eaton L, Wolmark N. Fifteen-year prognostic discriminants for invasive breast carcinoma. Cancer 2001;91:1679-87.
2. Fowble B. Ipsilateral breast tumor recurrence following breast-conserving surgery for early-stage invasive cancer. Acta Oncol 1999;38 Suppl 13:9-17.
3. Veronesi U, Marubini E, Del Vecchio M, Manzari A, Andreola S, Greco M et al. Local recurrences and distant metastases after conservative breast cancer treatments: partly independent events. J.Natl.Cancer Inst. 1995;87:19-27.
4. Fortin A, Larochelle M, Laverdiere J, Lavertu S, Tremblay D. Local failure is responsible for the decrease in survival for patients with breast cancer treated with conservative surgery and postoperative radiotherapy. J.Clin.Oncol. 1999;17:101-9.
5. Favourable and unfavourable effects on long-term survival of radiotherapy for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 2000;355:1757-70.
6. Whelan TJ, Julian J, Wright J, Jadad AR, Levine ML. Does locoregional radiation therapy improve survival in breast cancer? A meta-analysis. J.Clin.Oncol. 2000;18:1220-9.
7. Van de SJ, Soete G, Storme G. Adjuvant radiotherapy for breast cancer significantly improves overall survival: the missing link. Radiother.Oncol. 2000;55:263-72.
8. Overgaard M, Jensen MB, Overgaard J, Hansen PS, Rose C, Andersson M et al. Postoperative radiotherapy in high-risk postmenopausal breast-cancer patients given adjuvant tamoxifen: Danish Breast Cancer Cooperative Group DBCG 82c randomised trial. Lancet 1999;353:1641-8.
9. Overgaard M, Hansen PS, Overgaard J, Rose C, Andersson M, Bach F et al. Postoperative radiotherapy in high-risk premenopausal women with breast cancer who receive adjuvant chemotherapy. Danish Breast Cancer Cooperative Group 82b Trial. N.Engl.J Med 1997;337:949-55.
10. Ragaz J, Jackson SM, Le N, Plenderleith IH, Spinelli JJ, Basco VE et al. Adjuvant radiotherapy and chemotherapy in node-positive premenopausal women with breast cancer. N.Engl.J Med 1997;337:956-62.

Management of Invasive Local Recurrence in DCIS; Local and Systemic Therapy Patrick I. Borgen, M.D., Chief, Breast Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York

Ductal carcinoma in situ represents the most rapidly expanding segment of the breast cancer population in the United States. This expansion, driven by dramatic improvements in utilization of screening mammography, has resulted in a significant evolution of the treatment of ductal carcinoma in situ (DCIS). In broad terms this has paralleled the evolution of the treatment of invasive carcinoma of the breast. That is, breast conservation therapy (wide local excision with or without radiation therapy) is now considered the appropriate and preferable management option for a majority of patients with localized DCIS. The role of radiation therapy in DCIS has been addressed in a number of prospective randomized trials, most notably the NSABP B17 Trial, which showed not only a substantial reduction in relapses, but in particular a substantial reduction in invasive relapses. Along with other studies, this trial changed the paradigm for the majority of patients with ductal carcinoma in situ, and emphasized that these patients are at risk for local regional relapse.

There is a wide range of reported local-regional relapse rates ranging from 5% to nearly 20%. Overwhelmingly the site of recurrence is at or near the site of previous tumorectomy. (See figure 61-1) Age is also an important consideration in predicting the likelihood of local relapse with younger patients experiencing a significantly higher risk than older patients. Our group has previously shown that decreased volume of resection in younger patients compared to older patients may be a contributing factor to this phenomenon. (See figure below)

One relatively constant figure that has been reported from a majority of studies is the fact that fully 50% of recurrences are invasive in nature. Treatment of recurrent invasive disease in the breast after DCIS can be divided into surgical treatment and systemic treatment. Both are greatly influenced by the treatment approach taken with the index DCIS lesion.

Figure 61-1
Location of local recurrence in relation to the quadrant of the original carcinoma. (Reproduced by permission from Osborne MP, Borgen PI, Wong GY, Rosen PP, McCormick B. Savage mastectomy for local and regional recurrence after breast-conserving operation and radiation therapy. Surg Gynecol Obstet 1992, 174:189-194. By permission of Surgery, Gynecology & Obstetrics, now know as the Journal of the American College of Surgeons.)

Surgical Management of Local Recurrent DCIS

The range of recurrence types after BCT for DCIS ranges from small microscopic DCIS to inflammatory breast cancer and the surgical approach depends heavily on this factor. Treatment is also dependent on the treatment the patient has already received. In the past, the dogma was to recommend total mastectomy (salvage mastectomy) for all patients with any form of relapse. This has been called into question, and it is not unreasonable to, in effect, re-conserve the breast by performing a wide local excision, particularly if radiation therapy has not already been administered. It has been increasingly our practice to offer breast conservation therapy in these patients. (See algorithm below) For unifocal invasive relapses, we perform a sentinel lymph node biopsy at the time of the surgical excision of the primary tumor. There is no evidence that prior surgery diminishes the success or accuracy of sentinel lymph node biopsy in breast cancer. More extensive areas of DCIS or multiple disease areas involving more than one quadrant of the breast are best treated with mastectomy. Similarly, patients who are previously irradiated, in the majority of cases, are recommended to undergo a mastectomy following local relapse in the breast. Approximately 75% of our patients elect immediate breast reconstruction either with subpectoral tissue expanders or autogenous tissue transfer. Post mastectomy radiation therapy is currently done for patients with four or more positive axillary lymph nodes, involvement of skin, or involvement of pectoralis major.

Elegant studies in the 1980’s and 90’s indicated that the overwhelming majority of patients who had invasive cancer initially and who recurred in the breast were operable because they demonstrated no evidence of systemic disease. We can extrapolate from this to recurrences after conservative treatment of DCIS where the majority of patients will, in fact, have no evidence of systemic disease.

Systemic Therapy Considerations

Systemic therapy options for invasive relapses after conservative treatment of DCIS very closely parallel management strategies for patients who present with invasive carcinoma de novo. The prognosis and treatment are determined primarily by the status of the axillary lymph nodes and secondarily by tumor characteristics such as size, lymphovascular invasion, ER/PR status and Her-2/neu status. Patients who relapse locally in the breast, who are currently on tamoxifen represent a special therapeutic dilemma, which will be discussed. There is growing evidence concerning the role of aromatase inhibitors as anti-estrogen therapy in breast cancer, but specific studies in DCIS, and in particular recurrent DCIS, have not been reported.

