Clin Chem Lab Med 2012;50(1):5–21 2011 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.822 Circulating levels of HER-2/neu oncoprotein in breast cancer
Rafael Molina *, Jose M. Escudero, Montse Mu ñ oz,
Jose M. Aug é and Xavier Filella
HER-2/neu, also known as c-erbB-2/neu, is an oncogen Laboratory of Biochemistry (Oncobiology Unit) , School of located in chromosome 17 which encodes HER-2/neu, a trans- Medicine, Hospital Clinic, Barcelona , Spain membrane glycoprotein with tyrosine kinase activity belong-ing to the epidermal growth factor receptor (EGFR) family Abstract
(1, 2) . The HER-2/neu oncogene was originally described in a chemically induced rat neuroblastoma and shown to HER-2/neu, also known as c-erbB-2/neu, is an oncogene transform due to a point mutation in the DNA. In contrast to located in chromosome 17 which encodes HER-2/neu, a the rat HER-2/neu oncogene, the human homolog has been transmembrane protein belonging to the EGFR family. The implicated in neoplastic progression by gene amplifi cation external domain of this protein is released by the cell and and protein overexpression rather than by point mutation. can be studied in serum by immunoassay. HER-2/neu in The HER-2/neu gene is either amplifi ed or overexpressed in serum is a specifi c tumor marker and only slight eleva- 15 % – 30 % of invasive breast cancers but not in normal adult tions may be found in the absence of malignancy, mainly or fetal breast (3, 4) . in association with liver diseases. Likewise, the highest concentrations of this oncoprotein are found in patients with breast cancer, but lower concentrations may be found HER-2/neu in tissue
in other malignancies, particularly ovarian, prostate and Fluorescent in situ hybridization (FISH) is the reference lung cancer (mainly adenocarcinomas). HER-2/neu assay method to evaluate HER-2/neu status, but most studies sensitivity in patients with untreated primary loco-regional use immunohistochemistry (IHC). The advantages of IHC breast cancer is < 10 % and seems to be related to overex- include its wide availability, simplicity and relatively low pression in tissue as well as to the most important prog-nostic factors: tumor size and nodal involvement. Serial cost. Its disadvantages include subjectivity in evaluating the HER-2/neu determinations after surgery seem to be use- staining score, possible loss of HER-2/neu protein as a result ful in the early diagnosis of recurrence, mainly in patients of tissue storage and fi xation and variable results depending with HER-2/neu overexpression in tissue, but additional on both the antibody and staining procedure used. IHC crite- studies are necessary to confi rm these results. HER-2/neu ria are score 0 – no or up 10 % membrane staining, score 1 + sensitivity (proportion of patients with abnormal values) in – partial and/or faint membrane staining present in > 10 % of patients with metastasis is around 40 % – 45 % , with a clear tumor cells, score 2 + – weak to moderate complete membrane relationship to tissue overexpression and to site (higher in staining present in more than 10 % of tumor cells, and score visceral metastases) and number of metastases. The clini- 3 + – strong, complete membrane staining present in more cal utility of HER-2/neu in patients with advanced disease than 10 % of tumor cells. In contrast to IHC, FISH can theo- is mainly for therapeutic monitoring. Likewise, in most retically provide a more objective scoring system. It also has of the studies published, a relationship has been found the advantage of a built-in internal control consisting of two between serum HER-2/neu levels (either pretreatment or HER-2/neu gene copies in the non-malignant cells within the at follow-up) with tumor response. specimen. The disadvantages of FISH include its high cost, the need for a fl uorescent microscope and the inability to pre-serve the slide for storage and review. In addition, reading Keywords: breast cancer; CA 15.3; carcinoembryonic anti-
the fl uorescent signal in a suffi ciently large number of cells gen (CEA); HER-2/neu; oncoproteins; tumor markers. for reliable individual scoring is diffi cult. Several guideless have recommended IHC for determining HER-2/neu status in breast cancer (calibrated against FISH). For samples with an IHC score of 2 + , confi rmation by FISH should be carried *Corresponding author: Rafael Molina, MD, PhD, Oncobiology Unit, Laboratory of Biochemistry and Molecular Biology, Hospital The clinical applications of HER-2/neu in tissue are mainly Clinic, Villarroel 170, Barcelona 08036, Spain related to its prognostic or predictive value (6 – 8) . Most pub- E-mail: [email protected] Received July 25, 2011; accepted November 20, 2011; lished reports conclude that either HER-2/neu gene amplifi - previously published online January 4, 2012 cation or overexpression correlates with adverse outcome in Brought to you by Swets Download Date 9/19/12 5:00 PM 6 Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer patients with breast cancer, mainly in node positive patients beyond progression improves the activity of subsequent (5 – 8) . The most important clinical reason for determining chemotherapy in patients with HER-2/neu overexpression HER-2/neu levels in breast cancer is as a predictive factor. (16, 25) . However, it is accepted that only around 50 % of Most, though not all, of the studies published to date have HER-2/neu positive patients selected remain resistant to concluded that HER-2/neu overexpression is associated with Tmab-based therapy (7, 13, 20, 21, 31, 32) . It is important relative resistance to hormone therapy or CMF (cyclophos- to consider that the prediction of response to Tmab is also phamide, methotrexate and fl uoruracil) and a higher rate of important because there are secondary effects (cardiotoxic- response to anthracycline-based therapy, paclitaxel, docetaxel, ity) and because it is an expensive treatment (21) . Reasons to platinum salts and vinorelbine (6, 7, 9 – 14) . However, accord- explain this low response may be: ing to the present information and level of evidence, differ- 1. Related to the mechanism of action or as a result of ab- ent guidelines on breast cancer do not recommend analysis errant activation of signaling pathways downstream from of HER-2/neu for this purpose (5 – 7) . The American Society the receptor: loss of phosphatise and tensin homolog, acti- of Clinical Oncology (ASCO) panel does consider that vation of insulin-like growth factor homolog, mutation of HER-2/neu may identify patients who could particularly phosphoinositide-3 kinase, etc (7, 32 – 34) . benefi t from anthracycline-based therapy but that levels of Technical problems: the limitations of IHC are inter- HER-2/neu should not be used to exclude patients from laboratory variability, differences between techniques anthracycline treatment (6, 7) . (IHC-FISH), or characteristics of the sample (antigen loss) Therapeutic strategies have been developed to block . Different studies have shown a clear correlation HER2/neu signaling pathways, including monoclonal anti- between the evaluation of HER-2/neu amplifi cation and bodies and small molecule tyrosine kinase inhibitors (1, 4, 7, IHC 3 + (36, 37) . However, there are important discrep- 12) . Several monoclonal antibodies against the extracellular ancies between the two methods in patients classifi ed as domain of the HER-2/neu protein have been described that 2 + by IHC. Most of these studies have indicated that only inhibit the proliferation of human cancer cells overexpressing 30 % – 50 % of IHC 2 + tumors had amplifi cation, while oth- Her2/neu. One of these antibodies was fused to the frame- ers have reported amplifi cation in more than 90 % of the work region of human IgG 21 , called trastuzumab (Tmab), also tumors (38) . Inter-laboratory variability may also be an known as Herceptin ® and used in patients with metastatic important problem. In a multicentric study with IHC 3 + breast cancer (MBC) (1, 4, 7, 12, 13) . It has been assumed or IHC 2 + FISH + as inclusion criteria (in the local labora- that patients without HER-2/neu positive cancers will not tory), Cameron et al. (15) reported that 23 % of the 315 benefi t from Tmab (7, 15) . However, it is interesting to indi- studied patients were negative when the analysis was per- cate that only 15 % – 25 % of patients with MBC treated with formed in the central laboratory. These discrepancies are Tmab alone showed response (7, 16 – 18) . However, the most important because lapatinib was only found to be effective important contribution of Tmab and related treatments is their in these persistently positive samples but not in the 74 pa- combination with chemotherapy. Several studies have dem- tients considered as negative. onstrated that Tmab improves response rates, time to progres- 3. Another possible reason to explain the low rate of re- sion (TTP) and survival in patients with MBC when it is added sponse is that HER-2/neu