Our understanding of all breast cancer, particularly DCIS is largely descriptive. Progress in the Human Genome Project holds great promise for helping us move towards a more functional understanding of the disease. Approximately 19,000 genes and tens of thousands more tentatively described as expressed sequence tags (EST) have been identified. It is likely that many genes that might be useful for diagnosis andor prognostication in breast cancer have yet to be recognized in this group. The advent of cDNA microarray technology now allows for the efficient measurement of expression of virtually every gene in the human genome. Disease classification and treatment approaches will evolve as me move towards molecular classification of human malignancies.

Our current general algorithm for the systemic treatment of invasive carcinoma of the breast is presented below. Specific therapeutic agents will be discussed.

References:

1. Osborne MP, Borgen PI, Wong GI, et al, Salvage mastectomy for local and regional recurrence after breast conserving surgery and radiation therapy. Surg Gyn Obstet 1992; 174:189-194
2. Van Zee K, Borgen PI, Memorial Sloan-Kettering Cancer Center experience, in Ductal Carcinoma In-Situ, Silverstein eds. 1997, 455-467
3. Van Zee K, Liberman LL, McCormick B, et al, Long-term follow-up of DCIS treated with breast conservation : effect of age. Cancer 1999;86(9);1757-1767
4. Fisher B, Costantino J, Redmond C, et al, Lumpectomy compared to lumpectomy with radiation therapy for the treatment of intraductal breast cancer. N Engl J Med 1993; 328:1581-1586
5. Lagios M, Dcut carcinoma in-situ: pathology and treatment. Surg Clin N Am 1990;70:853-871
6. Swallow CJ, Van Zee K, Sacchini V, Borgen PI, Ductal carcinoma in situ of the breast: progress and controversy. Current Problems in Surgery 1996; 33(7)553-600
7. Hudis CA, Borgen PI, Systemic treatment for stage I and stage II breast cancer. In BoslGJ and Brennan M (eds) Surgical Oncology Clinics of North America, Adjuvant Therapy of Cancer. 1997;6(4):683-698
8. Van Zee K, Tan LK, Calvano JE, Rosen PP, Borgen PI, p53 mutations and HER-2/neu amplification in microdissected ductal carcinoma in situ.
9. Alizadeh A, Ross T, Perou C, Towards a novel classification of human malignancies based upon gene expression patterns. Journal of Pathology 2001;195:41-52

Treatment of Axillary Recurrence Gordon F. Schwartz, M.D., MBA, Professor of Surgery, Jefferson Medical College, Philadelphia, PA

Axillary recurrence following appropriately planned and executed procedures to treat carcinoma of the breast are infrequent. Whether axillary recurrence is newly recognized cancer in axillary lymph nodes intentionally or unintentionally left behind at the time of axillary dissection accompanying breast conservation or mastectomy, or in the soft tissues of the axilla as recurrence or metastasis, may significantly affect choice and efficacy of treatment.
As treatment of axillary nodes has changed in response to the “staging versus treatment” argument about the value of axillary dissection, with less rather than more extensive dissection of the axilla performed currently rather than a generation ago, the expectation that a greater incidence of axillary recurrence would be observed has not been validated. This does not negate the importance of a meticulous dissection when treatment of the axilla is indicated.
However, axillary recurrence does occur; when it does it is difficult to treat. Documenting the nature of the recurrence may be crucial in determining therapy. Most often, axillary recurrence is noticed by the patient as a mass of fullness in the axilla. Even more rarely is recurrence in a Rotter’s (interpectoral) node, which would present as a fullness in the infraclaviucular space behind or adjacent to the edge of the pectoralis major muscle. Occasionally, pain, decreased motion, or even newly noted arm edema may be signs of axillary recurrence. In general, axillary recurrence is observed most commonly in the first three years after initial treatment. Because axillary recurrence may be the first sign of systemic disease, the diagnosis of axillary recurrence should be made by FNA whenever possible, so that a complete patient evaluation is performed before any surgical treatment is planned. When axillary recurrence is accompanied by supraclavicular node disease, systemic metastasis is almost always present. CT scan or MRI of the axillary and supraclavicular areas may help document patient status as well as outline the extent of disease if a surgical procedure is contemplated.
In the absence of systemic disease, when only a single site (axilla) can be implicated, a combined surgical and radiation therapy approach is often appropriate, especially if the disease can be documented as nodal rather than soft tissue disease. The usual scenario for this diagnosis is a patient with an inadequate node dissection during mastectomy or as part of breast conservation. When a node dissection had not been part of the original treatment plan, and secondary nodal disease is the diagnosis, a formal node dissection alone may be appropriate. If a surgical procedure had been performed and the recurrence is in nodes left behind, a meticulous dissection of the axilla may be enough, but, depending upon the extent of the disease, axillary (and supraclavicular radiation) may be added. Rarely, the recurrence is in an interpectoral node; both surgery and radiation are then appropriate because of the difficulty performing an en bloc dissection of this area. Adjuvant chemotherapy (or Tamoxifen) would usually be considered in all of these patients. Residual nodal disease may be well controlled by a combination of surgery and radiation therapy, and long survival is often achieved in these patients with only axillary node disease. Unfortunately, patients with only small-volume nodal disease are not the rule, so that the majority of patients with axillary recurrence ultimate succumb to metastatic breast cancer.
If the recurrence is soft tissue rather than nodal, surgery is usually not very effective in eliminating further recurrence. Margins are vague, but large volume disease may be controlled locally, at least for some time, so that the combination of debulking surgery followed by radiation therapy, and adjuvant chemotherapy, are the usual recommendations. Removing “all” of the axillary disease surgically may be impossible, even macroscopically, but the debulking surgery and radiation may offer major improvement in the quality of patients’ lives, since chest wall and axillary recurrence become a source of extreme discomfort and embarrassment because of difficulty maintaining hygiene as the disease progresses.
An emerging problem in the next ten years may be an increase in the incidence of axillary node recurrence in patients who have undergone a “negative” sentinel lymph node biopsy. Because this procedure is still in its relative infancy, without lengthy follow-up data, the true incidence of false negative sentinel node biopsy is uncertain. Thus far, with close to ten years experience in the procedure by some investigators, axillary recurrence has been anecdotal. Hopefully that will continue as the procedure is more widely adopted in this country and abroad.

Should adjuvant therapy be given to all patients? Daniel F. Hayes, M.D., Director, Breast Oncology Program, University of Michigan Comprehensive Cancer Center, MI

Based on predictive factor categories (chemotherapy: age, ? ER, HER2; endocrine therapy: ER, PgR), the relative benefits from specific adjuvant systemic therapy (AST) can now be estimated 1,2. For individual patients, the absolute benefits for individual patients based on their prognostic (nodal status, tumor size, grade, and to a lesser extent, ER, PgR, and HER-2) and predictive factor profiles 3. Therefore, a woman can be informed about her odds of reducing the chance of developing subsequent, incurable recurrence from specific therapies. The decision about whether to accept one type of AST or another is complex. Preliminary studies have suggested that different patients, caregivers, and societies make decisions differently based on social, cultural, and economic factors 4,5. Furthermore, the endpoint (reduction in mortality, metastasis, local-regional recurrence, or new primary) substantially modifies these decisions. In summary, the answer to the question is no, but a thoughtful discussion with each patient will allow her to be the respondent.

1. Early Breast Cancer Trialist’s Collaborative Group: Tamoxifen for early breast cancer: An overview of the randomised trials. Lancet 351:1451-1467, 1998
2. Early Breast Cancer Trialist’s Collaborative Group: Polychemotherapy for early breast cancer: an overview of the randomized trials. Lancet 352:930-42, 1998
3. Ravdin PM, Siminoff L A, Davis GJ, et al: Computer program to assist in making decisions about adjuvant therapy for women with early breast cancer. J Clin Oncol 19:980-91, 2001
4. Coates AS, Simes RJ: Patient assessment of adjuvant treatment in operable breast cancer. New York, NY, John Wiley & Sons Ltd, 1992
5. Lindley C, Vasa S, Sawyer T, et al: Quality of life and preferences for treatment following systemic adjuvant therapy for early stage breast cancer. Journal of Clinical Oncology 16:1380-87, 1998

Overview of clinical studies on liposome therapy I. Craig Henderson, M.D., Adjunct Professor of Medicine, University of California, San Francisco, CA

Liposomal encapsulation of the anthracyclines results in reduced cardiotoxicity. Two formulations of doxorubicin have been widely studies, Doxil and Myocet, but only the former has been approved for marketing in the U.S. The differences between the two formulations result from the addition of polyethylene glycol or PEG to the outer surface of Doxil. Although all liposomal formulations have a longer half life than doxorubicin, the half life of Doxil is particularly long and exceeds 50 hours. The optimal dose-schedules for the two drugs is quite different. Myocet is usually administered every 3 weeks at a dose of 75 mg/m2 as a single agent or 60 mg/m2 in combination. Doxil is usually given every 4 weeks at a dose of 40-50 mg/m2 as a single agent and 30-35 mg/m2 in combination every 4 weeks. Both of these drugs have been compared as a single agent in randomized trials with other anthracyclines. The data are difficult to interpret, but in general the activity of the liposomal formulations is very nearly the same as that of doxorubicin or (in the case of Myocet) epirubicin. Both drugs have substantially and significantly less cardiotoxicity than doxorubicin. Both drugs have been evaluated in combinations. Trials of the liposomal drugs plus Herceptin are underway, but no definitive results are available yet or likely to be available soon. The toxicity profiles of the two drugs differ substantially. Except for cardiotoxicity, the side effects and dose limiting toxicity of Myocet is similar to that of doxorubicin. Doxil is associated with almost no hair loss or nausea/vomiting; myelosuppression is reduced compared to doxorubicin. The dose limiting toxicity from Doxil is palmar plantar erythrodysesthesia, but this can be substantially reduced by using lower doses with longer intervals. This class of drugs holds considerable promise for less toxic regimens for the management of breast cancer, especially for those who are particularly adverse to toxicities, for those who are at higher than average risk for cardiotoxicity, and in combination with drugs that synergize with the anthracyclines in inducing cardiotoxicity.

Her2 Determinations In Decision Making For the Treatment of Breast Cancer Peter M. Ravdin, Associate Professor of Medical Oncology, University of Texas, Health Science Center, San Antonio, TX

This talk will review this rapidly evolving area of interest. The talk will show how the ASCO guidelines for the use of Her2 might be challenged and review each of the 7 ASCO guideline statements with an eye as to whether these guidelines might be revised again in the near future. Admittedly one of the areas in the ASCO tumor guidelines that have gotten more complex and evolved over successive iterations is the topic of the use of Her2 as a tumor marker. The most recent ASCO guidelines suggest:

1. That Her2 expression level should be measured on every primary breast cancer, and suggested that measures of Her2 amplification might also be of value.

Is there any role for using Her2 by IHC? Examination of the role of FISH testing is now emerging as a more useful test and that measures of expression are being eclipsed. But need Her2 be determined prior to needing to know this information at the time of recurrence?

2. It was recommended that Her2 overexpression be used to identify patients most likely to benefit from Trastuzumab (Herceptin) treatment of metastatic, recurrent, or treatment refractory breast cancer (but not yet in adjuvant therapy). FISH measures of amplification are certainly more predictive.

3. It was recommended that Her2 determinations not be used determine whether CMF was appropriate adjuvant therapy. This seems to be holding up and is a good example how underpowered studies with subset analysis can be misleading.

4. It was felt that Her2 overexpression might identify patients who would particularly benefit from anthracycline based adjuvant therapy, but that the lack of Her2 overexpression should not be used to select against anthracycline based adjuvant therapy.

A curiously hedged statement. The data will be reviewed and be found to be provocative but still not definitive in support of the use of predictive power of Her2 determination to select for or against the use of anthracyclines in adjuvant therapy.

5. It was recommended that Her2 determinations not be used to determine whether endocrine therapy was appropriate in adjuvant therapy or the treatment of metastatic disease.

Although this seems to be true, Her2 positive patients do seem to benefit less from tamoxifen less than Her2 negative patients even in studies where all patients were estrogen receptor positive. Interesting preliminary studies suggest that Her2 positive patient tumors do not have this relative resistance to aromatase inhibitors. If true then Her2 might be used to select patients who particularly benefit from aromatase inhibitors.