status is currently routinely to chemotherapy (7, 11, 13, 19 – 21) . Moreover, Tmab is rarely studied in tissue from primary breast cancer (PBC) with used in monotherapy. Tmab had synergistic effects when used the assumption that the result is also representative of the in combination with cisplatin and carboplatin, docetaxel, and metastatic lesions. However, the primary tumor and subse- ionizing radiation and additive effects when used with doxo- quent metastases may have different levels of HER-2/neu rubicin, cyclophosphamide, methrotrexate and paclitaxel expression (39 – 41) . Several studies have suggested ge- . Likewise, different randomized trials netic heterogeneity (up to 70 % of polyclonality) in breast have established that adjuvant Tmab treatment increases the cancer (42 – 44) . survival among women with HER-2/neu positive early breast cancer (4, 7, 17, 25) . Lapatinib is a small molecule inhibitor Few studies have compared HER-2/neu status in the pri- of the intracellular tyrosine kinase domain of both EGFR and mary tumor and in the metastases and these are usually car- HER2/neu receptors (26) . Lapatinib interacts with intracel- ried out in a small number of patients due to the diffi culty in lular domains and does not require the presence of the full obtaining biopsies from metastatic sites. There are some dis- receptor for activity and may overcome some mechanisms of crepancies, but most published studies have reported a good Tmab resistance. Different studies have reported the safety correlation, higher than 80 % (37, 38, 45 – 57) . However, it is and effi cacy of lapatinib alone or in combination with other interesting to point out that the study including a signifi cantly chemotherapy agents, such as capacetabine, paclitaxel or higher number of patients showed discrepancies in 34 % of the endocrine therapy in patients with HER-2/neu overexpres- cases (52) while other studies published reported discrepan- sion in tissue (16, 27 – 29) . In contrast to Tmab, lapatinib had less cardiac toxicity and is administered orally, but the most Discrepancies between HER-2/neu status in the primary important toxicity is diarrhea, nausea and skin rash (27, 30) . and metastatic tumor may also be infl uenced by treatment, This benefi cial effect of Tmab paradoxically continues neoadjuvant or adjuvant. Results range from no modifi ca- beyond progression. Different studies have shown that con- tions to 17 % of discrepancies (22, 54, 57, 58) . Hayes et al. tinued treatment with Tmab or the inclusion of lapatinib, (59) studied 19 patients with MBC and reported a phenotype Brought to you by Swets Download Date 9/19/12 5:00 PM Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer conversion from HER-2/neu negativity to HER-2/neu posi- suggesting that the liver may have a role in the catabolism tivity during the course of therapy in a subset of patients. of this oncoprotein. No false-positive results were found in Pectasides et al. (57) investigated whether there were changes another group of patients with benign diseases. Motoo et al. in HER-2/neu status in shortly after Tmab treatment in 16 (83) also reported abnormal serum levels in patients with liver patients with Her-2/neu overexpression and found that six diseases. Similar results, indicating the high specifi city ( < 5 % (37 % ) patients had lost HER-2/neu overexpression. of false-positive results) of HER-2/neu have been reported (39, 76, 78, 80, 81, 84 – 88) . Overexpression of HER-2/neu in tissue is found in tumors HER-2/neu in serum
other than breast, such as lung, ovary or prostate cancer (89, 90) . Molina et al. (70) studied 489 patients with active The HER-2/neu oncoprotein is composed of three domains: advanced cancer of different origin and reported abnormal the internal tyrosine portion responsible for intracellular sig- levels in 19.6 % of the patients including patients with breast, naling, the transmembrane portion and the external portions, ovarian, colorectal, primary liver or bladder cancer. However, namely the extracellular domain (ECD) (60, 61) . The ECD is these elevated serum HER-2/neu levels in patients with non- a glycoprotein with a molecular weight of 97 – 115 kDa. It is breast malignancies were associated mainly with liver metas- shed by proteolytic cleavage by tissue ADAM metalloprotei- tases. Excluding patients with liver metastases, abnormal nases into culture supernatants of breast cancer cell cultures serum HER-2/neu levels were found in only 17.4 % of malig- and the remaining membrane-bound internal domain is acti- nancies (excluding breast), always being lower than 30 ng/mL: vated constitutively (35, 62 – 65) . ECD is a membrane bound, prostate, ovarian and lung cancer (mainly adenocarcinomas). N-terminally truncated HER-2/neu fragment with in vitro Similar results were published by Leitzel et al. tyrosinase activity (35, 66) . ECD production is increased by elevated serum levels ( > 5 U/mL) in 3.2 % of 31 ovarian can- tissue metalloproteinases activators and decreased by their cers and in only two of 124 (1.6 % ) patients with other malig- inhibitors (35, 67) . nancies. Other publications have reported similar results Several ELISAs to detect the HER-2/neu ECD in sera and some of these authors associated abnormal HER-2/neu (serum HER-2/neu) of breast cancer patients have been serum levels with poor prognosis in patients with pancreatic, described (41, 68 – 74) . All these assays are useful for evaluat- colorectal, prostatic carcinomas or non-small cell lung can- ing serum HER-2/neu as a tumor marker, although the lack of cer (74, 86, 92 – 100) . However, most of these studies clearly standardization among immunoassays makes comparison of indicated that only scarcely high abnormal serum HER-2/neu results diffi cult. However, two assays (Oncogene Science and serum levels may be found in patients with malignancy other Siemens automated Centaur) are the most used with a high than breast cancer, being habitually lower than 25 ng/mL correlation between them having been reported (75 – 78) . (99, 101 – 106) . In summary, HER-2/neu in serum is a specifi c tumor marker and only slight increases may be found in the absence Serum HER-2/neu in healthy subjects, benign
of malignancy, mainly in association with liver diseases. diseases and malignancies other than those
Likewise, only scarcely high serum levels may be found in of the breast
malignancies (ovarian, NSCLC, prostate, colorectal) other than breast cancer (85, 92 – 107) . Serum HER-2/neu levels in the normal population as well as the cut-off value used depend on the technique (41, 69, 76, 77) . Schwartz et al. (75) studied 120 healthy women and found ele- Serum HER-2/neu in breast cancer
vated values ( > 15 ng/mL) in 1.6 % of premenopausal (mean: 8.69 ± 1.72) and in 5 % of postmenopausal women (mean: Serum HER-2/neu in primary untreated locoregional
8.45 ± 4.05). These results were confi rmed by other authors breast cancer
and indicate that serum HER-2/neu serum levels using the Siemens assay or the Oncogene assay range between 1 ng/mL Carney et al. (40) reviewed 25 studies on a total of 2623 and 15 ng/mL and confi rm that 15 ng/mL is the reference cut- patients with PBC and found that approximately 18.1 % of the point (39, 71, 79 – 82) . Fehm et al. (72) evaluated HER-2/neu patients had elevated HER-2/neu serum levels (41, 73, 81, with a home-made assay in 62 healthy people and reported a 82, 108 – 111) . Lower sensitivity results (9.5 % ) have recently mean of 58.2 ± 12 fmol/mL, and a cut-off of 120 U/mL. In the been obtained by our group in the largest study published to same article they reported an equivalence between their assay date (112) . The wide range of results reported in the Carney and the Triton assay (1 U triton assay is equal to 5 units of review is surprising: from 3.1 % in stage I – II reported by their method) and suggested a cut-off of 25 U/mL. Narita et al. (84) to 40 % found by Mansour et al. (80) or HER-2/neu is a specifi c tumor marker, and only scarcely Kandl et al. (113) . These differences may be related to the high levels have been found in non-malignant diseases. techniques used or the cut-offs established. For example, Molina et al. (70) studied HER-2/neu serum levels in 112 Fontana et al. (108) reported a high sensitivity of 21 % in 125 patients with benign diseases of different origin. Elevated patients with PBC, although they used a cut-off of 8 ng/mL serum HER-2/neu levels were found in 38.5 in contrast to that usually used with this technique 15 U/mL. patients with liver cirrhosis (always lower than 30 ng/mL), With the same technique but using 20 ng/mL as the cut-off, Brought to you by Swets Download Date 9/19/12 5:00 PM 8 Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer Narita et al. (84) reported 3.1 % in stage I – II and 29.4 % in found that HER-2/neu serum levels were the most important stage III – IV. Imoto et al. (110) reported a sensitivity of 38 % prognostic factor in