6. It was recommended that Her2 determinations not be used determine whether the use of taxanes was appropriate in adjuvant therapy or the treatment of metastatic disease.

This seems to be holding up because this question has not been adequately tested.

It was felt that the data was insufficient to recommend the use of Her2 overexpression to identify patients with a higher risk of relapse.

An area of strong controversy particularly for overexpression. Interestingly a review of the current literature suggests that Her2 amplification may be a better test with greater prognostic import.

1. 2000 Update of Recommendations for the Use of Tumor Markers in Breast and Colorectal Cancer: Clinical Practice Guidelines of the American Society of Clinical Oncology. Robert C. Bast, Jr, Peter Ravdin, Daniel F. Hayes, Susan Bates, Herbert Fritsche, Jr, John M. Jessup, Nancy Kemeny, Gershon Y. Locker, Robert G. Mennel, and Mark R. Somerfield JCO Mar 15 2001: 1865-1878.

Optimal Chemotherapeutic Regimens: Duration, Timing, and Schedule Clifford Hudis, M.D., Chief, Medical Oncology of Breast Center, Memorial Sloan-Kettering Cancer Center, New York City, NY

The most advantageous means of timing and sequencing surgery, radiation therapy, and systemic therapy is uncertain and is the subject of ongoing clinical trials. Because chemotherapy successfully shrinks locally advanced breast cancers and can allow local control surgery in the majority of cases, there has been enthusiasm for broader use of neo-adjuvant therapy. Neo-adjuvant therapy has been shown to allow more frequent breast conservation in patients with initially resectable disease but this earlier use of chemotherapy per se does not influence the risk of relapse or death, although it may provide early information on prognosis. Based on this data, neo-adjuvant chemotherapy can be most easily defended for patients with initially unresectable breast cancer and for those who refuse to undergo mastectomy but for whom a limited excision and radiation therapy would be acceptable. On the other hand, it is possible that the risks of over treatment for low risk patients (i.e. chemotherapy given for largely in situ carcinomas) could outweigh the benefits of this approach.

The optimal duration and make-up of chemotherapy, whether given post-operatively or in the neo-adjuvant setting is not known. Six months of CMF is a gold standard based on the worldwide meta-analysis performed at Oxford University but a variety of other regimens could be superior or equally effective and less toxic. Three months of AC was equivalent to CMF in two NSABP trials while six months of CAF or CEF has been superior in most trials. How six months of therapy consisting of sequentially dosed AC and a taxane will fit in remains to be confirmed. Numerous clinical trials are exploring these issues and will be reviewed.

New Trends In Adjuvant/Neoadjuvant Chemotherapy Terry Mamounas, M.D., M.P.H., F.A.C.S., Associate Professor of Surgery, Northeastern Ohio Universities College of Medicine , Medical Director, Aultman Cancer Center, Canton, OH

The benefit from adjuvant chemotherapy has been convincingly demonstrated in patients with stage I and II breast cancer. In these patients adjuvant chemotherapy has been shown to substantially reduce the risk for recurrence and death.1 However, despite significant progress, several issues regarding adjuvant/neoadjuvant chemotherapy still remain outstanding2 and these will be the focus of the presentation.

Optimal Anthracycline-Containing Regimens

The 1995 Oxford Overview analysis demonstrated that, when compared to CMF alone, anthracycline-containing regimens produce somewhat greater reduction in recurrence and mortality.1 Randomized trials have shown a threshold effect for doxorubicin and cyclophosphamide, in the AC combination with no additional benefit seen with doses over 60mg/m2 of doxorubicin and 600mg/m2 of cyclophosphamide.3-5 However, there is still uncertainty as to which anthracycline-containing regimen is optimal. Randomized trials by the NSABP6, 7 have shown equivalence in efficacy between six cycles of the conventional CMF regimen and four cycles of AC. On the other hand, results of a U.S. Intergroup (INT 0102) trial8 and an NCIC trial9 have demonstrated statistically significant improvement in disease-free survival and overall survival with six cycles of CAF or six cycles of CEF when compared to six cycles of CMF in node-negative and node-positive breast cancer patients respectively. In both these trials the observed proportional reduction in mortality with the use of the anthracycline-containing regimen was in the range of 20-25%. The proportional reduction in mortality with anthracycline-containing versus non anthracycline-containing regimens observed in the 1995 overview was 11% but this analysis included various anthracycline-containing regimens (AC, EC FAC, FEC, etc.) given for varying number of cycles (4 to 12). Although it is uncertain whether some anthracycline-containing regimens are more active than others, the data indirectly suggest that six cycles of CAF or CEF might be more active than 4 cycles of AC. Proposed adjuvant trials by the U.S. cooperative groups will attempt to definitively address some of these questions.

Role of Taxanes as Adjuvant/Neoadjuvant Therapy

The role of taxanes in the adjuvant setting and the optimal way of integrating them with the other chemotherapy agents is still controversial and evolving. So far, two large adjuvant trials in node-positive patients (CALBG 9344 and NSABP B-28) and one large neoadjuvant trial (NSABP B-27) in patients with operable breast cancer have produced results using the sequential administration of paclitaxel or docetaxel following AC. Mature results from the first adjuvant trial show a small but statistically significant improvement in disease-free survival (13% reduction in the odds of recurrence) and a small, not statistically significant improvement in overall survival (14% reduction in the odds of death) with the addition of paclitaxel to AC.10 Most of the benefit was seen in receptor negative patients (25% reduction in the odds of recurrence and 22% reduction in the odds of death) although this subset analysis was unplanned. Preliminary results from the second trial (with 34 months of median follow up) do not show a benefit in disease-free or overall survival with the addition of paclitaxel to AC.11 However, a similar but non-significant trend to that seen in CALGB 9344 in favor of adding paclitaxel was seen patients that did not receive tamoxifen (most likely ER-negative). The NSABP B-27 trial compared neoadjuvant or adjuvant docetaxel following neoadjuvant AC to neoadjuvant AC alone. The addition of neoadjuvant docetaxel significantly increased clinical complete response rates (from 40% to 65%), pathologic complete response rates (from 13.7% to 25.6%) and the percentage of patients with histologically negative axillary nodes (51.5% vs. 59.5%)12 indicating additional antitumor activity with sequential docetaxel following AC. However, outcome results from this trial are not available yet. Two ancillary studies to the B-27 trial are exploring the potential relationship between serum/tumor biomarkers and response to preoperative AC and/or docetaxel chemotherapy and outcome.