disease free survival (DFS) (p < 0.0007), in 73 patients with PBC using 5.4 ng/mL as the cut-off with followed by tumor size. Similar results have been published another method. Another important explanation for the dis- by Schippinger et al. (124) in 313 patients treated with adju- crepancies observed between the different studies may be the vant chemotherapy. Ludovini et al. (78) studied 256 patients number of patients studied and their characteristics. Fehm et stage I – III and found that HER-2/neu was related to DFS in al. (72) found 31 % of sensitivity evaluating 52 patients and = 0.0001) and multivariate analysis (p = 0.009) 12.3 % in another study including 211 patients with PBC (71) . together with stage, nodes and PgR but not in overall sur- The studies with the highest sensitivity were performed in vival (OS). Recurrence was observed in 39.1 small subset of patients and/or including patients stage IV patients with abnormal HER-2/neu serum levels in contrast to (72, 81, 114) . In summary, most studies including more than 7.3 % (17/233) in patients with normal levels. In 211 patients 100 patients have reported a sensitivity of between 6 % and with advanced tumors Fehm et al. (72) found a relationship 12.3 % (71, 75, 78, 111 – 113, 115, 116) . between HER-2/neu and DFS but not OS in univariate and Tumor marker sensitivity is habitually related to tumor multivariate analysis (p = 0.01). Molina et al. (88) reported stage, with higher serum concentrations in larger tumors that patients with elevated presurgical HER-2/neu serum lev- or in patients with nodal involvement. Schwartz et al. (75) els had a 3.3-fold higher risk of recurrence than patients with reported a relationship with tumor stage, with abnormal val- normal levels. However, in another study (112) including a ues in 1.7 % of cases with stage I, 3 % in stage II and 16.7 % in higher number of patients and longer follow-up they found stage III. Ludovini et al. (78) studied 256 stage I – III patients that HER-2/neu serum levels was an independent prognostic and reported a relationship with histological grade (p = 0.003), factor only in patients with neoadjuvant treatment or in those stage (p = 0.008), nodal involvement (p=0.035) and negativity with HER-2/neu overexpression in tissue. Mehta et al. (125) of both steroid receptors [estrogen receptor; (ER) p = 0.016; found that abnormal HER/neu serum levels were associated progesterone receptor (PgR) p = 0.007]. HER-2/neu serum with poor outcome in patients with more than three nodes, levels are related to stage, tumor size or nodal involvement in treated with adjuvant chemotherapy but not in other groups. most (71, 75, 79, 80, 84, 87, 110 – 13) but not all of the stud- In summary, HER-2/neu sensitivity in patients with ies published (82, 116 – 118) . Correlation with steroid recep- untreated PBC is lower than 12 % and seems to be related tors is less frequently studied but most groups have reported to overexpression in tissue as well as to the most important the highest values in steroid receptor negative patients (39, prognostic factors: tumor size, nodal involvement, ER, PgR 78, 109, 120) . In a study evaluating 885 patients with PBC and histological grade. Further studies are required to con- found similar results, correlation with fi rm the prognostic value of HER-2/neu in serum, in the total tumor size (p = 0.014), nodal involvement (p = 0.028), histo- group as well as in patients with HER-2/neu overexpression logical grade (p = 0.022) and steroid receptor status (p = 0.001) but only in those patients with IHC positivity in tissue. These results were similar to previous studies by our group (88, 119) HER-2/neu in early detection of recurrence
and those reposted by Isola et al. (86) , with a clear relation-ship between tumor size and nodal involvement in patients About 40 % – 50 % of patients with breast cancer develop dis- with overexpression in tissue. tant metastases within 5 years after radical primary treatment. HER-2/neu sensitivity has been compared with the most Experimental studies have shown greater response to treat- frequently used tumor markers in breast cancer: carcinoem- ment with smaller tumor masses. According to this hypothe- bryonic antigen (CEA) and/or CA 15.3. Most of studies have sis, early detection of MBC seems to be an essential requisite reported a lower sensitivity with this oncoprotein (5, 68, 72, for successful therapy in patients with breast cancer (5, 126, 87, 88, 108, 118, 119, 121) . However, there are some discrep- 127) . In an interesting review including 1773 breast cancer ancies, with most showing a very high HER-2/neu sensitiv- patients, Carney et al. (40) showed that monitoring of HER-2/ ity, mainly due to the use different cut-off for HER-2/neu as neu levels after surgery in HER-2/neu tissue-positive patients previously indicated in this article (110, 122, 123) . In the last may be benefi cial in the early detection of breast cancer recur- study including 883 patients with PBC our group (112) found rence (73,86,122,127 – 130) . However, most of these publica- the sensitivity to be 19.7 % , 15.9 % and 9.5 % for CA 15.3, tions indicated the sensitivity of this oncoprotein at diagnosis CEA and HER-2/neu, respectively. However, all the stud- of recurrence (86, 128) or in some selected groups of patients ies published have suggested that the combination of tumor but not in a true follow-up evaluating sensitivity and speci- markers increases the sensitivity. Molina et al. (112) reported fi city in a large population of patients with breast cancer abnormal levels of one of these tumor markers or another in 34.5 % of the total group of patients with PBC, being 43.8 % in Sugano et al. (117) studied HER-2/neu, CEA and CA 15.3 node positive patients, 57.8 % in those with T3-4 tumors and one month before diagnosis of recurrence in 36 patients with 70.7 % in those with larger tumors and nodal involvement. relapse and found increased levels in 58.3 Fewer studies have evaluated HER-2/neu serum levels as a 36.1 % , respectively. Fehm et al. (72) retrospectively analyzed prognostic factor in patients with PBC, with most but not all serial serum samples of 52 patients with PBC who had devel- suggesting (109) prognostic value in the total group or in some oped metastases during follow-up. Tumor marker sensitivity subset of patients. Mansour et al. (80) studied 75 patients and 6 months before the diagnosis of metastases was 27 % (13/49) Brought to you by Swets Download Date 9/19/12 5:00 PM Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer and 16 % (8/49) for HER-2/neu and CA 15.3, respectively approach offers the use of a very good tumor marker in a and 50 % (22/44) and 32 % (14/44) at 3 months before the selected group of patients, those with HER-2/neu tissue posi- diagnosis of metastases. HER-2/neu sensitivity at diagnosis tivity in the primary tumor. The use of tumor markers may was 62 % , rising to 83 % with the simultaneous use of the two be an intermediate way to obtain an early diagnosis, with low cost, doing imaging techniques (excluding mammography) Isola et al. (86) retrospectively studied HER-2/neu serum only in those patients with serial increasing tumor markers levels in a group of 27 patients with recurrence during fol- low-up and found elevated serum levels in 10 of the patients In summary, few retrospective studies have been performed (37 % ) 6 months before diagnosis of relapse. False-positive to demonstrate the utility of serum HER-2/neu in the early results were found in 8 % (14/166) of the patients without diagnosis of recurrence. Although these results as well as the relapse. However, some of these patients with false-pos- sensitivity of this oncoprotein in advanced breast cancer have itive results had only a peak, with decreasing levels in the suggested its utility in the diagnosis of recurrence, mainly in follow-up. Molina et al. (121, 130) retrospectively studied patients with tissue overexpression. Additional studies are CEA, CA 15.3 and HER-2/neu in 200 patients after surgery necessary to confi rm these results. and relapse was diagnosed during follow-up in 89 patients (loco-regional: 17; metastases: 72). Criteria to suspect recur- Serum HER-2/neu in MBC
rence were two serial increases (every result > 25 % of the pre-vious one) of tumor markers and with at least one result > 10 In a review of 50 studies in a total of 5044 patients with MBC, ng/mL, 60 U/mL or 20 ng/mL for CEA, CA 15.3 or HER-2/ Carney et al. (40) reported an mean sensitivity (percentage of neu, respectively. None of the 111 patients without recurrence patients with abnormal levels) of 45.6 % (range: 23 % – 80 % ) had abnormal levels according to the criteria used (specifi city for HER-2/neu in serum (14, 68 – 70, 73, 75, 77, 118, 123, 100 % ). By contrast, HER-2/neu was the fi rst sign of recur- 131 – 144) . In another review including 3254 patients from 23 rence (clinical or radiological) in 28.4 % of the patients (lead studies Leary et al. (145) reported an overall prevalence of time: 4.5 ± 2.4 months) with recurrence (Figure 1 ). One tumor 36.5 % , ranging from 23 % to 62 % . The wide range of results marker or another was the fi rst sign of metastases in 76.3 % of published is again surprising. The possible explanations for patients with distant recurrence, 11.2 % more than using only these differences are similar to those described in PBC. In CEA and CA 15.3 (121, 130) . HER-2/neu utility in the early this review we reported 13 studies including more than 100 diagnosis of recurrence was related to site of recurrence with patients, and in nine with a total number of 2420 patients the highest sensitivity and median concentrations of all tumor using 15 ng/mL as the cut-off sensitivities of between 30 % markers in patients with liver metastases, and the lowest in and 42 % were reported (71, 76, 78, 102, 124, 140, 146, 147) . patients with loco-regional recurrence. By contrast, four studies including a total of 1030 patients The advantage of HER-2/neu over other tumor markers in using 20 or 30 ng/mL as the cut-off described sensitivi- breast cancer is the relationship between tissue overexpres- ties ranged from 19 % to 30.9 % (69, 86, 148, 149) . Another sion and serum levels of this oncoprotein. HER-2/neu serum two studies reported sensitivities higher than 60 % but they levels were the fi rst sign of recurrence in 80 % of patients included selected patients, those with tissue overexpression with overexpression in tissue (primary tumor) in contrast to or only patients with PgR + (123) . Molina et al. (121, 146) only 9 % in those without overexpression (121, 130) . This reported a sensitivity of 39.7 % using 15 ng/mL as the cut-off and 18.9 % using 30 ng/mL. It is also interesting to indicate that HER-2/neu serum levels in patients with MBC are sig-nifi cantly higher than in other malignancies, and the range of Serum HER-2/neu concentrations results described may be of up to 6.000 ng/mL. Differences in sensitivity of HER-2/neu may also be related to patient characteristics, mainly the site of recur- rence. Molina et al. (146) found that sensitivity as well as median concentrations of HER-2/neu were clearly related to the site of recurrence, the lowest sensitivity being observed in patients with loco-regional recurrence (15 % , mean 10.4 ng/mL), intermediate sensitivity in patients with bone metas-tases (26 % , mean 28 ng/mL) and the highest sensitivity in patients with liver metastases (48 , mean 180 ng/mL). Likewise, the highest HER-2/neu concentrations were found in patients with more than one site involved and liver metas- tases (68 % , mean 471 ng/mL). Ali et al. (123) also reported abnormal HER-2/neu serum levels in 24 % of patients with only one metastatic site vs. 37.2 % in those with more than Progressive increase in HER-2/neu serum levels in a one site. Similar results have been reported by other authors patient with loco-regional breast cancer treated with radical surgery and without evidence of disease before clinical or radiological detec- indicating the lowest sensitivity in loco-regional recurrences tion of relapse. CT, computed tomography. and the highest in patients with metastases, mainly of the liver Brought to you by Swets Download Date 9/19/12 5:00 PM 10 Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer (38, 84, 86, 115, 123, 140, 144, 145, 147, 150 – 153) . Minor Correlation between tissue and serum HER-2/neu
discrepancies have been habitually reported in studies with a low number of patients (65, 111, 112, 114) . There is also Tumor markers are synthesized by malignant cells and thus, agreement in the relationship of HER-2/neu serum levels and a correlation may be present between tumor marker expres- steroid receptors, with signifi cantly higher levels in ER – or sion in tissue and in serum. HER-2/neu serum levels may be PgR – tumors (71, 84, 88, 114, 139, 146, 149) . related to the release by breast tumor cells, but as has been HER-2/neu has been compared with CA 15.3 and CEA previously indicated and similarly to other tumor mark- in patients with advanced breast cancer. Most of the stud- ers, other factors are related to the presence of abnormal ies published have clearly indicated that the sensitivity of HER-2/neu serum levels, as for example tumor extension. HER-2/neu is lower than that obtained with other tumor mark- HER-2/neu sensitivity in early PBC is low, being 8 % – 12 % ers (73, 117, 118, 126, 138) . Ali et al. (123) studied CA 15.3 less than 50 % than in tissue, having a tissue/serum correla- and HER-2/neu in 566 MBC patients and found abnormal CA tion which is diffi cult to fi nd. Different publications have 15.3 levels in 60 % of the patients, in contrast to 30 % found evaluated this correlation with contradictory results, mainly with HER-2/neu. This study also reported that abnormal CA due to the previously indicate reasons: different techniques 15.3 are more frequently found in patients with abnormal and cut-offs (serum or in tissue) or for the selection of stud- HER-2/neu (80 % , 135/168 patients) than in those with HER-2/ ied patients. Several studies have reported a correlation (78, neu in the normal range (51 % , 202/398 patients). Similar 80, 87, 88, 108, 109, 112, 117, 141, 142, 155) while others results were reported by Colomer et al. (74) with abnormal deny it (82, 143, 152, 156, 157) . Ludovini et al. (78) studied HER-2/neu serum levels in 17/28 patients with CA 15.3 posi- 256 stage I - III patients and found a tissue/serum relationship tive in contrast to only in 5/27 patients with CA 15.3 negative of 87.1 % . Abnormal HER-2/neu serum levels were found in (p = 0.0032). However, most authors accept that both tumor 38.1 % (16/42) IHC + subjects (3 + or 2 + FISH + ) in contrast to markers are complementary, with a better sensitivity achieved only 3.3 % (7/214) in IHC- patients (p < 0.0001). By contrast, using CA 15.3 and HER-2/neu simultaneously (78 % ) or CEA Kong et al. (82) studied 86 patients with loco-regional PBC (126) . Robertson et al. (137) studied 67 patients and reported and did not fi nd a correlation with abnormal serum levels in elevated HER-2/neu or CA 15.3 serum levels at diagnosis of 6.2 % and 4.5 % in those with or without tissue overexpression metastases in 84 % of the patients, but during therapy elevated (IHC 3 + or 2 + FISH + ). It is interesting to note that seven of markers rose to 96 % . the eight studies including more than 100 patients reported a Fehm et al. (120) studied the prognostic signifi cance of tissue/serum correlation with signifi cantly higher HER-2/neu serum HER-2/neu and CA 15.3 at the fi rst diagnosis of MBC serum levels in patients with tissue overexpression (78, 88, and reported a signifi cantly shorter median survival of patients 112, 117, 124, 134, 143) . The only study without a correla- with elevated HER-2/neu serum levels (8 months) than of tion was performed using other technique, with the cut-off those with normal levels at diagnosis (18 months) (p < 0.01). being diffi cult to evaluate comparatively (156) . Other stud- HER-2/neu was the most important independent prognostic ies have evaluated only patients with tissue overexpression factor for survival even when adjusted for tumor load. Ali and found a very high HER-2/neu serum sensitivity, ranging et al. (123) found a shorter survival in patients with elevated from 24 % to 69 % , being signifi cantly higher than that previ- HER-2/neu values (513 vs. 869 days, p < 0.0001) or CA 15.3 ously described in unselected patients (8 % – 12 % ), indirectly (689 vs. 939 days, p < 0.0001) on univariate and multivariate suggesting a higher sensitivity in patients with tissue overex- analysis. Bramwell et al. studied HER-2/neu serum pression (21, 111, 113, 144, 158) . In summary, these results levels at baseline and during follow-up in 107 patients with suggest that a tissue/serum correlation with abnormally high newly diagnosed metastases and found that baseline values HER-2/neu serum levels mainly in patients with tissue HER-2/ was an independent prognostic factor together with the site neu overexpression. Moreover, only a proportion of patients of recurrence. Kandl et al. (113) studied 79 MBC patients and with loco-regional PBC (30 % – 60 % ) with overexpression in found that HER-2/neu serum levels at the time to diagnosis of tissue has abnormal HER-2/neu serum levels. These differ- relapse was related to survival, with a median survival of 21 ences are mainly due to the fact that HER-2/neu serum levels months in patients with abnormal serum levels in contrast to are related to tumor extension and sensitivity in early stages a median of 64 months in patients with normal values. Other is low. In our experience evaluating 883 patients with PBC, authors have suggested similar results (72, 86, 115, 120, 138, only 20 % of patients with tissue overexpression had abnor- 140, 147, 153, 154) , although in some articles it is diffi cult mal serum levels. This proportion increased in patients with to distinguish the prognostic from the predictive value (123, greater tumor size (25.5 % ) or nodal involvement (87, 121) . 