One issue that has emerged regarding the sequential anthracycline-taxane trials is whether the observed benefit might be the result of administration of additional cycles of chemotherapy in the experimental group (4 vs 8 cycles) and not necessarily the result of administration of non-cross resistant chemotherapy. Two trials have attempted to address this issue. In the first randomized trial from the M.D. Anderson Cancer Center the role of paclitaxel was evaluated in the neoadjuvant/adjuvant setting. This trial compared four cycles of preoperative/post-operative paclitaxel to four cycles of preoperative/postoperative FAC. In both groups, four additional cycles of adjuvant FAC were given postoperatively. Thus, in terms of DFS and overall survival, this study effectively compared eight cycles of FAC with four cycles of paclitaxel followed by four cycles of FAC, testing whether the sequential administration of two clinically non-cross resistant regimens (given for four cycles each) might be more advantageous than the administration of one of the regimens given for the same total number of cycles (eight cycles of FAC). Results from the neoadjuvant part of that trial on 174 patients,13 demonstrated a similar rate of overall clinical response and similar extent of residual disease at the time of surgery between the two treatment groups. Preliminary outcome results for 524 patients were recently disclosed14 and demonstrated that the 4-year DFS was 85% for paclitaxel compared with 81% for FAC (p=0.2). Although there was a trend toward a better outcome in patients who received the noncross-resistant regimens (paclitaxel X 4 followed by FAC X 4) compared with those who received FAC X 8, the difference was not statistically significant.

This concept was also tested by a somewhat different approach in a smaller randomized trial conducted in Aberdeen, UK.15,16 In this trial, patients with large operable ((4 cm) or locally advanced (T3-4, TxN2) breast cancer were given four cycles of preoperative cyclophos-phamide, vincristine, doxorubicin, prednisolone (CVAP) and, if they responded, were randomly assigned to receive four more cycles of pre-operative CVAP or four cycles of preoperative docetaxel. Patients who did not respond were given four cycles of docetaxel. After comple-tion of chemotherapy, final tumor response was assessed and appropriate surgery, which included assessment of pathologic response, was performed. Of 167 patients who were given initial chemotherapy with CVAP, 102 (61%) had a clinical response and were judged to be suit-able for randomization. Median follow up was 38 months. Those who continued on four more cycles of CVAP had a final clinical response rate of 66%, whereas those who were given docetaxel had a significantly higher final clinical response rate of 94%. More importantly, com-plete pathologic response in the randomized patients was 18% with CVAP X 8 and significantly higher (34%) with CVAP X 4/ docetaxel X 4. This difference in pathologic response rates translated to a survival improvement. In patients randomized to receive further CVAP, the 3-year survival was 84%, while in those randomized to docetaxel, 3-year survival was 97% (p=0.02; log-rank test). In patients randomized to receive further CVAP, the 3-year disease-free interval was 77% while in those randomized to receive docetaxel, the 3-year disease-free interval was90% (p=0.03; log-rank test).

Finally, combinations of anthracyclines and taxanes have been found to be very active in phase II-III trials in patients with advanced breast cancer and several adjuvant trials have compared or are in the process of comparing doxorubicin-taxane combinations (with or without cyclophosphamide) to AC, FAC or AC followed by taxane. These trials will, hopefully, shed light on the optimal way of integrating taxanes into the adjuvant setting.

References

1. Early Breast Cancer Trialists’ Collaborative Group: Polychemotherapy for early breast cancer: an overview of the randomized trials. Lancet 1998; 352:930-42.
2. 2. 2000 NIH Consensus Development Conference on Adjuvant Breast Cancer Treatment: November 1-3, 2000, Bethesda, MD.
3. Fisher B, Anderson S, Wickerham DL, et al: Increased Intensification and Total Dose of Cyclophosphamide in a Doxorubicin-Cyclophosphamide Regimen for the Treatment of Primary Breast Cancer: Findings from National Surgical Adjuvant Breast and Bowel Project B-22. J Clin Oncol 1997; 15:1858-69.
4. Fisher B, Anderson S, DeCillis A, et al: Further Evaluation of Intensified and Increased Total Dose of Cyclophosphamide for the Treatment of Primary Breast Cancer: Findings from National Surgical Adjuvant Breast and Bowel Project B-25. J Clin Oncol 1999; 17:3374-88.
5. Henderson IC, Berry D, Demetri C, et al.: Improved disease free survival (DFS) and overall survival (OS) from the addition of sequential paclitaxel (T), but not from the escalation of doxorubicin (A) dose level in the adjuvant chemotherapy of patients (PTS) with node-positive primary breast cancer (BC). Proc Am Soc Clin Oncol 1998; 17:101a, abstract.
6. Fisher B, Anderson S, Tan-Chiu E, et al: Tamoxifen and Chemotherapy for Axillary Node Negative, Estrogen receptor-Negative Breast Cancer: Findings from the National Surgical Breast and Bowel Project B-23. J Clin Oncol 2001; 19:931-42.
7. Fisher B, Brown A, Dimitrov N, et al: Two Months of Doxorubicin-Cyclophosphamide With or Without Interval Reinduction Therapy Compared with Six Months of Cyclophosphamide, Methotrexate, and Fluorouracil in Positive-Node Breast Cancer Patients With Tamoxifen Nonresponsive Tumors: Results from the National Surgical Adjuvant Breast and Bowel Project B-15. J Clin Oncol 1990; 8:1483-96.
8. Hutchins L, Green S, Ravdin P, et al: CMF versus CAF with and without tamoxifen in high-risk node-negative breast cancer patients and a natural history follow up study in low-risk node-negative patients: First results of Intergroup Trial 0102. Proc Am Soc Clin Oncol 1998; 17:1a, abstract.
9. Levine MN, Bramwell VH, Pritchard KI, et al: Randomized Trial of Intensive Cyclophosphamide, Epirubicin, and Fluorouracil Chemotherapy Compared With Cyclophosphamide, Methotrexate, and Fluorouracil in Premenopausal Women With Node-Positive Breast Cancer. J Clin Oncol 1998; 16:2651-58.
10. Henderson IC: 2000 NIH Consensus Development Conference on Adjuvant Breast Cancer Treatment: November 1-3, 2000, Bethesda, MD.
11. Mamounas EP: 2000 NIH Consensus Development Conference on Adjuvant Breast Cancer Treatment: November 1-3, 2000, Bethesda, MD.
12. Bear H: The effect on primary tumor response of adding sequential Taxotere to Adriamycin and cyclophosphamide: preliminary results from NSABP protocol B-27. Breast Cancer Res Treat 2001; 69:210, Abstract 5.
13. Buzdar AU, Singletary SE, Theriault RL, et al: Prospective evaluation of paclitaxel versus combination chemotherapy with fluorouracil, doxorubicin, and cyclophosphamide as neoadjuvant therapy in patients with operable breast cancer. J Clin Oncol 1999; 17:3412-17.
14. Thomas E, Buzdar A, Theriault R, et al: Role of paclitaxel in adjuvant therapy of operable breast cancer: preliminary results of prospective randomized clinical trial. Proc Am Soc Clin Oncol 2000; 19:74a, abstract.
15. Hutcheon AW, Ogston KN, Heys SD, et al: Primary chemotherapy in the treatment of breast cancer: significantly enhanced clinical and pathological response with docetaxel. Proc Am Soc Clin Oncol 2000; 19:83a, abstract.
16. Hutcheon AW, Heys SD, Miller ID, et al: Improvements in survival in patients receiving primary chemotherapy with docetaxel for breast cancer: a randomised controlled trial. Breast Cancer Res Treat 2001; 69:298, Abstract 506.