131, 139, 148) . HER-2/neu sensitivity in serum of patients with advanced In summary HER-2/neu sensitivity in patients with MBC disease is signifi cantly higher than in PBC, and it is easier is signifi cantly higher than in patients with PBC, 35 % – 50 % . to fi nd a tissue/serum correlation (40, 113, 120 – 122, 146, This sensitivity is related to the site of metastases, being sig- 155, 159, 160) . Most studies have found signifi cantly higher nifi cantly higher in patients with visceral metastases, mainly HER-2/serum levels in patients with tissue overexpression liver metastases, and the lowest in patients with loco-regional (40, 41, 112, 113, 117, 142, 143, 146, 149, 161, 164) . Only relapse. HER-2/neu also seems to be related to steroid recep- one study (113) including only 24 patients, using 10 ng/mL as tor status and to be a prognostic factor, with a shorter OS in the cut-off reported no correlation with abnormal HER-2/neu patients with abnormal serum levels. serum values in 60 % of patients with tissue overexpression Brought to you by Swets Download Date 9/19/12 5:00 PM Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer 11 and 42.8 % in those without abnormal values. Discrepancies In summary, there is a relationship between HER-2/neu among the different studies suggesting a correlation (includ- in tissue and in serum, with signifi cantly higher serum lev- ing 1333 patients) are mainly in relation to the sensitivity els in patients with tissue overexpression. Approximately obtained in patients with tissue overexpression (ranging from 70 % – 80 % of patients with tissue overexpression and 25 % 43.4 % to 100 % ) and specifi city or abnormal serum levels of those without presented abnormal serum levels, using 15 in patients without overexpression (ranging from 45.7 % to ng/mL as the cut-off. These results are around 50 % – 60 % in 95 % ). Discrepancies in sensitivity are not as high as the wide tissue positive and 10 % in tissue negative subjects when 25 range of results previously indicated suggest because the two ng/mL is used as the cut-point. Discrepancies between tissue studies reporting the lower sensitivities used high cut-points, and serum may be related to the previously indicated tech- 20 ng/mL or 37 ng/mL (82, 149) . Likewise, another four stud- nical problems (IHC variability) or differences between the ies including more than 50 selected patients with metastases tumor characteristics in the PBC and MBC. Another impor- and tissue overexpression (total number of patients 787) also tant reason to explain differences may be related to the rela- reported HER-2/neu sensitivities from 69 % to 73.3 % (74, tionship to HER-2/neu serum levels and tumor stage or site of 144, 158, 163, 164) . metastases that is not present in tissue evaluation. IHC per- An important point to consider is the substantial proportion formed in the primary tumor may be compared as a picture of of women without HER-2/neu overexpression that release the tumor while HER-2/neu serum levels may be compared signifi cant amounts of HER-2/neu. The proportion of patients with a movie, as shown in Figure 2 . If we use 30 ng/mL as in this situation ranges from 5 % to 55 % according to the dif- the cut-off to suggest treatment, patients will be considered ferent authors. This wide range of results may be partially due as without overexpression, while in the follow-up this patient to the cut-off used in tissue as well as in serum. Authors with a may show very high HER-2/neu serum levels suggesting high proportion of patients without tissue overexpression and overexpression. Most tumor marker guidelines suggest that abnormal HER-2/neu serum levels use 15 ng/mL (113, 161) the correct use of tumor markers implies not to use cut-points while most studies with a higher specifi city use 20 ng/mL or but rather follow-up to correctly interpret the results. Similar more as the cut-point (41, 74, 82, 84, 117, 142, 149) . In a study suggestions may be made with HER-2/neu in the selection of including 113 MBC patients Molina et al. (87) found elevated treatment as well as a tumor marker. serum levels ( > 15 ng/mL) in 25 % of IHC – patients in contrast to 74.4 % in IHC + patients. It is interesting to point out that serum HER-2/neu levels of > 25 ng/mL were observed in only HER-2/neu in therapy monitoring
1.5 % of patients without tissue overexpression compared to 60 % in patients with tissue overexpression. It is generally well accepted that serial determination of tumor Another possible explanation is the fact that patients with markers is useful to evaluate the effi cacy of therapy (Figure IHC 2 + and FISH negative have scarce overexpression but 2 ) (5) . Robertson et al. (137) prospectively studied 67 patients without amplifi cation. These patients with a small proportion with metastases at diagnosis and reported that changes in the of HER-2/neu positivity cells or with overexpression with- markers were in line with an often predated therapeutic out- out amplifi cation may be considered as negative by IHC but come as assessed by the UICC criteria for both remission and may explain small increases in HER-2/neu in serum, mainly progression. Pegram et al. (14) evaluated serum HER-2/neu in patients with multiple metastases. Esteva et al. (164) com- changes during treatment with cisplatin and a murine Mab pared serum HER-2/neu levels in 103 patients with metas- 4D5, a Herceptin ® precursor, and found that serial measure- tases and did not fi nd any differences between those (31 ments of this oncoprotein in serum were useful in disease patients) with an IHC score of 2, (mean of 182.8 ng/mL) and monitoring, especially in determining treatment failure. 72 patients with a score of 3 + (mean of 201.8 ng/mL). Similar Esteva et al. (164) retrospectively studied the concentrations results were reported by Köstler et al. (144) in 55 patients with tissue overexpression (2 + and 3 + ). These results suggest Serum HER-2/neu concentrations that scarcely high abnormal HER-2/neu serum levels may be found in patients without tissue overexpression, but very high levels are only found in patients with tissue overexpression. Finally, it has been reported that liver function may be impor- tant in the HER-2/neu catabolism, and patients with liver metastases and scarcely high HER-2/neu serum levels ( < 30 ng/mL) may refl ect catabolic problems more than tissue over-expression. Kong et al. (143) studied 195 patients and using ROC curves found that a cut-off of 37 ng/mL allows HER-2/ neu serum specifi city of 95 % and 62 % of sensitivity in rela- tion to tissue status. This cut-off may be different in patients with liver metastases (133 ng/mL) than in those without (32 ng/mL). Using this cut-off the sensitivity was 51 % in patients with liver metastases and 55 % in those without, with Figure 2 Serial HER-2/neu serum determinations in a patient with
a specifi city related to tissue overexpression of 95 % . breast cancer and bone metastases during systemic treatment. Brought to you by Swets Download Date 9/19/12 5:00 PM 12 Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer of CA 15.3 and serum HER-2/neu and found that both tumor (142) . Multivariate analysis indicated that HER-2/neu serum markers were useful in the follow-up of 103 patients with levels and performance status were independent prognostic overexpression in the primary tumor treated with Herceptin ® - factors. Similar studies have been performed by other authors based therapy. The correlation between serum levels and clin- (lower response rate, shorter TTP and OS in patients with ical status was 0.793 for HER-2/neu and 0.627 for CA 15.3, abnormal HER-2/neu serum levels), using fi rst or second line rising to 0.829 with the use of both. Other authors have also hormonotherapy (141, 150, 169) . reported the utility of HER-2/neu in therapy monitoring (69, However, other studies have shown different results. 72, 77, 114, 132, 138, 142, 153, 166 – 168) . studied 67 stages III or IV patients treated with fi rst-line tamoxifen and did not fi nd any relation- HER-2/neu as therapeutic predictive factor
ship between serum levels and response rates when patients were subdivided according to ER status, but they did observe It has been suggested that HER-2/neu in tissue may be a pre- an association with poor OS. Hayes et al. (139) studied 242 dictive factor in both hormonal therapy and chemotherapy. MBC and did not fi nd any relationship between pretreatment This option has been scarcely studied in PBC with HER-2/ HER-2/neu levels and the rate of response to either endo- neu serum levels. Schippinger et al. (124) studied 108 patients crine therapy (megestrol acetate) or chemotherapy. In con- with locally advanced PBC treated with neoadjuvant antracy- trast, univariate and multivariate analysis showed a worse cline-based chemotherapy and found higher tumor response OS in patients treated with megestrol acetate but not with in those patients with higher baseline HER-2/neu serum lev- chemotherapy (mainly anthracycline-containing regimens) els (median in CR 10.95 ng/mL, moderate responders 9.35 with raised HER-2/serum levels. In summary, most studies, ng/mL and no responders 8.35 ng/mL), but not with the mainly those with a higher number of patients (more than HER-2/neu status in tissue (IHC/FISH). By contrast Mazouni 1900 patients), suggest that elevated HER-2/neu serum levels et al. (111) sequentially studied HER-2/neu serum levels in 39 predict poor response to hormonotherapy as well as a shorter patients with loco-regional PBC treated by chemotherapy and TTP and poor survival (69, 123, 140, 141, 148, 150, 154, 169, Tmab (29 patients) and did not fi nd any relationship between 170) , meanwhile other studies indicate poor prognosis but no the baseline concentrations and clinical response. However, relationship with the tumor response (139, 162) and fi nally they did suggest the use of a decrease in the baseline levels of other studies did not fi nd any prognostic value (68) . 9 % in weeks 3 and 6 to predict complete response (p = 0.04). As previously indicated, HER-2/neu overexpression in tis- Similar results were reported by Köstler et al. (144, 159) sue seems to be a predictive factor for chemotherapy with in two different studies, with no relationship being found higher or lower response according to the drugs used. HER-2/ between basal levels and response but with utility in the serial neu serum levels as a predictive factor may also be related. A HER-2/neu serum determination . It is interesting to fi rst look at the results published is surprising, with articles indicate that patients with abnormal levels and decreasing suggesting higher response, TTP or OS in patients with abnor- serum concentrations at day 15 had a longer TTP than those mal HER-2/neu serum levels (69, 163, 170, 174) , while others with no decreasing levels (p < 0.001). Objective response was report a higher response, TTP or OS in patients with normal found in 67 % (10/15) of patients with abnormal levels and a levels (120, 131, 168, 171 – 175) and fi nally, other publica- decrease of 66.1 % or more in contrast to 13 % (2/15) found in tions have found no relationship with tumor response (113, patients with a lesser decrease. 131, 135, 139, 171, 175) . The reasons for these discrepancies Ali et al. (123) studied 566 MBC patients who were ER + may be related to the fact that most of these studies used basal and/or PgR + (496 patients) or of unknown receptor status HER-2/neu serum levels to evaluate tumor response, which, (69 patients) and found increased HER-2/neu levels in 30 % as previously indicated, may be related to several patient char- and CA 15.3 in 60 % . Patients were randomized to receive acteristics, such as the site of metastases, HER-2/neu overex- megestrol acetate or aromatase inhibitors. Patients with pression, ER status, etc., the use of different techniques and abnormal HER-2/neu had lower response (18.8 % vs. 36.4 % ), a cut-off from 10 ng/mL (113) to 27 ng/mL (74, 135, 160) . shorter TTP (89 days vs. 176 days) and a shorter survival HER-2/neu sensitivity is clearly related to the site of recur- (513 days vs. 869 days; p = 0.0001) on univariate and mul- rence. The use of a low cut-off will imply the consideration of tivariate analysis. Lipton et al. studied 562 patients patients with liver or visceral metastases without overexpres- with advanced breast cancer and ER + tumors who were ran- sion as positive, because low HER-2/neu serum levels may domized to receive letrozole or tamoxifen. They reported be found ( < 30 ng/mL) in patients with liver diseases, with that patients with elevated HER-2/neu serum levels treated or without tissue overexpression. Harris et al. (163) studied with letrozole had a higher tumor response (39 % vs. 26 % ; the relationship between HER-2/neu serum levels and tumor p = 0.008) and a longer TTP (12.2 months vs. 8.5 months; response in 191 women who received either doxorubicin or 0.019) than those treated with tamoxifen, but only in CMF-based therapy and found that patients with elevated patients with normal HER-2/neu serum levels. Similar results HER-2/neu serum levels had a higher probability of response were obtained in another study by this group including 226 only in those receiving a regimen containing doxorubicin patients with recurrence and steroid receptor positivity, with a (64.1 % vs. 30.8 % ), with a trend toward a longer TTP. This lower objective response to letrozole (14 % vs. 30 % ), shorter study refl ects that the chemotherapy used is important to eval- TTP (4 months vs. 14 months) and a shorter OS (p < 0.0005) in uate the possible HER-2/neu predictive value. Muller et al. patients with HER-2/neu serum levels higher than 20 ng/mL (174) studied 105 patients with MBC treated with epirubicin Brought to you by Swets Download Date 9/19/12 5:00 PM Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer and cyclophosphamide or paclitaxel, and found no relation- Serum HER-2/neu concentrations ship between HER-2/neu basal serum levels and response (positive 42.4 % vs. 43.9 % in negative) in the total group, but shorter OS (p = 0.0092) in those patients with abnormal levels. However, a higher response and OS (p = 0.0092) were found in patients treated with cyclophosmide and HER-2/neu serum negative (response 49.5 % ) than in those with abnormal serum levels (29.4 % ). Sandri et al. (168) found no relation-ship between basal pretreatment HER-2/neu serum levels and tumor response in 39 patients with MBC treated with low doses of cyclophosphamide and methotrexate. By contrast, when the evolution of HER-2/neu in serum (basal/2 months) was compared, higher tumor response, TTP (p and OS (p = 0.0091) were found in patients with decreasing HER-2/neu serum levels than in those with increasing levels. In summary, it is diffi cult to obtain conclusions of these three studies, with different conclusions, but that the use different Figure 3 Tumor marker utility in the evaluation of tumor response
cyclophosphamide based chemotherapy as well as different and evolution in a patient treated with trastumab-based therapy. criteria (evolution/basal) may explain the different results. The use of HER-2/neu in serum as predictive factor in MBC treated with taxanes has been evaluated, albeit also with patients with PBC HER-2/neu + treated with Tmab and FEC contradictory results (69, 74, 150, 171 – 174) . Some of these and found that a decrease of 9 % from Week 3 to 6 was asso- studies using dose-intensifi ed paclitaxel monotherapy or dif- ciated with pathological complete remission. Similar results ferent combinations with paclitaxel have reported higher were found by Köstler et al. (159) in 16 PBC patients with response in patients with abnormal HER-2/neu serum levels tissue overexpression treated by Tmab and chemotherapy. (69, 170, 174) and others in those with negative HER-2/neu However, these studies clearly showed that HER-2/neu in serum levels with a shorter TTP (74, 172, 173) . These discrep- serum can not substitute tissue overexpression for the low ancies are also found when other chemotherapy regiments are sensitivity in early stages but may select patients with tissue used (131) with publications indicating a shorter OS in patients overexpression and a higher probability of response. with positivity in serum (113, 120, 131) or not (135) or by The relationship between HER-2/neu serum levels and contrast, lower response in those with abnormal values (120) . tumor response in MBC patients is confusing. Some stud- In summary, the results obtained are contradictory and ies have reported no correlation between baseline HER-2/ do not clarify the predictive value of HER-2/neu in serum. neu serum levels and response (158, 159, 161, 164, 175, 176, Different chemotherapy regimens have been studied in a 180) , while others have described a higher response (135, 138, lower number of patients making conclusions impossible. 144, 170, 177, 178) or higher TTP (144, 164) in patients with Likewise, the different criteria used do not facilitate under- high baseline levels and other groups have reported a higher standing the different conclusions made. The use of basal response or TTP in those with normal baseline levels (133, HER-2/neu serum levels is diffi cult because these may be 160, 164, 166 – 168, 176) . Baseline HER-2/neu serum levels different according to the tumor stage or sites of metasta- may be related to several factors indicated previously. Luftner ses. The best system to use tumor markers is one which is et al. (170) indicated that the probability of tumor response dynamic, habitually used and suggested for different publica- correlated with HER-2/neu serum levels: 23.1 % in patients tions and guidelines, as was previously indicated in the use of with normal levels ( < 15 ng/mL), 35.5 % in those with inter- HER-2/neu in therapy monitoring (Figure 3 ) (5, 6) . However, mediate levels (15 – 50 ng/mL) and 46.7 % in those with high response may only be observed in patients with abnormal lev- levels ( > 50 ng/mL). Most of these studies use 15 ng/mL as the els. A patient with normal HER-2/neu serum levels indicates cut-point, but Burstein et al. (176) suggested a higher response a tumor with low release of this antigen and modifi cations in in those with negative HER-2/neu, using 30 ng/mL as the the normal range do not indicate tumor response. cut-off. The number of patients included was also very differ-ent (20–322 patients), and only four studies included more than 100 patients, and only one suggested the utility as predictive Herceptin ® treatment