New molecular diagnostics to aid in choosing therapy Debu Tripathy, M.D., UCSF Carol Franc Buck Breast Care Center, University of California, San Francisco, CA

The benefits of system therapy for breast cancer are typically defined through clinical trials with the broadest possible eligibility criteria. Therefore, the benefits such as response rate and time to disease progression for metastatic disease, or disease-free and overall survival for early stage disease represent population averages. Only in the case of hormonal therapy and HER2/neu-targeted therapy is there an estab-lished basis for tissue testing in order to choose therapy. However, retrospective analyses of large clinical trials have in some cases

suggested that specific host and tumor tissue markers might influence outcome in a treatment-specific fashion. These analyses are confounded by several facts:

* Subsets of interest are often small and hence the statistical power is limited and not definitive
* Assays and interpretation for specific protein or genetic markers have not been standardized and this makes it difficult to compare or combine studies
* Some marker may behave as prognostic markers in that they predict outome independent of therapy or are predictive in that they only predict a differential outcome in regards to a specific therapy. Many markers actually have both prognostic and predictive properties

The following markers are either established or under study for predictive or prognostic value

Predictive markers have the potential to aid in choosing optimal therapy not only to maximize the benefit but to spare toxicity to those not likely to have a therapeutic response. The obvious example is the well documented lack of benefit of hormonal therapy such as tamoxifen and aromatase inhibitors in patients whose tumors are negative for both estrogen and progesterone receptors. There is now growing evidence that a certain threshold of HER2/neu expression as defined by either immunohistochemical score or gene amplification predicts response to trastuzumab (Herceptin) such that those below the cutoff should both receive this drug and avoid the associated cardiotoxicity risk.

New molecular and protein diagnostics that predict responses to specific therapies are being pursued intensively although none are ready for routine use. In general, predictive markers must have certain properties, the extent of which will determine their clinical utilite. These include:

* Discriminatory power (odds of response or disease free/overall survival associated with presence of marker)
* Prevalence of the marker
* Reprodicibility and feasibility of assay
* Well defined cutpoint to define positivity of markers

Components of signaling pathways that are related to the drug of interest or that deal with the metabolism or cell transport of the drug are candidate markers for study. Specific markers being assessed include the following. Note that some studies are attempting to assess tumor tissue either at baseline or after therapy while others are assessing host factors, such as such as drug metablism or membrane transporters, that would affect the anti-tumor activity of the drug of interest

This list represents general classes of genes and proteins. No specific marker has yet been validated prospectively. New technology that allows high throughput analysis of a broad array of genes or proteins has facilitated the development of specific genetic prognostic and predictive markers. In animal tumor xenograft studies, discreet genes are upregulated and downregulated differentially based on responsiveness to specific chemotherapeutic agents. However, the statistical complexity of analyzing very large number of markers requires that some selectivity be applied. For example, rather than analyzing 20 to 30 thousand genes, it is more practical to focus on genes sets of functional relevance such as those outlined on the table above. Statistical tools such as hierarchical clustering can identify groups of genes that are associated with a particular phenotype such as responsiveness to specific therapy. Thus, even without knowing the function of the genes in such a “cluster”, specific profiles might be defined that would predict sensitivity or resistance to therapy, particularly therapy that targets a specific biological pathway.

Most large cooperative group trials now have an extensive effort to collect tumor tissue blocks. Retrospective analyses for predictive markers that are generated from smaller pilot studies can be validated with these resources. Ultimately, prospective trials that determine therapy on the basis of predictive markers will need to be done to determine the true value of any marker. As cancer treatments evolve to biologically targeted therapies, this will be even more critical.

Assessing Hormone Receptors: How Accurate Are Our Measurements? D. Craig Allred, M.D., Professor of Pathology, Breast Center, Baylor College of Medicine, Houston, TX

Estrogen receptors (ER) and progesterone receptors (PR) are the most important biomarkers in breast cancer. Determining their status is essential in deciding how to treat all patients with breast cancer. Until about 5 years ago, standardized biochemical ligand binding assays (LBAs) were used to assess ER and PR in nearly all laboratories. Over the past 5 years, however, the LBAs have been essentially replaced by immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tumor tissue. Is this good?

There are several economical, logistical, and technical advantages to using IHC over the LBA, which is why most laboratories changed over. Like any procedure, however, IHC must be performed properly to obtain accurate results. Unfortunately, because of the diverse and often sub-optimal methodologies being used in laboratories around the world, up to 20% of the IHC results are probably inaccurate (1-4), which is unacceptable. Most of the inaccuracies are false-negatives, meaning that each year perhaps 40,000 newly diagnosed breast cancer patients are being denied the potential benefits of hormonal therapy in this country alone.

The College American Pathologists (CAP) recently approved the use of IHC for assessing ER and PR in routine clinical practice without providing specific guidelines as to how the tests should be performed (5). However, “expert” panels of pathologists, oncologists, and surgeons have published general guidelines for assessing and judging the worth of tumor biomarkers (5-8). These guidelines all agree that markers used in routine practice should be clinically validated, technically validated, and useful. Clinical validation means that the test being used to measure the marker identifies groups of patients with significantly different risks of relapse, survival, or treatment response that ideally have been demonstrated in multiple randomized studies. Technical validation means that the test is specific, sensitive, reproducible, calibrated to patient outcome, and interpreted in a relatively uniform manner from laboratory to laboratory. Useful means that the results are actually used by physicians to make treatment decisions.

It is difficult to know exactly what methodologies are being used in the thousands of laboratories performing IHC tests for ER and PR. If the peer-reviewed medical literature is any indication, then methods and quality vary enormously, and it is quite likely that problems in the real world on a daily basis are much larger than indicated by scientific publications which, themselves, are problematic.

There are about 50 published studies assessing the relationship between ER status by IHC and patient response to hormonal therapy in one setting or another (9, 10). The design and quality of these studies vary considerably, but nearly all reported a significant correlation between a positive test (i.e. detectable ER expression in tumors) and favorable response to hormonal therapy, which is encouraging and suggests that clinical validation for ER by IHC is close if not already achieved. From a technological point of view, however, these studies used diverse and largely obsolete methodologies such as relatively insensitive antibodies on frozen tissue sections. Such methods are nearly useless in today’s laboratories where testing is virtually restricted to formalin-fixed tissue and newer more sensitive antibodies with little or no scientific track record. A handful of recent studies have been published which go a long way towards validating some of the newer reagents and methodologies (9, 11-14). If adopted widely, which has not happened, overall accuracy and reproducibility of ER testing would improve dramatically.

The problems with testing for PR by IHC are much larger than for ER. Far fewer clinical studies have been published, their results have been mixed, and their methodologies diverse and based almost exclusively on frozen tissue and older antibodies that are no longer available (9, 10). To date, no substantial studies have been published validating the performance of newer PR antibodies in fixed-archival tissue, so there are few if any methodologies for laboratories to emulate.

On a national and global scale, when measured against the scientific principles and guidelines for assessing tumor biomarkers that have been published (refs), the clinical and technical validation of IHC tests for ER and PR have not been achieved, yet they are being performed daily in thousands of laboratories and oncologists are using the information to treat patients with breast cancer. It is a near certainty that a significant proportion (up to 20%) of patients are being mistreated because of inaccurate results (usually false negatives), which will contin-ue until methodologies are adequately validated and implemented on a large scale. There are many unequal reagents and methodologies for laboratories to chose from and their choices currently appear to be influenced more by vendor advertising and cost than scientific validity.

The CAP currently has plans to implement educational programs for laboratories to help alleviate some of these problems. Until then, laboratories performing these tests should describe their methods in some detail (especially naming the antibodies being used), report results explicitly (% positive cells, etc.), and interpret results as “positive” or “negative” only if they have been calibrated to clinical outcome. Ideally they should adopt published validated methodologies that can be referenced in their reports. Oncologists using these tests in treating patients should be wary of “negative” results unless the laboratories they rely on use validated methodology and, if not, should probably have them repeated in laboratories that do.

References

1. Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 1999;17:1474-1481.
2. Rhodes A, Jasani B, Barnes D, Bobrow L, Miller K. Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems. J Clin Pathol 2000;53:125-130.
3. Rhodes A, Jasani B, Balaton A, Miller K. Immunohistochemical demonstration of oestrogen and progesterone receptors: correlation of standards achieved on in house tumours with that achieved on external quality assessment material in over 150 laboratories from 26 countries. J Clin Pathol 2000;53:292-301.
4. Rhodes A, Jasani B, Balaton A, Barnes D, Anderson E, Bobrow L, et al. Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Am J Clin Pathol 2001;115:44-58.
5. Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GC, et al. Prognostic factors in breast cancer. College of American Pathologists consensus statement 1999. Arch Pathol Lab Med 2000;124:966-978.
6. McGuire WL. Breast cancer prognostic factors: Evaluation guidelines. J Natl Cancer Inst 1991;83:1-9.
7. Hayes DF, Bast RC, Desch CE, Fritsche H, Kemeny NE, Jessup JM, et al. Tumor marker utility grading system: a framework to evaluate clinical utility of tumor markers. J Natl Cancer Inst 1996;88:1456-1466.
8. Panel AE. 1997 update of recommendations for the use of tumor markers in breast and colorectal cancer. J Clin Oncol 1998;16:793-795.
9. Allred DC, Harvey JM, Berardo MD, Clark GC. Prognostic and predictive factors in breast cancer by immunohistochemical analysis (Review). Mod Pathol 1998;11(2):155-168.
10. . Mohsin SK, Allred DC. Immunohistochemical biomarkers in breast cancer (Review). The J Histotechnol 1999;22:249-261.
11. . Alberts SR, Ingle JN, Roche PR, Cha SS, Wold LE, Farr GH, et al. Comparison of estrogen receptor determinations by a biochemical ligand-binding assay and immunohistochemical staining with monoclonal antibody ER1D5 in females with lymph node positive breast carcinoma entered on two prospective clinical trials. Cancer 1996;78:764-762.
12. Barnes DM, Harris WH, Smith P, Millis RR, Rubens RD. Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. Br J Cancer 1996;74:1445-1451.
13. Clahsen PC, van de Velde CJH, Duval C, Pallud C, Mandard AM, Delobelle-Deroide A, et al. The utility of mitotic index, oestrogen receptor and Ki-67 measurements in the creation of novel prognostic indices for node-negative breast cancer. Eur J Surgical Oncol 1999;25:356-363.
14. Elledge RM, Green S, Pugh R, Allred DC, Clark GM, Hill J, et al. Estrogen receptor (ER) and progesterone receptor (PgR) by ligand-binding assay compared with ER, PgR, and pS2 by immunohistochemistry in predicting response to tamoxifen in metastatic breast cancer: A Southwest Oncology Group Study. Int J Cancer 2000;89:111-117.