factor (20, 158, 164, 165, 178) . Discrepancies may be related to the fact that abnormal Her-2/neu serum levels refl ect tissue Tmab treatment has been used in neoadjuvant treatment, adju- overexpression but also tumor extension, with the highest val- vant treatment as well as in advance breast cancer therapy. ues in patients with more advanced stage and worse progno- There are only two small studies that evaluate the HER-2/neu sis. In theory, a patient with normal HER-2/neu serum levels serum levels in patients with neoadjuvant treatment including may refl ect a lack of overexpression (less response) but may Tmab and no correlation was found between baseline ECD also indicate smaller tumor mass (better response). By con- with response, although serial determinations of this marker trast, abnormal HER-2/neu serum levels may indicate over- were related (113, 159) . Mazouni et al. (111) evaluated 39 expression (higher response) but also larger tumor mass or Brought to you by Swets Download Date 9/19/12 5:00 PM 14 Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer visceral metastases (mainly in liver), that may explain a worse Tmab therapies are only used in patients with HER-2/ prognosis. In summary, the data published do not suggest the neu overexpression in tissue. Response in other patients is use of basal HER-2/neu as criteria for response in patients rarely studied and is habitually lower than 15 % . However, with tissue overexpression. To use these criteria different the selection of patients without tissue overexpression and cut-points will be required according to tumor extension response may be important. Previously we have indicated that and the site of metastases. 10 % – 30 % of patients without tissue overexpression had A higher homogeneity has been published in relation to abnormal serum levels. The use of ECD as the criteria for the use of serial HER-2/neu serum levels as a predictive fac- selection of patients for Tmab-based therapy, mainly in those tor (Figure 3 ). Most publications have reported a relation- without overexpression has rarely been studied. Fornier et al. ship between serial HER-2/neu and tumor response and TTP (161) reported response in 37 % of the 19 patients with IHC/ or OS (41, 134, 170, 178, 180) , including publications that FISH negativity and abnormal serum HER-2/neu. Ardavanis did not fi nd any relationship between basal HER-2/neu and et al. (177) studied 22 MBC patients with abnormal serum response (145, 161, 164, 177) or those with higher response HER-2/neu without HER-2/neu overexpression in tissue in patients with negative basal results (160, 165, 166, 177) . treated with Tmab and docetaxel or paclitaxel and reported a However, there are some discrepancies (158) . Lennon et al. signifi cant decrease in the serum HER-2/neu concentrations (158) evaluated serial HER-2/neu serum levels in 144 breast in four of fi ve patients with PR, in seven of 11 patients with cancer patients (IHC 2 + or 3 + ) treated with six different regi- stable disease and in only two of the 11 patients with pro- ments in four clinical trials including different treatments gression. Ali et al. (165) studied 53 IHC – patients (1 + or 2 + ) (from Tmab monotherapy to different combinations with or or unknown results and found that a signifi cant decrease was without Tmab), and mixed patients with MBC (sensitivity related to response. These studies included a small number of 62.2 % – 71.2 % ) and patients with locally advanced tumors patients but suggest the possibility to using Tmab in patients (sensitivity 31 % – 33 % ). The relationship between HER-2/neu without tissue overexpression and with abnormal HER-2/neu serum levels and tumor stage has been previously indicated serum levels. Comparison between HER-2/neu in serum at and it is very diffi cult to use HER-2/neu as a predictive fac- baseline and at 30 days may indicate a subgroup of patients tor in patients without metastases due to the lower sensitiv- with response to this therapy. Those with signifi cantly decreas- ity obtained. However, this study clearly showed that serial ing levels may continue treatment while in those with similar HER-2/neu serum levels were not related to tumor response, levels treatment (1 month) may be avoided. Other studies are with decreasing levels (day 42) in most of the patients with necessary to confi rm these preliminary data. abnormal basal levels. Patients with normal baseline levels Most evaluations of HER-2/neu in serum as a predictive only showed a slight change with therapy and not related factor are compared with overexpression in tissue with the to response. Lennon et al. (158) reported that only patients aim of determining whether evaluation in serum may substi- receiving Tmab monotherapy showed a clear relationship tute tissue evaluation. The results previously indicated clearly between response to therapy and initial changes in HER-2/ show that this is not possible; HER-2/neu in tissue is and may neu serum levels. be the selection criteria for Tmab treatment. However, only Most publications suggest the predictive value of serial 50 % – 60 % of patients with tissue overexpression respond to HER-2/neu serum levels but differ according to the criteria used, combined therapy. Most studies support the contention that such as response or the timing to determine the tumor marker serial HER-2/neu in patients with overexpression may suggest (Figure 3 ). Most studies suggest a 55 % – 77 % decrease as cri- patients without tumor response, at least those with progres- teria for response (41, 134, 144, 145, 160, 161, 170, 178, 180) . sion, and some suggest that this information may be obtained Ali et al. (165) retrospectively studied the data of 307 MBC in 15 days or 1 month. To avoid secondary effects in patients patients from seven institutions, most being 3 + (76.6 % ) or 2 + without benefi t of therapy may be one of the most important (10.42 % ) treated with Tmab-based therapy and found that evo- utilities of HER-2/neu in serum. However, this approach lution of serum HER-2/neu is an independent prognostic factor may be studied only in patients with abnormal serum levels. (p < 0.0001) with response in 57 % of patients with > 20 % (ROC Likewise, clear criteria, according to the time of serum deter- analysis) of decline (day 30) in contrast to 28 % in those with a mination should be established to discriminate response or decline < 20 % (p > 0.01), with a longer duration of response (369 days vs. 230 days) (p = 0.008) and TTP (320 days vs. 180 days, In summary, there are important discrepancies in the use p < 0.0001). Tmab-based therapy may produce important second- of basal HER-2/neu serum levels as a predictive factor for ary effects, and for this reason some authors have evaluated the Tmab therapy indicating that different factors may infl u- kinetics early after initiation of therapy, in an attempt to avoid ence the results. Most studies (41, 69, 115, 134, 160, 161, treatment and risk in patients without response. Köstler et al. 164 – 166, 177) support the use of serial determination of this (144) studied the kinetics of HER-2/neu in the 40 MBC patients tumor marker as a predictive factor, while others do not (158) . with abnormal levels treated by Tmab and chemotherapy and High HER-2/neu levels are rarely found in other situations found that those with decreasing concentrations higher than excluding breast cancer and they are related mainly to tis- 66.1 % on day 15 showed a higher response (67 % ) than those sue overexpression and tumor stage making their inclusion with a lower decrease (response 13 % ) (p = 0.009). Likewise, in dynamic criteria logical. The data presented previously patients with increasing levels (at least 10 % ) on days 15 or 22 clearly indicate that HER-2/neu serum levels cannot sub- had a signifi cantly higher risk of progression (p = 0.008). stitute HER-2/neu evaluation in tissue. However, the two Brought to you by Swets Download Date 9/19/12 5:00 PM Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer techniques may be complementary. Results in tissue decide Research funding: None declared.
treatment and HER-2/neu serum levels may be important Employment or leadership: None declared.
decision criteria in patients with increasing levels to fi nalize Honorarium: None declared.
unnecessary treatment and avoid toxicity. Likewise, patients with abnormal HER-2/neu serum levels and negativity in tissue may be reevaluated or the possibility of Tmab treat- References
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Willsher PC, Beaver J, Pinder S, Bell JA, Ellis IO, Blamey RW, serum tumor markers as predictive factors and cardiac surveil- et al. Prognostic signifi cance of serum c-erbB-2 protein in breast lance algorithm. J Clin Oncol 2003;21:2889 – 95. cancer patients. Breast Cancer Res Treat 1996;40:251 – 5. 177. Ardavanis A, Kountourakis P, Kyriakou F, Malliou S, Mantzaris Harris LN, Trock B, Berris M, Paik S. The role of I, Garoufali A, et al. Trastuzumab plus paclitaxel or doc- ERBB2 extracellular domain in predicting response to etaxel in HER-2-negative/HER-2 ECD-positive anthracycline Brought to you by Swets Download Date 9/19/12 5:00 PM Molina et al.: Serum levels of HER-2/neu oncoprotein in breast cancer and taxane-refractory advanced breast cancer. Oncologist regimen for human epidermal growth factor receptor-2 over- 2008;13:361 – 9. expressed metastatic breast cancer. Proc Am Soc Clin Oncol 178. Lieberman GL, Gourlay P, Klein S, Bruno R. Pharmacokinetic- pharmacodynamic relationships of single agent weekly trastu- Gasparini G, Sarmiento R, Amici S, Longo R, Gattuso zumab in patients with HER-2-overexpressing MBC. Proc Am D, Zancan M, et al. Gefi tnib (ZD1839) combined with Soc Clin Oncol 2003;22:20 (abstract 76). weekly epirubicin in patients with metastatic breast c á ncer. 179. Yeung K, Gupta R, Haidak D, Katzen H, Greer J, Quader A phase I study with biological correlate. Ann Oncol ML. Weekly herceptin and one hour taxol infusion 2005;16:1867 – 73. Rafael Molina Graduate is author or co-author of about 30 original published articles in Medicina (University of and about 100 communications and lectures to national and Barcelona in 1979) and spe- international symposiums. Since June 2004 he is Member of cialist on Clinical Pathology Tumor Markers Committee in the SEQC (Spanish Society of (Hospital Clinic 1983). Since Clinical Chemistry). Since 2009 he is member of the ISOBM 1996 is Assistant Professor of (International Society for Oncology and Biological Medicine). Biochemistry and Molecular Since 2010 he is member of the executive committee of the Biology of the University of Catalan colon cancer screening programme.
Barcelona and from 2002, president of the Biochemist Xavier Filella is doctor of Section of the Catalonian Medicine from the University Academy of Medical of Barcelona. He completed Sciences, Vicepresident and their studies on Clinical Board member of the ISOBM (International Society of Biochemistry in the Hospital Oncodevelopmental Biology and Medicine) and mem- Clinic of Barcelona (1985- ber of the editorial board of Tumor Biology and Journal of 1988), and there he was spe- Biological Markers. Likewise, Dr. Molina is the Chairman cialized in the fi eld of tumor an active member of the European Group on Tumor markers markers. At the present, he (EGTM) and has been contribute to the publication of guide- is Senior Consultant in the lines about the tumor marker use in Breast cancer and lung Department of Biochemistry cancer in this group and in the NACB (National Academy of and Molecular Genetics Clinical Biochemistry). Dr. Molina is author of 175 original (CDB) in the Hospital Clinic of Barcelona. He is leader of the research line on the study of tumor markers in prostate can- Josep M. Augé-Fradera is cer. He also is responsible of the Immunoassay Unit of Core graduated in Medicine at Laboratory. He is member of the International Society for the University of Barcelona Oncology and Biological Medicine and the European Gropu on 1999. Professional on Tumor Markers. He has published more than 130 origi-training carried out at the nal articles, and he has an accumulated impact factor of more Department of Biochemistry than 390 promised patients. He published numerous papers and Molecular Genetics, about tumor markers in several journals as J Int Cancer, Eur Hospital Clinic (University J Cancer, The Prostate or Urology. Specialist in respiratory of Barcelona). He specialized medicine and leader of the research line on the study of pul- in Clinical Biochemistry on monary infi ltrates in immunocompromised patients. He also July 2005. Since July 2005 evaluates the role of immunomodulating drugs, such as cor- is Member of Oncobiology ticosteroids, in the treatment of severe community-acquired Unit of the Hospital Clinic pneumonia. Every two years I organized a Course about the (Barcelona) and in charge of clinical use of Cytokines. This year I have participated in the the Barcelona screening colon cancer programme. As member organization of the X International Symposium on Biology of the Oncobiology Unit, he participates in the organization and Clinical Usefulness of Tumor Markers, of that I have been of a biennial Symposium (XIII edition on 2011) and Courses scientifi c secretary. I published various papers about PSA and on Tumor Markers (X edition on 2012). The Oncobiology cPSA in several journals as J Int Cancer, Eur J Cancer, The Unit is reference in clinical evaluations on Tumor Markers. Prostate or Urology.
He has participated in more than 35 national and interna-tional symposiums and collaborated in about 25 courses. He Brought to you by Swets Download Date 9/19/12 5:00 PM



JBC Papers in Press. Published on May 31, 2011 as Manuscript M111.253674 OPTOGENETIC CONTROL OF MOTOR COORDINATION BY Gi/o PROTEIN-COUPLED VERTEBRATE RHODOPSIN IN CEREBELLAR PURKINJE CELLS. Davina V. Gutierrez2, Melanie D. Mark1, Olivia Masseck1, Takashi Maejima1, Denise Kuckelsberg1, Robert A. Hyde2, Martin Krause1, Wolfgang Kruse1, and Stefan Herlitze1,2

Medicina, Psicologia, Biologia, Normativa e Scienze varie: tutto cio' che fa cultura SCIENZA E PROFESSIONE Anno 7 numero 10 Sospeso il Rosiglitazone (Avandia, Avandamet Avaglim) Incertezze sul rapporto rischio-beneficio. I pazienti dovranno essere guidati a modificare la cura, ma senza allarmismi. Multe effettuate sclerosi multipla Nulle dal 2007 in vero utile se il can-