Estrogen receptor function: The laboratory and clinical data on Faslodex. C. Kent Osborne, M.D., Professor of Medicine & Cell Biology, Baylor College of Medicine, Houston, TX

Estrogen receptor (ER) is an important diagnostic and treatment target in breast cancer. ER is activated by ligand-binding and undergoes a confirmational change that permits association with coregulatory proteins. Phosphorylation of ER by ligand-binding or through the action of MAP kinases also influences receptor function. The receptor can then influence gene expression by both classical and non-classical mechanisms. There is some evidence that ER can also act on the membrane to stimulate growth factor pathways. Coactivator proteins are important for ER function and an abundance of coactivators can increase the estrogen agonist activity of drugs such as tamoxifen, which have a mixed activity spectrum. Tamoxifen-stimulated growth, due to its increasing agonist effects, is one mechanism for tamoxifen resistance in patients. This type of resistance generated great interest in pure antiestrogens that have no estrogen agonist qualities. Fulvestrant (Faslodex) is a steroidal antiestrogen with pure antagonist qualities. It binds to the estrogen receptor with high affinity and blocks gene expression more completely than tamoxifen. In preclinical models Faslodex was a more potent anti-tumor agent than tamoxifen or estrogen withdrawal, and Faslodex was shown to inhibit tamoxifen-stimulated tumors. This activity of Faslodex in tamoxifen-resistant patients was confirmed in a Phase II trial, and subsequent Phase III trials demonstrate that it is at least as effective as aromatase inhibitors in patients who are resistant to tamoxifen. Faslodex represents a new class of endocrine agents that should help clinicians and patients in the management of hormone responsive breast cancer.

Mechanisms of resistance to endocrine therapy Professor Anthony Howell, CRC Department of Medical Oncology, University of Manchester, UK

Increases in our knowledge of the molecular and cell biology of the breast and breast tumours are giving new insights into potential mechanisms of endocrine sensitivity and resistance. The normal breast is relatively resistant to the major stimulatory hormone oestradiol possibly because of the separation between the non-dividing ER positive cell and adjacent proliferating ER negative cell.1 An early event in the malignant process is the ability of the ER positive cell to divide and adapt to the prevailing serum oestradiol concentration.2 The Oxford overview and the other studies indicate that the ER (or PR) is a prerequisite for endocrine responsiveness.

A major research focus has been on the mechanism of resistance to the antioestrogens. However, with an increase in the importance of aromatase inhibitors, it is important to consider resistance to them when detected. When detected, endocrine responsive breast cancers are growing in response to the prevailing serum concentration of oestrogens particularly oestradiol (E2). Reducing (or increasing) the concentration of E2 or blocking its interaction with ER inhibits tumour cell growth. In the adjuvant situation some micrometastases appear to be inhibited lifelong by this approach whereas in advanced breast cancer objective responses or stabilisation of disease occurs for a finite period. Mechanisms of resistance are studied in the clinic and in breast tumour cell lines and animal models. Studies have reported a large number of potential mechanisms of resistance but we need to focus on the most promising. These show alteration of cell signalling pathways to the ER as a major cause of resistance. For example, by depriving MCF-7 cells of E2 in-vitro Santen and his colleagues3 have shown that the cells adapt and grow in response to very low concentrations of E2 but also the signal transduction enzyme is phosphorylated and can stimulate growth in an ER dependent and non-dependent ways. Treating these cells with physiological concentrations of E2 causes apoptosis and may be analogous to responses to high dose oestrogens in-vivo. Reversible tamoxifen resistance to tamoxifen may be related to growth factor activation of intracellular mediators (eg, AKT2 and MAPK)4,5 which can phosphorylate the ER and cause growth stimulation in the presence of tamoxifen-occupied ER. Another mechanism of resistance to both tamoxifen and fulvestrant (ICI 182,780) is seen when MAPK inhibits (by phosphorylation) the activity of p27, a protein which blocks cell cycle progression. Recent studies indicate that antioestrogens are inactive when p27 is low or inactivated.6 Antioestrogens and high dose oestrogens may actively stimulate tumour growth in patients. One potential mechanism is binding of the AE/ER complex to other transcription factors (fos and jun) which may then secondarily stimulate growth via API sites in the promotor regions of growth regulatory genes. The importance of the cell and molecular biology studies concerning mechanisms of resistance are that we can see methods for abrogating them, for example, by adding signal transduction inhibitors to endocrine therapy.

1. Clarke et al, Cancer Research 57: 4987, 1997
2. Shoker et al, Am J Pathol 155:1811, 1999
3. Song et al, JNCI 93: 1714, 2001
4. Kurokawa et al, Cancer Research 60: 5887, 2000
5. Mei et al, Cancer Research 61: 5985, 2001
6. Donovan et al, J Biol Chem 276: 40888, 2001
7. Paech et al, Science 227: 1508, 1997

The potential mechanisms of four clinical scenarios will be discussed: 1. Response to tamoxifen and a second response to an aromatase inhibitor (AI) or fluvestrant (ICI 182,780: Faslodex). 2. Response to a single endocrine therapy and no response to a second therapy. 3. Response to an AI and then oestrogen and 4. Antioestrogen stimulated growth. 1. Failure after initial response to tamoxifen is associated with increased intratumoural expression of EGFR, c-erbB2, TGF( and activated (phosphorylated) MAP kinase. Tamoxifen occupied ER can be phosphorylated serine 118 by the activated MAP kinate3 pathway and on serine 167 by activation of the p13 kinase/AKT pathway4 via cell surface growth receptors. In-vitro studies show that tamoxifen is ineffective when these pathways are activated but active when the pathways are specifically inhibited. Both fulvestrant and AIs prevent ER dimerisation receptor phosphorylation suggesting this may be the mechanism of a second response. 2. For response to antioestrogens, it is necessary for the cell cycle inhibitor p27 to bind to cyclin E1. In-vitro experiments indicate that activated MAP kinase inhibit p27 activity and produces complete endocrine resistance to both tamoxifen and to fulvestrant.5 3. AIs lower serum oestradiol. Recently it has been shown in MCF-7 cells in-vitro and in the nude mouse model that the cell response to low E2 is to increase ER concentration and activity. Importantly it was shown that relatively high concentrations of E2 caused Fas mediated apopotosis and may be the mechanism of response to high dose oestrogens.6 4. Withdrawal responses to tamoxifen and high dose oestrogens have been reported suggesting both therapies may stimulate cell growth. Several groups have shown increased fas/jun activity at API sites in tamoxifen resistant tumours and in-vitro antioestrogens stimulate growth through this mechanism in the presence of ER( and ER(.7 Elucidation of the molecular biology of endocrine resistance is exciting and highly important since it is then possible to devise ways to abrogate resistance using other inhibitors.

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